Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies. Snakebite is a significant public health issue, as more than 5.4 million people are bitten annually, resulting in as many as 1.8 million envenoming incidents and 138,000 deaths, which primarily affect impoverished communities in the rural tropics and subtropics across Africa, the Middle East, Americas, Asia and Australasia ,3. SinceBecause the toxin composition of venomous snakes varies extensively among species ,7, the pSnakebite coagulopathy is the consequence of certain venom toxins consuming and/or increasing the abnormal activation of key clotting factors , ultimately leading to a loss of clotting capability ,12,13. ASerum-derived, polyclonal antibody-based antivenoms remain the gold standard therapeutic option for treating snakebite envenoming . DespiteAs a result of the aforementioned limitations, several attempts have been made to improve the cross-reactivity and dose efficacy of snakebite treatments over recent years, both via the optimisation of conventional polyclonal antivenoms  ,26,27 anCalloselasma rhodostoma venom [Deinagkistrodon acutus [Bothrops atrox [Macrovipera lebetina [E. coli) or yeast . However, some snake venom toxins can be challenging to successfully express in such systems because of the frequency of cysteine residues and disulfide bonds, resulting in significant obstacles relating to correct protein folding, solubility and yield [E. coli lack glycosylation apparatus, limiting their use if effector functions are needed [Crotalus durissus terrificus venom) [The expression of recombinant proteins has been historically undertaken in a wide variety of different host systems, including bacteria, yeast, plant, insect and mammalian cells. Several snake venom proteins, including ancrod from ma venom , acutin n acutus , batroxops atrox , and faclebetina  have prend yield ,34, evennd yield ,36. Furte needed , while ee needed . Consequs venom) . Mammalis venom) ,40,41, aCalloselasma rhodostoma, batroxobin from Bothrops atrox and RVV-V from Daboia russelii) were expressed in HEK293F mammalian cells for use as immunogens to generate experimental antivenoms. To this end, following functional validation of the resulting recombinantly expressed toxins in fibrinogenolysis experiments, a murine immunisation regimen was undertaken to generate polyclonal antibodies against each of these three toxins. Thereafter, we explored the in vitro immunological recognition and inhibitory capability of the resulting antibodies against the different recombinant toxin immunogens and a panel of crude snake venoms. Our findings demonstrated that polyclonal antibodies generated against specific venom toxins exhibited broad in vitro cross-reactivity with a geographically diverse array of native snake venoms and were capable of inhibiting certain toxin functional activities, including fibrinogenolysis and the prolongation of plasma clotting times. More broadly, our findings suggest that the recombinant expression of key, functionally important, snake venom toxins, could be a valuable approach to generate new pathology-specific antivenoms against snakebite or to enhance existing antivenoms via increasing antibody titres against specific pathogenic toxins.In this study, three fibrinogenolytic SVSPs sourced from distinct medically important viperid snake venoms (ancrod from C. rhodostoma (Genbank: L07308.1) [B. atrox (Genbank: J02684.1) [D. russelii (Genbank: MF289120.1) [http://www.ncbi.nlm.nih.gov/genbank/ (accessed on 7 May 2022)) and the SignalP-5.0 Server ) was used to detect and remove the presence of signal peptides. Integrated DNA Technologies  commercially synthesised sequences of inserts  for cloning into the expression vector  , batroxo02684.1)  and RVV-89120.1) . Coding n vector .®Inc., Hitchin, UK), 5 µL of each restriction enzyme EcoRI  and NheI   and 100 μL of nuclease-free water, followed by incubation for one hour at 37 °C in a water bath. Following brief centrifugation, dephosphorylation of the resulting digested vector was performed by the addition of 20 µL of shrimp alkaline phosphatase , 20 µL of 10× CutSmart buffer  and 60 mL nuclease-free water, followed by a further one-hour incubation step at 37 °C. Thereafter, samples were briefly centrifuged again and incubated for 30 min at 65 °C to deactivate the alkaline phosphatase. DNA inserts   were prepared using 500 ng of insert, 10 µL of 10× buffer 2.1: , 5 µL of the EcoRI  and NheI  restriction enzymes, and 100 mL of nuclease-free water, followed by incubation for one hour at 37 °C in a water bath and brief centrifugation thereafter.For the generation of plasmid vectors containing the toxin-encoding DNA inserts, we used commercially available pFUSE-hlgG1-Fc2 Plasmids (4194 bp) . Restriction digests were performed using 5 µg of the pFUSE-hlgG1-Fc2 vector, 10 µL of 10× buffer 2.1 . The running conditions were 50 V, 240 mA for 80 min, with downstream visualisation using a Gel Doc EZ Gel Documentation System (Bio-Rad).Digests for both vector and inserts were examined on 0.8% agarose  in 1× TBE buffer , pH 8.3). The solution was heated in a microwave until fully dissolved before the addition of 9 µL of ethidium bromide  and polymerisation in a Bio-Rad gel system. Gels were immersed in 1× TBE buffer prior to the loading of 50 μL and 200 μL of vector and insert, respectively, alongside 10 μL (0.5 μg) of 1 kb DNA ladder , as per the manufacturer’s instructions. The resulting insert and vector DNA concentrations were quantified by Nanodrop and the samples stored at −20 °C until downstream use.http://www.insilico.uni-duesseldorf.de/Lig_Input.html (accessed on 7 May 2022)) and prepared in ligation reactions consisting of 1 µL of 10× ligase buffer , 100 mM MgCl2, 100 mM DTT and 10 mM ATP) and 1 µL of T4 ligase (supplied in 10 mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol) , with the final reaction volume adjusted to 10 mL with nuclease-free water. Control reactions were also performed in parallel and consisted of vector alone without the insert. Ligation reactions were incubated at 16 °C overnight.The inserts were ligated into the vector using a 3:1 molar ratio. The necessary volumes of each insert for ligation with 1 µL of vector were calculated , following the manufacturer’s instructions. Thereafter, bacteria were streaked onto LB plates  , 4.5 g agar , 300 mL water, pH 7.5–7.8, containing 75 µL Zeocin  before incubation at 37 °C overnight. A single bacterial colony for each sample was then selected and used to inoculate 5 mL of LB media, before shaking incubation at 37 °C for 6–8 h at 220 rpm. Erlenmeyer flasks (125 mL) were prepared with 50 mL of LB media supplemented with 12.5 µL Zeocin . A total of 1 mL of resulting bacteria was added to each flask before shaking incubation at 37 °C and 220 rpm overnight. Thereafter, glycerol stocks were prepared using a 300 µL aliquot of the overnight bacterial culture mixed with 700 µL sterile 50% glycerol before storage at −80 °C. After that, overnight cultures were centrifuged at 6000× g for 10 min at room temperature and plasmid DNA was purified using the Hi-Speed Plasmid Midi Kit , following the manufacturer’s instructions. To determine the resulting DNA yield, DNA concentrations were measured via Nanodrop . DNA plasmids were then commercially sequenced via Sanger sequencing  to confirm insert fidelity.Next, ligated DNA was transformed using TOP10 2. Cells were originally seeded at 2 × 105/mL and grown over three days to 1–2 × 106/mL. Cell cultures were counted and checked for at least 90% viability using a Luna II automated cell counter . The cells were seeded at 7 × 105/mL for transfection, equivalent to 2.1 × 107 cells total in 30 mL medium. Cells were left to grow for 16–24 h, then 30 µg of DNA was placed into a sterile Eppendorf tube for each Erlenmeyer flask (125 mL). An equivalent volume of Freemax transfection reagent was added to a separate tube, and the volumes of each made up to 600 mL with Optipro SFM transfection medium (Gibco). Both 600 mL aliquots were mixed together and incubated for 12 min at room temperature. The mixture was then added to swirling cells in the Erlenmeyer flasks (125 mL), and the cells were left to grow for seven days at 37 °C, 8% CO2 on a shaking platform. After seven days, cell cultures were pooled and centrifuged at 500× g for five minutes to pellet the cells. Supernatants were filtered through a 0.22 µm pore membrane and stored at 4 °C before protein purification. Proteins were purified by hydrophobic affinity chromatography using an ÄKTA pure protein purification system . Supernatant was loaded onto a 1 mL HiTrap™ Chelating Sepharose™ Nickel column  in 40 mM imidazole in Na phosphate buffer 20 mM with 150 mM NaCl pH 7.4 and eluted with 500 mM imidazole, 150 mM NaCl, 20 mM sodium phosphate buffer pH 7.4. The proteins were eluted into 2 mL fractions, which were pooled and dialysed in Slide-A-Lyzer G2 Dialysis Cassettes (1–30 mL) 2 K molecular weight cut-offs (MWCOs) . This step was performed via three changes of 1× PBS (pH 7), first for two hours at room temperature, secondarily at room temperature overnight and then for two hours at room temperature again. Finally, the protein was concentrated using 30 K MWCOs concentrator tube  and centrifuged at 1684× g for 15–30 min. Protein concentrations were quantified by Bradford protein assay  by using 96-well flat-bottomed plates (NUNC) and a LabTech LT-4500 microplate absorbance reader at 595 nm.For protein expression, we used HEK293F cells and the Freestyle 293 Expression System . Cells were maintained in suspension, in Freestyle serum-free media, with culture conditions of 37 °C, 125 rpm and 8% CONext, we used each recombinantly expressed snake venom toxin in immunisation experiments. Prior to immunisation, we performed quality control experiments on the recombinant toxins, namely SDS-PAGE gel electrophoretic assessments of purity, and fibrinogenolytic and clotting time assessments of functional activity, using the methods outlined in full below. The murine immunisation experiments were undertaken with the approval of The Animal Welfare and Ethical Review Boards of the Liverpool School of Tropical Medicine and the University of Liverpool, under project license (#P58464F90) granted from the UK Home Office, and in accordance with the UK Animals (Scientific Procedures) Act 1986.Murine immunisation experiments broadly followed a recently outlined approach . ExperimCalloselasma rhodostoma (captive bred), Daboia russelii (Sri Lanka) and Bothrops atrox (Brazil). Lyophilised snake venoms were sourced from the historical venom collection stored in the herpetarium of the Centre for Snakebite Research and Interventions (CSRI) at the Liverpool School of Tropical Medicine. All experimental animals received 1 µg of toxin/venom at each immunisation , with the groups receiving the mixture of recombinant toxins or venoms receiving equal amounts  of the different toxins/venoms per contributing sample. All immunogens were prepared in sterile PBS and mixed in a 1:1 ratio with the Sigma Adjuvant System , to a final injection volume of 40 µL.Experimental animals were allowed to acclimatise for one week, and thereafter groups of five female CD-1 mice  were immunised with either: (i) recombinant ancrod, (ii), recombinant batroxobin, (iii) recombinant RVV-V, (iv) a 1:1:1 mixture of the three recombinant toxins, or (v) a positive control group consisting of a 1:1:1 mixture of three crude snake venoms sourced from g for 10 min. The resulting sera were stored at −20 °C until further use.Each experimental animal was immunised a total of five times, with secondary immunisations occurring two, four, eight and twelve weeks after primary immunisation. For immunisation, animals were briefly anaesthetised with an inhalational anaesthetic . Whilst under anaesthesia, the dorsal fur was shaved at the injection site, and all animal groups were subcutaneously injected with 1 µg immunogen in a total volume of 40 µL, with the first two immunisations delivered to a single injection site and thereafter split across two sites . The exception to this was the batroxobin immunogen group, for which the immunogen dose was increased during immunisation  and mice received 2 µg at the third and fourth immunisations (weeks 8th and 12th). Interim blood samples to profile immune responses were collected via tail tip sampling on weeks three, six, ten and a terminal sample was collected following euthanasia at week 14. All blood samples were allowed to clot for a minimum of two hours at room temperature and centrifuged at 23,548× g for 60 min at 4 °C. The resulting supernatant from each sample was dialysed separately using 35 mm 3.5 K MWCO SnakeSkin Dialysis Tubing  and sodium citrate buffered saline , with two changes of SCS, first at room temperature for one hour, and then secondarily at 4 °C overnight. Following dialysis, the final IgG samples were lyophilised using a Labogene lyophiliser and stored at 4 °C prior to reconstitution in PBS.The terminal murine serum obtained for each group were pooled and then IgG purified to produce the resulting experimental antivenoms . Pre-immunisation serum samples were processed in an identical manner to serve as the negative control . IgG was purified from pooled mice serum  using 14In addition to the immunogens (the three recombinantly expressed SVSP toxins and corresponding snake venoms), an additional ten snake venoms  and 7 µL tetra-methyl-ethylene-diamine (TEMED); stacking gel, 2.5 mL H2O, 1 mL 1 M Tris pH 6.8, 350 µL 40% bis-acrylamide, 30 µL 10% APS and 5 µL TEMED. Next, 10 µL of each recombinant toxin or venom (1 mg/mL) was mixed 1:1 volume/volume with reducing buffer  and heated for 10–15 min at 100 °C. Thereafter, 10 µL was loaded onto the gel, alongside 5 µL of broad molecular mass protein marker , and the samples were run at 200 volts for 55 min using a Mini-PROTEAN Electrophoresis System . The resulting gels were then stained at a final concentration of 0.1% (w/v) Coomassie blue R350  glacial acetic acid and 20% (v/v) methanol) overnight at room temperature. Gels were destained  for at least 120 min at room temperature and images captured using a Bio-Rad Gel Doc EZ Gel Documentation System.For gel electrophoresis, 15 well 15% SDS-PAGE gels were hand-cast, using the following approach: resolving gel, 3.75 mL HFor immunoblotting experiments, SDS-PAGE gel electrophoresis was performed as described above, except with the use of pre-stained protein marker (10–225 kDa)  and, instead of Coomassie staining, proteins in the gel were transferred onto 0.2 µm nitrocellulose membranes using a Trans-Blot Turbo Transfer System . Following confirmation of the transfer by reversible Ponceau S staining, membranes were blocked for non-specific binding using 5% non-fat dried milk in TBST , and left overnight at 4 °C on a rocker set at slow speed. Subsequently, blots were washed three times over 15 min with TBST, before the addition of primary antibodies  diluted to 1:5000 in 5% non-fat dried milk in 50 mL TBST for two hours at room temperature. The immunoblots were then washed in triplicate with TBST as described above and incubated for two hours at room temperature with appropriate secondary antibodies  diluted 1:2000 in 50 mL PBS. The immunoblots were then rewashed with TBST and developed by adding DAB substrate  by placing the membrane into the substrate for 30 s before washing with deionised water.To analyse the changes in molecular masses of glycosylated/deglycosylated recombinant toxins, we treated each recombinant protein with PNGase F. Briefly, recombinant toxins (14 μg) were mixed with 2 μL of 10× Denaturing Buffer  and ddH20 to make a 20 μL total reaction volume, before incubation at 100 °C for five minutes. Next, 4 μL of 10× G7 Reaction Buffer (500 mM Sodium Phosphate pH 7.5), 4 µL of 10% triton™ X-100 , 1 μL PNGase F  and 1 μL ddH20 were added and samples left to digest overnight at 37 °C. Reaction products were then analysed by reduced SDS-PAGE gel electrophoresis as described above, with 40 µL 2 × PLOB reduction buffer and 60 µL PBS added to the 30 µL of each deglycosylated sample.Microtiter 96 well ELISA plates  were coated with coating buffer  containing 100 ng per well of each immunogen and incubated overnight at 4 °C. Plates were then washed three times with TBST before adding 5% non-fat milk in 100 mL TBST. Following incubation at room temperature for two hours, the plates were washed another three times with TBST. Next, 120 µL of the primary antibodies (1 mg/mL) were added to the plate in duplicate at an initial dilution of 1:50 in 5% non-fat milk in TBST, followed by five-fold serial dilutions across the plate and incubation at 4˚C overnight. The plates were then rewashed with TBST and incubated for two hours at room temperature with appropriate secondary antibodies  diluted to 1:2000 in PBS. The plates were then rewashed with TBST before the addition of substrate  in citrate buffer  containing 0.015% hydrogen peroxide, Sigma-Aldrich, UK). Plates were gently mixed and incubated at room temperature for 15 min before the signal was read spectro-photometrically at 405 nm on a FLUOstar Omega microplate reader.C. rhodostoma venom, anti-ancrod was used). Next, ten well 8% SDS-PAGE gels were hand-cast using the following approach: 10 mL resolving gel ; 4 mL of 4% stacking gel . Thereafter, 10 µL of each sample was loaded on the gel and analysed under reducing conditions as described earlier. The negative control consisted of human fibrinogen only .To assess the fibrinogenolytic activity of the immunogens and their inhibition by the resulting experimental antivenoms, we used an SDS-PAGE gel electrophoresis approach using human plasma fibrinogen . The recombinant toxin immunogens  and various crude venoms  were incubated with human fibrinogen  for two hours at 37 °C. All immunogen samples were also used in neutralisation experiments following a pre-incubation step at 37 °C for 15 min with 1.75 µg (1 mg/mL) of the corresponding toxin-specific experimental antivenom  to serve as quality controls for the various reagents employed in the methods described below. To this end, normal and abnormal test controls were implemented for the experiments described below prior to experimentation. These consisted of 1) Diagen control plasma for fibrinogen: normal (RCPN070) and abnormal (RCPA080) and 2) control plasma for aPTT and PT: normal (IQCN130), abnormal 1  and abnormal 2 . All QC samples were aliquoted into 500 µL, stored at −80 °C, and defrosted in a water bath for five minutes at 37 °C immediately prior to use. To act as positive controls for the experimental antivenoms generated here, we used commercially available antivenoms, specifically the Thai Red Cross monovalent equine antivenoms directed against the Malayan Pit Viper C. rhodostoma  and the Russell’s viper D. siamensis  as controls for C. rhodostoma and D. russelli venom, respectively, and the Instituto Butantan polyvalent equine SORO anti-botropico/crotalico antivenom  for B. atrox. Antivenoms were reconstituted with pharmaceutical grade water supplied by the manufacturer, protein concentrations measured using a Nanodrop , and then stored short term at 4 °C until use. Normal horse IgG  and normal mouse IgG  were used as negative controls for the commercial antivenoms and experimental antivenoms, respectively, throughout.Blood samples for clotting profiling experiments were obtained according to ethically approved protocols  from consenting healthy volunteers who confirmed that they had not taken any anticoagulant treatments for at least three months prior to blood collection. Blood samples were collected in tubes containing acid citrate dextrose adenine (ACD-A) as an anticoagulant. To prepare Fresh Frozen Plasma (FFP), fresh blood samples were centrifuged at 2500× C. rhodostoma venom, B. atrox venom or D. russelii venom, or saline solution as the negative control. All immunogen samples were also co-incubated at 37 °C for 15 min with 0.5 µg of either: (i) the corresponding toxin-specific experimental antivenom , (ii) the anti-toxin mix experimental antivenom, (iii) the anti-venom mix experimental antivenom, (iv) the corresponding commercial antivenom  and (iv) normal mouse IgG as a non-immunised control. Thereafter, samples were added to the glass test tubes and gently tilted at regular intervals for precisely five minutes at 37 °C. Finally, 50 µL of 25 mM calcium chloride (pre-incubated at 37 °C) was added to each tube, and the tube was gently tilted until the resulting clot time was recorded. All experiments were performed in duplicate.To measure the inhibitory capability of the experimental antivenoms against recombinant toxins acting on the intrinsic and common coagulation pathways, we quantified differences in the activated Partial Thromboplastin Time (aPTT) between toxin and venom samples in their presence and absence. To do so, 50 µL of Micronised Silica/Platelet Substitute Mixture  was placed in glass test tubes (10 × 75 mm) in a water bath at 37 °C and incubated for 60–120 s. Next, 50 µL of FFP was spiked with 0.6 ng (100 ng/mL) of ancrod, batroxobin, RVV-V, C. rhodostoma venom, B. atrox venom or D. russelii venom, or saline solution as the negative control. All immunogen samples were also co-incubated at 37 °C for 15 min with 0.5 µg of either: (i) the corresponding toxin-specific experimental antivenom , (ii) the anti-toxin mix experimental antivenom, (iii) the anti-venom mix experimental antivenom, (iv) the corresponding commercial antivenom  and (iv) normal mouse IgG as a non-immunised control. Next, samples were introduced to the thromboplastin and time measurements were commenced, with tubes gently tilted at regular intervals (returning to the water bath between tilting), and the time for the formation of a clot recorded. All experiments were performed in duplicate.To measure the inhibitory capability of the experimental antivenoms against recombinant toxins acting on the extrinsic coagulation pathway, we quantified differences in the prothrombin time (PT) between toxin and venom samples in the presence and absence of the different antivenoms. Measurements of PT were undertaken by first adding 100 µL of Calcium Rabbit Brain Thromboplastin  to a glass test tube (10 × 75 mm) and incubating at 37 °C for 60–120 s in a water bath. Next, 50 µL of FFP was spiked with 0.6 ng (100 ng/mL) of ancrod, batroxobin, RVV-V, C. rhodostoma venom, B. atrox venom or D. russelii venom, or 0.9% saline solution as the negative control. All immunogen samples were also co-incubated at 37 °C for 15 min with 0.5 µg of either: (i) the corresponding toxin-specific experimental antivenom , (ii) the anti-toxin mix experimental antivenom, (iii) the anti-venom mix experimental antivenom, (iv) the corresponding commercial antivenom  or (iv) normal mouse IgG as a non-immunised control. Samples were then diluted tenfold with 0.02 M imidazole buffer (pH 7.35), transferred to glass test tubes (10 × 75 mm) and warmed at 37 °C for 120 s. Thereafter, 100 µL of thrombin reagent  was added, and time measurements commenced. Tubes were gently tilted at regular intervals (returning to the water bath between tilting), and the time for the formation of a clot was recorded. To calculate the fibrinogen concentration (g/L) from the clotting time, calibration curve analysis was used. All experiments were performed in duplicate.The Clauss method is a quantitative, clot-based assay that measures the ability of thrombin to convert fibrinogen to a fibrin clot, followed by manual time measurements of clotting . Here wep ≤ 0.05). Specifically, we tested whether the experimental antivenoms exhibited significantly increased inhibitory profiles over the negative  controls. All statistical analyses were performed using Prism v8 software (GraphPad).To statistically test whether experimental antivenoms, commercial antivenoms or normal mouse control antibodies inhibited the pro-coagulant activity of each toxin/venom in the PT and fibrinogen consumptions assays, we used one-way ANOVA with Dunnett’s multiple comparisons test and a significance threshold of  for ancrod, suggesting the production of several different glyco-forms  provided clear evidence that these two recombinant toxins stimulated the production of antibodies by the end of the experimental time course that recognised native venom proteins , and a control group that received a native venom mixture. To quantify antibody binding levels to the immunogens, serum samples were collected at weeks 3, 6, 10 and 14  and assessed via ELISA. The mice responded to the recombinant immunogens in a variable manner, though gradual increases in antibody binding levels were typically observed following primary immunisation until the end of the experiment . The excnisation F. Howeveproteins E,G. Highproteins H, unlikeC. rhodostoma venom and the three-venom mixture (B. atrox and D. russelii venom were very low (We used endpoint ELISA (EPT-ELISA) experiments to quantify the binding levels detected between the resulting experimental IgG antivenoms and the toxins and venoms used as immunogens. Comparisons of the binding profiles revealed that responses to ancrod, RVV-V and the mixture of the three recombinant toxins were highly consistent across the different experiential antivenoms generated and stimulated the highest binding titres A–D. Nota mixture E,H, respvery low F,G, perhC. rhodostoma, B. atrox and D. russelii in a highly comparable manner   , and theus spp.) . The posus spp.) .α and β chains [α and β chains, or preferentially cleave either the α or β chains, of fibrinogen [α chain of fibrinogen . While thrombin can convert fibrinogen to fibrin via cleavage of the β chains , venom Tbrinogen . During brinogen . Co-incu, but not against batroxobin or RVV-V . Our results showed that each of the recombinant toxins and corresponding crude venoms substantially prolonged the PT, with the crude venoms resulting in increased clotting time delays over each of the corresponding recombinant toxins , likely or RVV-V .In contrast to these findings using the PT, none of the recombinant toxins or crude venoms under study affected the aPTT, which measures clotting dictated by the intrinsic and common coagulation cascades, and thus inhibitory effects could not be measured .C. rhodostoma, B. atrox and D. russelii) all dramatically reduced fibrinogen levels compared to the control   . Noticeactively) . HoweverAntivenom is the only specific treatment for snakebite envenoming, and though these therapeutics save countless lives each year, they have several limitations that need to be overcome to ensure that effective, safe and affordable treatment is accessible for tropical snakebite victims. One of the main limitations with current antivenoms is that only about 10–20% of the antibodies present in them are specific to the toxins found in the venom immunogens ,27. FurtE. coli-expressed insoluble inclusion bodies, even under conditions intended to favour disulfide bond formation [C. rhodostoma, batroxobin from B. atrox and RVV-V from D. russelli. Expression of each protein resulted in acceptable yields (≈0.7 g from 1 L culture), purity and, since many SVSPs are variably glycosylated [In this study, we explored the potential utility of using recombinantly expressed toxins to generate polyclonal antibody responses directed against the haemotoxic SVSP toxin family. The SVSPs are enzymatic proteases that are common toxins in snake venom systems and have been studied for many decades due to their ability to interfere with haemostasis in a variety of ways . Many SVormation ,36. Suchormation ,32,53,54osylated ,56, evidC. rhodostoma venom particularly noticeable, as the anti-ancrod, anti-RVV-V and the anti-toxin mix antivenoms produced end-point titration ELISA binding profiles nearly comparable to the antivenom mix control antivenom against this venom , and also reduced the consumption of fibrinogen and the prolongation of the PT measured in plasma spiking experiments . AlthougHowever, perhaps somewhat surprisingly, we failed to observe any discernible advantage in combining the three recombinant SVSP toxins together as immunogens. The ‘anti-toxin mix’ antivenom resulted in highly comparable binding titres and toxin specificities to the anti-ancrod and anti-RVV-V antivenoms against the various immunogens and the panel of native snake venoms used in this study , while tn = 5) and ethical constraints), our analyses of the binding and functional neutralisation of SVSP toxins and crude venoms were limited to in vitro experiments. While there remain considerable issues associated with current preclinical models used to assess antivenom efficacy, including their questionable relevance for modelling VICC [Despite the promise of the findings presented herein, it is important to note that there are several limitations associated with this study. The most important of these is that, due to the restricted blood sample volumes collected post-immunisation (which were due to the relatively small sample size per immunogen group (ing VICC , these iThe capability of toxin family-specific antivenoms to broadly protect against venom-induced lethality may be restrictive, particularly when attempting to neutralise snake venoms where distinct toxin families contribute substantially towards severe pathology. However, a previous study that used a recombinantly expressed neurotoxin as immunogen, specifically a chimeric consensus short-chain α-neurotoxin three finger toxin, resulted in an equine experimental antivenom capable of neutralising the lethal effects of both specific neurotoxins and a variety of crude neurotoxic snake venoms . Thus, iOverall, our findings further strengthen the potential value of using recombinant venom toxins as immunogens to stimulate focused and desirable antibody responses capable of neutralising specific venom-induced pathological effects. Such tools are a welcome addition to the diversity of experimental approaches currently being explored to circumvent limitations associated with current antivenoms, with the long-term goal of dramatically improving therapeutics for the world’s neglected tropical snakebite victims.


This pathway is augmented by NHE1. Additionally, hyperosmolarity inhibited mTORC1 activation and cell proliferation. Thus, activation of p38-MAPK and WNKs is important for RVI and for cell proliferation.p38-MAPK is a stress-response kinase activated by hyperosmolarity. Here we interrogated the pathways involved. We show that p38-MAPK signaling is activated by hyperosmotic stimulation in various solutions, cell types and colonic organoids. Hyperosmolarity sensing is detected at the level of the upstream activators of p38-MAPK: TRAF2/ASK1 (but not Rac1) and MKK3/6/4. While WNK kinases are known osmo-sensors, we found, unexpectedly, that short (2 h) inhibition of WNKs (with WNK463) led to elevated p38-MAPK activity under hyperosmolarity, which was mediated by WNK463-dependent stimulation of TAK1 or TRAF2/ASK1, the upstream activators of MKK3/6/4. However, this effect was temporary and was reversed by long-term (2 days) incubation with WNK463. Accordingly, 2 days (but not 2 h) inhibition of p38-MAPK or its upstream activators ASK1 or TAK1, or WNKs, diminished regulatory volume increase (RVI) following cell shrinkage under hyperosmolarity. We also show that RVI mediated by the ion transporter NKCC1 is dependent on p38-MAPK. Since WNKs are known activators of NKCC1, we propose a WNK Moreover, recent studies have shown that p38 plays an important role in coordinating cell size (mass) and cell cycle progression in animal cells: small cells display elevated p38 activity and spend more time in G1 phase than larger cells, an effect lost upon inhibition of p384.In mammalian cells, the Mitogen Activated Protein Kinase (MAPK) proteins belong to three major classes of Ser/Thr kinases: ERK, JNK, and p38 MAPK . They orchestrate different cellular functions including proliferation/growth, differentiation, survival and stress response6. In the absence of bicarbonate, RVI is propelled by influx of Na+ and Cl−, mediated by ion transporters such as the Na+/H+ antiporter (NHE1 or 2) or the cotransporter Na+/K+/2Cl− (NKCC1 or 2), followed by water8. Interestingly, hyperosmotic stress also activates p3811, and p38 was shown to be essential for RVI13Incubating cells in hyperosmotic solution leads to cell shrinkage followed by volume recovery by a process called regulatory volume increase (RVI)14. Moreover, we showed that stable knockdown of NKCC1 led to reduced cell size/volume, inhibition of p3814 and impaired RVI. We thus hypothesized that p38 may mediate the positive effect of NKCC1 on RVI.Our recent work demonstrated that NKCC1 provides a link between cells mass and cell volume regulation by regulating both cell volume and mTORC1 activation17. However, the relationship between p38 and WNK -mediated osmo-sensing and contribution to RVI is/are unknown.In addition to p38, WNK kinases (the upstream activators of SPAK/OSR1) are also known to be stimulated by hyperosmolarity-induced cell shrinkage and to regulate RVIsuppression of p38, via regulation of TAK1 or TRAF2/ASK1. This suppression is transient, however, as long-term inhibition of WNK inhibits p38, supporting the finding that both p38 and WNK work in concert  to promote RVI. Moreover, hyperosmolarity severely reduces cell proliferation likely by suppressing mTORC1. WNK inhibition also attenuates cell proliferation, while p38 inhibition has only a small effect, likely due to its maintenance of high mTORC1 activity.Thus, given the importance of p38 in regulating cell volume, cell mass and cell cycle, we investigated the mechanism and pathways responsible for the regulation of p38 by hyperosmolarity, and the possible link between p38 and the WNK kinases, especially in regulating RVI. Our work identified the upstream hyperosmotic sensors of p38 activation, and provides a surprising finding that a short-term exposure to hyperosmolarity leads to WNK kinases-mediated 11, as we confirm here as well , compared to 135 mM (315 mOsm) in the iso-osmotic controls. Our results show that all these hyperosmotic solutions were able to activate p38 (p-p38) and its downstream target MK2 (p-MK2)  is ~ 30 mM in the 580 mOsm NaCl solution, versus. ~ 5 mM [Na+]i in the 580 mOsm NMDG-Cl solution  Fig.  with esc21. Hence, we tested the effect of hyperosmolarity on the p38 upstream activators MKK3/MKK6 and MKK4 in HeLa cells. Our data show that active, phosphorylated p-MKK3/p-MKK6 and p-MKK4 levels were all elevated in hyperosmotic solutions  in HeLa cells, as also observed (for p-MKK3/p-MKK6) in HEK293 cells, Human Primary Bronchial Epithelial Cells and in colonic organoids  in response to hyperosmotic stress. Our results show that TRAF2 is phosphorylated and activated in hyperosmotic solutions  not only did not suppress p38 activation under hyperosmotic conditions, but actually enhanced it  with shRNA Fig. A. Collec28, and is inhibited by WNK129. As seen in Fig. 30. As indicated above, WNK463 also increased the association of TRAF2 with ASK1  by which WNKs inhibit p38 activation, we focused on TAK1, a kinase that under immune stimulation activates the p38 pathwayThese results suggest that WNKs inhibit the p38 pathway by suppressing TAK1, an activator of MKK3/6 and hence of the p38 pathway, as well as by reducing TRAF2/ASK1 interactions  WNK463 treatment stimulated p38 activity, we found that a long term WNK463 treatment actually Both p38 and WNK kinases are known osmo-sensors activated by hyperosmolarity, which promote RVI. This raises the question as to how they can both promote RVI if WNKs inhibits p38. To address this potential contradiction, we analyzed the contribution of p38, WNKs, or both, to RVI in our HeLa cells, the time course of their effect on RVI, and the correlation of the RVI time course to the time course of WNK-mediated p38 inhibition.For the RVI experiments, HeLa cells were incubated in hyperosmotic solution, which led to an immediate cell shrinkage, as expected , and proposed that hypertonicity induces p38 via Rac1–OSM–MEKK3–MKK3–p38 pathway24. However, work from another group demonstrates an opposite result: Knockdown of Rac1 or OSM by siRNA increases p38 phosphorylation rather than decreases it34. Our own work also showed that inhibiting Rac1 with the inhibitor NSC23766 did not abolish p38 activation in hyperosmotic solutions . Thus, it is not clear under what conditions, or cell types, is Rac1 an upstream osmo-sensor of p38.The activation of p38 by hyperosmolarity has been well studied in the past decades36, is the osmo-sensor for p38 activation. Surprisingly, in our studies TNFR1 depletion induced rather than inhibited p38 activation under hyperosmotic stress. Thus, the exact mechanism that promotes TRAF2/ASK1 complex formation induced by hyperosmolarity in our cells is so far unknown.Instead, we focused on other upstream activators of p38. Our result demonstrated that the interaction between TRAF2 and ASK1 (MAP3K5) is important for activating the p38 pathway in cells exposed to hyperosmolarity. We also investigated if TNFR1, a known upstream activator of the TRAF2/ASK1 complexWe also demonstrate that TAK1 (MAP3K7) is another important MAP3K that induces p38 activation in cells under hyperosmotic stress, although it is not yet known how TAK1 is activated by hyperosmolarity. In addition, we show, unexpectedly, that a short-term (2 h) treatment with WNK463 leads to p38 hyperactivation by inducing TAK1 phosphorylation, as well as by promoting ASK1/TRAF2 association, under hyperosmotic stress. This suggests that normally WNKs can inhibit p38 via suppressing TAK1 or ASK1/TRAF2 binding. However, in cells treated for 2 days with WNK463, TAK1 and p38 activation were reduced to levels similar to the untreated cells. MK2, the downstream target kinase of p38, was actually suppressed upon such long-term WNK463 treatment. The reason behind this observation is still unclear, but may represent a compensatory effect involving genomic or protein synthesis effects (see below). However, we cannot preclude the possibility that a long term WNK463 treatment affects other proteins aside from WNKs, which can, in turn, affect TAK1, p38 and/or MK2 activity. Of interest, in colonic organoids, WNK inhibition alone (even in the absence of hyperosmolarity) was sufficient to enhance MKK3/6 and p38 activation, revealing an exquisite sensitivity of p38 signaling to WNK in these cells. Currently, we do not know the biochemical pathway(s) involved, although it/they are clearly independent of SPAK/OSR1.https://www.biorxiv.org/content/10.1101/2022.01.10.475707v1) it was demonstrated that hyperosmotic stress leads to sequestration of WNK1&3 in membrane-less droplets due to molecular crowding by the reduced cell volume; this promotes signaling that activates NKCC1, to induce RVI. An obvious question is how under these conditions is WNK1 (or WNK3?) able to inhibit p38? Is it via activation of signaling involving pSPAK/pOSR1 that then activate NKCC1? Or is p38 also clustered in liquid droplets along with the WNK. Future experiments will address these questions.In a recent study  has no effect on RVI regulation. However, a long-term inhibition (2 days) of p38  or WNKs, attenuate RVI. This result suggests that WNKs and p38-mediated regulation of RVI requires other downstream components, and possibly also genomic effects that lead to enhanced transcription of key genes and/or increased translation of critical proteins, yet to be identified.37, is an activator of p38, suggesting a WNK- > NKCC1- > p38 pathway that promotes RVI in cells. However, we noted that WNKs inhibition did not lead to complete blockade of RVI. A possible explanation for this observation could be that p38 activation was not completely blocked by the long-term WNKs inhibition. In addition, earlier studies indicated that NHE1 activity can be induced by p3840. Thus, it is possible that a long-term WNK463 treatment, in which p38 is still active in our cells , promotes NHE1 activity, allowing partial RVI to proceed. Moreover, the short-term WNK463 treatment, which failed to inhibit RVI, may also be explained by an increase of NHE1 activity induced by the hyperactivated p38. Thus, in order to completely block RVI in our cells, both the WNKs and NHE1 have to be inhibited together  needed to restore cell volume. We recently showed that NKCC1 inhibits mTORC1 by blocking LAT1 and the Akt/Erk pathways (which activate mTORC1), and by promoting p38 (which inhibits mTORC1)ere Fig. A, and as16, and Subramanya, unpublished). Since we showed that in our own generated stable HeLa WNK1-KD cells p38 activity is elevated, the obvious question is why a long-term (2 day) treatment of cells with WNK463 leads to a reduction in p38 activation in our current study, while WNK1 knockout or knockdown results in elevated p38 activity. It is possible that long-term treatment with WNK463 has a strong effect on the other WNKs (e.g. WNK 3—known to be sensitive to hypertonic stress and involved in RVI41), which in-turn exert their effects on p38. Lastly, we cannot preclude the possibility that a long-term WNK463 treatment may also affect other proteins in addition to WNKs.A recent study showed that NKCC1-dependent RVI was impaired in WNK1 knockout HEK293 cells, especially when treated also with a NHE1 inhibitor  effect on inhibiting cell proliferation, only observed after 5 days. It is thus possible that the sustained mTORC1 activation following p38 inhibition is responsible for the almost normal, un-attenuated cell proliferation in our experiments. In agreement, in our previous study we demonstrated an increase of cell division rate/cell proliferation when NKCC1 was depleted14. Since NKCC1 depletion suppresses p38 activation, similar to p38 inhibition with a drug, this suppression promotes mTORC1 activation, as we show here and in our recent paper14; this, in turn, induces cell proliferation.Our work also shows that hyperosmotic stress strongly decreases cell proliferation rate, consistent with other studiesTaken together, our current study demonstrates that there is a tight connection between the WNK and the p38 pathways, with WNKs regulating the p38 pathway. Both pathways are important in regulating volume recovery (RVI) and cell proliferation.Reagents and solution: see Tables HeLa cells were transfected with human TNFR1 shRNA construct , ASK1 shRNA construct  and WNK1 shRNA construct . The cells were then selected with Puromycin (2 µg/ml) 24 h after transfection. Medium was changed every 2–3 days. Once the transfected cells were established and expanded, TNFRα, ASK1, or WNK1 knockdown was verified by immunoblotting.14), were washed in PBS, and treated with either iso-osmotic or hyperosmotic solutions ). Proteins were then separated on SDS-PAGE and immunoblotted with the indicated antibodies. All immunoblots were imaged using the Odyssey Imaging system  and quantified using Image Studio version 5.2 (LI-COR).HeLa, HEK293, MDCK, human primary bronchial epithelial cells, or mouse colonic organoids , WNK1 knockdown (KD) or NKCC1 KD) cells were grown in 6 well tissue culture plates. Cells were dissociated using trypsin and treated with either iso-osmotic or hyperosmotic NaCl solutions Table  for the + ([Na+]i) and K+ ([K+]i) measurements, cells were grown in 10 cm tissue culture plates and treated with the various osmotic solutions (Table 3. The [Na+]i and [K+]i were measured using a PinAAcle 900F Atomic Absorption Spectrometer (PerkinElmer) and then normalized to cell number and cell volume.For intracellular Nans Table  for 15 m4 cells per well in a 24-well plate. After the measurement of cell number on day 1, hyperosmotic cellular medium  > 0.05, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001, using unpaired student t-tests).All immunoblots were imaged using the Odyssey Imaging system and quantified using Image Studio version 5.2 (LI-COR). Each experiment was performed for at least three independent times. Statistic analysis was performed by using GraphPad Prism 8. Histogram bars represent mean ± s.e.m. Supplementary Information.


Trypanosoma cruzi and T. brucei, respectively, pose relevant health challenges throughout the world, placing 65 to 70 million people at risk each. Given the limited efficacy and severe side effects associated with current chemotherapy, new drugs are urgently needed for both diseases. Here, we report the screening of the Pathogen Box collection against cruzain and TbrCatL, validated targets for Chagas disease and Human African Trypanosomiasis, respectively. Enzymatic assays were applied to screen 400 compounds, validate hits, determine IC50 values and, when possible, mechanisms of inhibition. In this case, 12 initial hits were obtained and ten were prioritized for follow-up. IC50 values were obtained for six of them (hit rate = 1.5%) and ranged from 0.46 ± 0.03 to 27 ± 3 µM. MMV687246 was found to be a mixed inhibitor of cruzain (iK = 57 ± 6 µM) while MMV688179 was found to be a competitive inhibitor of cruzain with a nanomolar potency (iK = 165 ± 63 nM). A putative binding mode for MMV688179 was obtained by docking. The six hits discovered against cruzain and TbrCatL are of great interest for further optimization by the medicinal chemistry community.Chagas disease and Human African Trypanosomiasis, caused by  Despite being described over a hundred years ago, Chagas Disease  and Human African Trypanosomiasis  are still relevant public health problems . While CT. b. gambiense strain, with efficacy similar to NECT [T. b. rhodesiense strain [T. b. gambiense and T. b. rhodesiense, and either in early or late stages is highly demanded [Available treatments are suboptimal for both diseases. Benznidazole and Nifurtimox, the current chemotherapeutic options for CD, are only effective in the acute phase and are associated with significant adverse effects, which include gastric and neurological disorders ,11,12,13 to NECT ,24,25, ae strain . Therefodemanded .Trypanosoma cruzi, is a validated [TbrCatL, a homologous protease from Trypanosoma brucei, is also a validated target for discovering trypanocidal compounds [TbrCatL might be relevant for the parasite to cross the blood-brain barrier [TbrCatL inhibitors have been reported, such as thiosemicarbazones [Cruzain, a cysteine protease of alidated ,27,28,29alidated  for a realidated . TbrCatLompounds ,33. Cruzompounds  and is iompounds , cellulaompounds ,37, and ompounds . TbrCatL barrier , althoug barrier . Several barrier ,42, nitr barrier ,44, amin barrier , benzimi barrier ,47, viny barrier ,49, anal barrier , quinazo barrier , and car barrier . Likewisrbazones , bromoisrbazones , nitrilerbazones ,56, thiarbazones ,58, triarbazones , macrocyrbazones , vinyl krbazones , and vinrbazones . Simultarbazones ,60,63,64TbrCatL inhibitors [https://www.mmv.org/mmv-open/pathogen-box/about-pathogen-box, accessed on 3 February 2023). PB comprises of 400 drug-like compounds active against parasites that cause NTD and with low-cytotoxic. Some of the molecules originally included in the PB due to in vitro activity against other parasites other than trypanosomatids were later reported to show trypanocidal activity [One of the strategies for discovering new lead inhibitors for a target is screening diverse, curated compound sets. For instance, the Medicines for Malaria Venture (MMV) partnership organized the Malaria Box, a compound set for catalyzing NTD research . The Malhibitors . More reactivity ,67.TbrCatL as targets, we screened and validated hits from the PB against both enzymes. Out of 400 compounds, we describe six hits with IC50 in the low micromolar to nanomolar range against both enzymes, including one competitive inhibitor. These molecules will be very relevant for future medicinal chemistry efforts. Given the relevance of CD and HAT and the attractiveness of cruzain and www.mmv.org, accessed on 30 May 2020). HepG2 cytotoxicity data (CC20 and/or CC50) are also provided for approximately three-quarters of the compounds. Data on other assays for selected compounds were obtained from the CHEMBL database [PB data spreadsheets containing chemical structures, SMILES, molecular weight, cLogP and biological activity were retrieved from the Pathogen Box website (database .The 400 compounds from the Pathogen Box were supplied in 96-well plates containing 10 mM frozen dimethylsulfoxide (DMSO) solutions. Solid samples of the 10 following compounds were resupplied by Evotec upon request to the MMV: MMV688179, MMV688271, MMV667494, MMV634140, MMV690027, MMV690028, MMV688362, MMV085499, MMV687246, MMV688466, MMV687812, MMV688466, and MMV085499.TbrCatL, respectively. In vitro activity of proteases cruzain and TbrCatL was assayed as previously described [Km = 0.5 ± 0.1 μM against cruzain and Km = 0.5 ± 0.1 μM against TbrCatL [Allison Doak and Prof. Brian Shoichet  and Prof. Conor Caffrey  generously provided recombinant cruzain and escribed ,63. Brie50 curves were obtained from a non-linear fit of at least seven distinct compound concentrations, in triplicate. Reported IC50 values are the mean and SEM of two independent measurements.For the initial screening, compounds were assayed at 5 μM. Compounds were selected for further assays based on the combination of the inhibition observed, novelty, and diversity (see details below). IC50. When analyzing the effects on the inhibition of the concentration of Triton X-100 and pre-incubation of the compound with BSA, differences higher than 20% were considered [To assess the effects of the Triton X-100 concentration on cruzain inhibition, assays were performed using 0.001%, 0.01%, and 0.1% Triton X-100. To evaluate the impact of pre-incubation with bovine serum albumin (BSA), 50 µL of a solution of the compound in the assay buffer containing 0.001% of Triton X-100 was incubated with 4 mg/mL of BSA (Sigma-Aldrich) for 10′ in a 96-well plate. Next, 50 µL of a solution containing circa 2 nM of cruzain in the same buffer was added to each well and incubated for another 10′. Finally, 100 µL of a solution of 5 µM Z-FR-AMC in the same buffer was added to each well and the fluorescence was immediately read. The final compound concentration varies and was selected to be near the compound ICnsidered .50 value, two concentrations higher and two concentrations lower than the IC50) and in the absence of the compound. Non-linear regression to Michaelis-Menten models and linear regression for the Lineweaver-Burk plot were performed. iK was estimated by both methods. The effect of the inhibitor concentration on the Km app was evaluated using the General Linear F-test.The mechanisms of inhibition were determined using seven substrate concentrations  and five inhibitor concentrations   using DaThe synthesis and characterization of compounds 1–5  and 6–7 https://www.chemaxon.com, accessed on 8 December 2022), was the +2 protomer. MMV688179 geometry was optimized at ab initio level using HF/6-31G* in Psi4 [The crystallographic structure of cruzain was obtained from RSCB PDB (ID: 3KKU ) and had in Psi4 . Docking in Psi4 . The griTbrCatL, we initially screened the 400 compounds in the PB library at 5 μM against both enzymes  inhibited either enzyme by at least 40% %; 10′ inc: (58 ± 44)%, TbrCatL after 10′ incubation ((73 ± 1)% vs. (42 ± 3)%). MMV690028 was prioritized because of its TbrCatL inhibition (49 ± 4% without pre-incubation). MMV690027 was deprioritized because of its low cruzain inhibition ((6 ± 2)% without pre-incubation; (0 ± 6)% after 10‘ pre-incubation) and because it was supplied as a racemic mixture. MMV085499 and MMV687246 were prioritized based on the TbrCatL inhibition results after pre-incubation ((46 ± 5)% and (49 ± 4)%, respectively). MMV688179, MMV688271, and MMV688362 have been previously assayed against T. cruzi and T. brucei and showed IC50 between 0.2 µM and 14 µM [TbrCatL 10′ inc). Compound MMV676512 (TbrCatL velocity after 10′ pre-incubation). Thus, we also selected compounds MMV676409 and MMV676512 for further investigation. Hits were chosen for follow-up based on the combined analysis of their novelty concerning known inhibitors, chemical diversity within the set, and potency in the initial screening. We initially considered for further investigation the 12 compounds that inhibited either cruzain or cubation , Table 1nhibitor . The arynd 14 µM ,78 % inhibition), but not potent enough for IC50 determination. The remaining six compounds inhibited either or both enzymes to a 90–100% extent, and we determined their IC50 against both enzymes with and without pre-incubation , suggesting that none of the compounds is a time-dependent inhibitor. Furthermore, because none of these compounds bears electrophilic moieties, the inhibition likely is a fast-reversible, non-covalent one. Therefore, the IC50 and Ki values can capture the potency of these compounds. In addition, potencies towards cruzain and TbrCatL were similar for all compounds, except for MMV688179, which was the most potent cruzain inhibitor (IC50 0.46 ± 0.03 µM 0’ and 0.53 ± 0.03 10′ inc) but had 10-fold higher IC50 against TbrCatL (IC50 = 4 ± 1 µM 0’ and 5 ± 2 uM 10′ inc). MMV688362 was the most potent TbrCatL inhibitor (IC50 2.9 ± 0.8 µM 0’ and 2.3 ± 0.1 µM 10′ inc). Overall, IC50, overall the results do not suggest aggregation. Importantly, results for MMV688179, the most potent cruzain inhibitor, do not suggest aggregation properties near the IC50. To investigate if inhibition was due to colloidal aggregation, a common cause of unspecific enzyme inhibition in in vitro assays , we emplKi = 57 ± 6 µM .Inhibition mechanisms against cruzain could be determined for MMV688179 and MMV687246 . For MMV7 ± 6 µM A. The la7 ± 6 µM B. Accordof Kmapp F, for whMotivated by the high potency and competitive mechanism of MMV688179 against cruzain, we sought to obtain preliminary SAR data on this scaffold. We used a similarity search to find analogues bearing a similar diphenylfurane core in an in-house library (BraCoLi) and obtained seven analogues that were available in our stocks . UnfortuDue to the competitive mechanism and the high potency of MMV688179, we employed molecular docking to propose a binding mode for this molecule. Overall, the ten docking poses were similar and the best-scored one was selected by visual inspection as the possible binding mode of MMV688179. In this binding pose, one of the protonated guanidines is buried in the S2 subsite and an ion-ion interaction with Glu208 is suggested. The other protonated guanidine forms hydrogen bonds with the main chain of residues Gly20 and Cys22, both at the S1 subsite . In addiPlasmodium , Mycobacterium , and kinetoplastids , with the remaining 19% being active against other pathogens [50 of 27 µM against T. cruzi [50 against cruzain we report here, cruzain inhibition is possibly related to its trypanocidal activity. This may also be true for MMV688362 (EC50 against T. cruzi reported on PB: 13 µM). Both MMV688179 and MMV688362, however, are known to bind to the DNA minor groove, as shown by SPR, ITC, CD, and Tm experiments [T. brucei. Our findings regarding cruzain inhibition suggest, thus, a possible dual mechanism of action. To what extent the trypanocidal effect is derived from the cruzain or DNA binding effect is still to be evaluated by further studies. Finally, it is worth noting that multi-target drugs are an interesting strategy in medicinal chemistry, possibly leading to improved efficacy and overcoming drug resistance [Regarding NTD drug discovery, phenotypic approaches seemed to have been overshadowed by target-based ones over the second half of the 20th century. Their apparent historic contrast has then evolved and merged into complementarity . First-iathogens ,77. MostT. cruzi , and giveriments ,85, whicsistance ,87.TbrCatL we reported upon screening MMV’s Malaria Box [Pro, a cysteine protease from another family, yielding 2 hits (hit rate = 0.5%). Interestingly, MMV688179 was one of the hits with an IC50 of 1.6 µM against MPro [Pro activity is likely due to specific interactions with the enzyme. This is also in line with the lack of MMV688179 inhibition against two unrelated human proteins ferrochelatase and porphobilinogen deaminase [50 of MMV688179 is 11.6 µM [The overall hit rate of 1.5% (6 out of 400) observed in our screening was similar to the 1% (4 out of 400) hit rate against cruzain and aria Box . PB was eaminase . As the  11.6 µM , the sel 11.6 µM ,51, sugg 11.6 µM ) and hum 11.6 µM ), a highTbrCatL in the 2–4 µM range.It is worth noting that here we report MMV688179 to be a novel, non-covalent cruzain inhibitor bearing a competitive mechanism and nanomolar potency. Unfortunately, we could not determine the potency of a first round of analogues due to interference of the small molecules with the assay readout. Nevertheless, MMV688179 is an interesting candidate for hit-to-lead optimization. This is also true, to a lesser extent, for MMV688362 and MMV687812 which inhibited cruzain and TbrCatL. In summary, three main contributions arise from this work. First, our results shed light on possible trypanocidal mechanisms of some of the PB compounds. Second, we add to the evidence that screening diverse and curated, yet small libraries are a useful strategy for discovering new leads. Third, we provide the drug discovery community with novel hits for drug discovery targeting cruzain and TbrCatL, two validated targets for discovering leads against T. cruzi and T. brucei, respectively. From the 400 compounds in the library, we obtained 12 hits and validated six of them as inhibitors of cruzain and TbrCatL. Particularly, MMV687246 is a mixed inhibitor of cruzain with a iK of 57 ± 6 µM and MMV688179 is a competitive inhibitor of cruzain with a iK of 165 ± 63 nM. We also proposed a possible binding mode of MMV688179 to cruzain to aid further optimization efforts. Hit-to-lead optimization of MMV688179 should focus on increasing selectivity towards T. cruzi and improving its potency against cruzain may be an attractive strategy in that context. We believe that the molecules discovered in this work, especially MMV688179 are candidates for optimization. CD and HAT remain relevant, life-threatening diseases affecting mainly disadvantaged populations worldwide. Here, we screened Pathogen Box against cruzain and


Graphene oxides (GOs) are a popular graphene alternative. The goal of this study was to compare the biocompatibility of a diversity of well-characterized GOs. Our previous work advanced developmental zebrafish as a model to interrogate the interactions and biological responses following exposures to engineered nanomaterials (ENMs). Here, we investigated GO 250 nm × 250 nm (sGO), 400 nm × 400 nm (mGO), and 1 μm × 1 μm (lGO), partially reduced GO (prGO) 400 nm × 400 nm, and reduced GO (rGO) 400 nm × 400 nm and 2 μm × 2 μm, which first underwent extensive characterization under the support of the Nanomaterials Health Implications Research (NHIR) Consortium. GOs were stabilized in water (GOs), while prGO and rGOs were dispersed in sodium cholate. Zebrafish were statically exposed to up to 50 μg/mL of each material from 6 h post-fertilization (hpf) until 120 hpf. Toxicity was dependent on GO properties. mGO was the most toxic material; its effects manifested in the yolk syncytial layer (YSL). Additionally, sodium cholate stabilization significantly increased GO toxicity. The observed effects were size- and oxidation-state-dependent, revealing the importance of identifying the structure-specific toxicity of GOs. Graphene nanomaterials are composed of carbon atoms arranged in a two-dimensional hexagonal lattice, making them useful for applications in advanced electronics due to their high conductivity and large surface area . The higDue to their ease of modification throughout production, GOs have diverse medical applications. For example, GOs loaded with doxorubicin have been used to deliver the chemotherapeutics directly to cancer cells in vitro ,8. GOs aDanio rerio) are well established for toxicological applications due to their small size, low husbandry cost, rapid development ex utero, optical transparency early in development, and molecular and genetic similarity to humans .,22.21,22Pronase  was used to remove the chorion of embryos at 4 h post-fertilization (hpf) in an automated dechorionator . FollowiFollowing the procedure above, the toxicity of sodium cholate was determined using 96-well plates containing 100 μL per well of varying concentrations  . EmbryosIncidences of mortality and abnormal morphology were recorded at 24 hpf and 120 hpf for 18 endpoints: eye, snout, jaw, notochord, heart, somite, brain, fin, otic vesicle, yolk sac, trunk, body axis, swim bladder, pectoral fin, caudal fin, pigment . An obseThe photomotor response behaviors of 120 hpf larvae following GO exposure were assessed using a ZebraBox system that tracked larval movement, and motion analysis was performed by the ZebraLab software . Light/dark (visible/infrared) cycles were 3 min each and repeated 4 times, with only the last light cycle used for data analysis, as it typically has the highest signal:noise ratio. Behavior data analysis was performed using a modified version of a previously described protocol .n = 12). Statistical significance was determined using a t-test (α = 0.05) that compared UltraPure™ water-exposed  embryos to GO-exposed embryos.Zebrafish embryos exposed to 50 μg/mL of a GO in individual wells of a 96-well plate were screened hourly from 6–30 hpf (24 h) to identify the onset of significant embryo mortality .An acridine orange (AO)  assay was used to visualize an apoptotic response in 6 hpf zebrafish embryos following exposure to mGO. First, embryos were exposed to 50 μg/mL of mGO for 30 min, washed 3 times with 100 μL of UP, followed by an addition of 100 μL of AO to a final concentration of 5 μg/mL. The embryos were incubated in AO at room temperature for 1 h, washed 3 times with UP to remove AO residues, and euthanized with tricaine. Embryos were immediately imaged using a rhodamine filter and brightfield imaging in a Keyence BZ-X700 fluorescence microscope.Dichloro-dihydro-fluorescein diacetate (DCF-DA)  is a non-fluorescent molecule, but upon oxidation, typically through interactions with ROS, a fluorescent intermediate is produced . A DCF-Dt-test (α = 0.05) was used to test for significant mortality and abnormal morphology incidences compared to those of the GO + UP group. Further statistical analysis used a two-way ANOVA to detect the effect that the concentration and medium had on the viability of exposed embryos. Welch’s t-test was also employed to test the significance in percent differences from 1 h post-exposure to 24 h post-exposure in the FBS + mGO group against the UP + mGO group.FBS (VWR product number: 89510-194) and mGO co-exposures were performed with 4% FBS while mGO concentrations followed the standard 0 μg/mL, 2.32 μg/mL, 5 μg/mL, 10.7 μg/mL, 23.2 μg/mL, and 50 μg/mL curve (unpublished data). Each well of a 96-well plate contained 100 μL of mGO + 4% FBS solution, as well as a control plate that contained mGO mixed with UP water. All equipment was sanitized with 70% ethanol, and work was performed under a laminar flow hood to maintain a sterile environment. Mortality was monitored at the timepoint determined by the window of susceptibility, and morphology and mortality assessments were performed at 120 hpf. A Welch’s Initially, the toxicity of ascorbic acid (AA) was determined using 6 hpf embryos; previous studies showed that the maximum tolerated concentration of AA was ≤250 μM (exposures started at 3 hpf), 180–360 μM (exposures started at 4 hpf), or 100–200 μM (exposures started at 0 hpf) ,30,31. Wn = 24) were co-exposed, from 6–120 hpf, to 107 μM ascorbic acid  and 10.7 μg/mL mGO in a 96-well plate; 107 μM was chosen instead of 100 μM to reach a 2:1 (AA:mGO) ratio as closely as possible. The same plate also contained embryos (n = 24) exposed to mGO, AA, or UP alone. Embryo viability was recorded at 7 hpf, 24 hpf, and 120 hpf. Statistical significance was tested using Welch’s t-test (α = 0.05).Zebrafish embryos (p < 0.05)  . All subn = 32) at any of the concentrations investigated  (p < 0.05)  caused developmental toxicity dependent on the size of the nanomaterials. sGO exposures did not cause significant mortality to dechorionated embryos (0 μg/mL) A. mGO an < 0.05) B,C.p < 0.05), but sGO alone did not (GO co-exposures with 400 μg/mL of sodium cholate (denoted as -GOC) demonstrated increased incidences of teratogenicity, specifically mortality, compared to GO alone . sGOC co did not A. The lo did not B,C.p < 0.05), whereas 400 nm × 400 nm rGO exposures did not cause significant mortality at any of the concentrations tested  behavior in a size-dependent manner . Larval rGO exposures resulted in concentration-dependent and size-specific behavioral changes . In the p = 0.037), but prGOs (dispersed in sodium cholate) of the same size (400 nm × 400 nm) caused mortality by 11 hpf (p = 0.012). lGO exposures caused significant mortality by 9 hpf (p = 0.027), and rGO 2 μm × 2 μm exposures caused significant mortality by 10 hpf (p = 0.012). Therefore, the ranking of the nanomaterials based on their time until the onset of mortality was: mGO, lGO, rGO 2 μm × 2 μm, and prGO 400 nm × 400 nm.The onset of significant mortality was GO-specific. Exposure to 50 μg/mL of lGO, mGO, rGO 2 μm × 2 μm, and prGO 400 nm × 400 nm caused significant embryo mortality within 30 hpf . mGO expn = 22; p > 0.05). Exposures to 50 μg/mL mGO beginning at 8 hpf and 10 hpf did not cause significant mortality when compared to 6 hpf mGO exposures (n = 12 per treatment). Comparing the treatments to their unexposed counterparts of the same age, significant mortality occurred in 6 hpf embryos exposed for 1 h , but not in the 8 hpf and 10 hpf embryos exposed for 1 h .Later-life-stage embryos (8–48 hpf) exposed to 50 μg/mL mGO had a higher survival rate than earlier-life-stage embryos. No significant incidences of mortality occurred in 24 hpf and 48 hpf embryos exposed to 50 μg/mL mGO or lGO for 30 min, 1 h, 2.5 h, and 3 h , and FBS-mediated protection against mortality was heightened in higher concentrations  of mGO at both timepoints investigated (ANOVA: p < 0.001). The difference between 7 hpf and 30 hpf in FBS-mediated protection was significant for mGO + FBS exposures at 23.2 μg/mL when compared to mGO + UP exposures under the same conditions (p < 0.0001).Since most ENM toxicity assessments are performed on cultured mammalian cells that are commonly supplemented with FBS, we sought to compare the zebrafish endpoint results more directly to that of mammalian culture endpoints by adding FBS in the GO exposures. When FBS was added to mGO prior to 6 hpf zebrafish exposures, FBS mitigated mGO-induced mortality in a time-dependent manner . At 7 hpAscorbic acid (AA), a radical scavenger molecule, did not reduce mGO-associated mortality. AA and mGO were combined in a 1.76:1  mixture prior to 6 hpf embryo exposure. The AA + mGO mixture did not reduce embryonic mortality compared to mGO alone.Consistent with prior studies, GOs exhibited size- and oxidation-state-dependent toxicity in early-life-stage zebrafish ,33,34. HThe presence of sodium cholate in GO dispersions significantly increased their teratogenicity. Sodium cholate is an established dispersant and stabilizer for nanoparticles and insoluble organic compounds for both in vivo and in vitro systems ,39,40. TOur results suggest that GO-induced mortality was driven by damage to the yolk sac. Exposure to mGO that began at 50%, 75%, and 100% epiboly  seemed to be negatively correlated with YSL disruption. Other studies have shown that chemicals and nanomaterials, such as estradiol and GO, accumulate in the yolk sac . GraphenThe GO oxidation level likely played a role in the rapidity of mortality, where fully oxidized GO exposures  caused more rapid mortality than the less-oxidized materials (prGO 400 nm × 400 nm and rGO 2 μm × 2 μm). Interestingly, there were size-specific differences in GO toxicity, even in materials such as mGO and lGO that possess the same molecular structures. According to Komeily-Nia et al., the carbon:oxygen (C:O) ratio determines the density of radical carbons in GO structures . The NHIThe interactions between FBS and mGO significantly reduced embryonic mortality. FBS contains a variety of compounds such as amino acids, standard cell culture medium, and vitamins . The mixGO toxicity in the developing zebrafish depended on the size and oxidation level. GO 400 nm × 400 nm (mGO) was the most toxic. The mechanism may be lipid peroxidation and membrane damage by immobilized free radicals, especially in the more oxidized GOs ,43. This


The values measured by two radiologists with different levels of experience were evaluated in order to assess the inter-reader reproducibility assessment (ICC = 0.52 for PP and DP). Further, we performed a population-based analysis and a drug-oriented subgroup analysis in 54 DOX-treated and 28 EPI–TRAS-treated patients. In the general cohort of women treated with any of the two drugs, we observed in the lapse T0–T1 a relative increase (RI) of 25% vs. 20%  as well as in the lapse T0–T5 an RI of 17% vs. 15% . The DOX-treated patients reported in the lapse T0–T1 an RI of 22% (p < 0.0001) in PP and an RI of 16% (p = 0.018) in the DP, with ECV values remaining stably high at T5 both in PP  and in DP  highlighting a possible hallmark of a persisting CTX sub-damage. On the other hand, ECV measured in EPI–TRAS-treated women showed an RI in T0–T1 of 18% (p = 0.001) and 29% (p = 0.006) in PP and DP, respectively, but the values returned to basal levels in T5 both in the PP (p = 0.12) and in DP setting (p = 0.13), suggesting damage in the first-year post-treatment and a possible recovery over time. For the 82 patients, an echocardiography was performed at T0, T1= 12 m + 3 m and T5 = 60 m + 6 m with LVEF values at T0 (64% ± 5%), T1 (54% ± 6%) and T5 (53% ± 8%). WB-CT-derived ECV values could provide a valid imaging marker for the early diagnosis of cardiotoxic damage in BC patients undergoing oncological treatments. We detected different patterns during the follow-up, with stably high values for DOX, whereas EPI–TRAS showed a peak within the first year, suggesting different mechanisms of cardiac damage.Breast cancer patients undergoing neoadjuvant chemotherapy with anthracyclines or trastuzumab can suffer cardiotoxic issues. Nowadays, the markers of cardiac damage are still not reliable, and extracellular volume (ECV) calculated from CT could be a promising cardiotoxic marker. Eighty-two patients, treated with two different chemotherapy regimens based on doxorubicin (DOX) or epirubicin-trastuzumab (EPI–TRAS), were retrospectively selected and the variations in extracellular volume (ECV) values were measured and analyzed. Whole Body CT (WB-CT) scans were acquired after 1 min, in the portal phase (PP), and after 5 min, in the delayed phases (DP), at the baseline (T In the era pre-COVID-19, cardiovascular disease and cancer diseases were the most common causes of death in the world and, in particular, in the middle-upper and higher income countries .It has been estimated that a woman, without additional genetic factors, has a lifetime risk of one in eight about 12.4%) to receive a diagnosis of breast cancer (BC) , with a % to receThe main adjuvant and neoadjuvant chemotherapy regimens involve  the use The aim of this study was (i) to investigate a possible validation of myocardial ECV measured from total body CT as an early imaging marker of myocardial damage, evaluating whether neoadjuvant chemotherapy (NACT) in BC patients is associated with an increase in ECV value even in the absence of clinical, echocardiographic or electrocardiographic signs of cardiac damage; (ii) to analyze ECV values one year after the end of treatment and five years later with the same objective; (iii) to evaluate whether ECV values in patients who received Adriamycin as chemotherapy are different from those who received epirubicin and trastuzumab.0), at the first-year post-therapy (T1) and at the fifth-year post-therapy (T5) during the oncological follow-up.We retrospectively selected 102 whole body computed tomography (WBCT) about female patients affected by BC who underwent neoadjuvant chemotherapy (NACT) with an anthracycline as backbone therapy between January 2010 and July 2016 at Tor Vergata University Hospital, Rome, Italy. All patients’ heart condition and oncological status was assessed at baseline  with a histological diagnosis of BC and with a therapeutic decision to perform NACT, without any further restrictions.Exclusion criteria were as follows: (i) the presence of a pre-existing cardiac disease; (ii) previous cardiotoxic treatments, in particular chemotherapeutic treatments, and chest irradiation therapies ; (iii) hematocrit examinations performed outside the indicated time interval (4 weeks before or after the CT examination); (iv) cases in which chemotherapy dose reduction was necessary; and (v) patients with a relapse or metastases from BC or a second tumor who underwent to additional oncological treatment.2) or epirubicin  following the guidelines for locally advanced BC. Patients with BC were considered for treatment schemes, such as AC-TXT  and FEC , alternatively. In Her-2 positive BC patients, trastuzumab (TRAS) was delivered weekly during association with FEC scheme . Chemotherapy doses were adjusted for body weight and body surface area according to clinical guidelines.The patients were treated with polychemotherapeutic protocols with a neoadjuvant purpose. The schemes used were AC-based using either DOX  using a multi-step spiral acquisition and with cranial–caudal scan direction. The scans included the abdomen, part of the thorax, the heart and the skull. The acquisitions were performed during scheduled oncologic follow-ups; therefore, without ECG gating. The imaging acquisition adhered to a standard protocol requiring a baseline scan and three subsequent scans after administration of 100–120 mL of iodinated contrast  followed by 30–50 mL of saline (injection rate of 3 mL/s).Arterial phase, generally about 15–18 s after contrast injection;Portal phase, 1 min after contrast injection;Delayed phase, about 5 min after contrast injection.The post-contrast scans were acquired applying the bolus tracking technique: a threshold of 120 HU was set to start the scan using a region of interest (ROI) placed in the descending aorta at the thoracic-abdominal passage. Three phases were obtained:Radiation doses were reported using the following formula: dose length product (DLP) expressed in mGy × cm .WB-CT images were retrospectively evaluated by two radiologists with at least 5 years of experience in cardiovascular radiology. Each operator chose the best slice to visualize an axial view of the four cardiac chambers.The regions of interest were placed in the interventricular septum and in the ‘blood’ pool of the left ventricle and they were measured in PP and DP, respectively, drawing them of equal proportions.ROIs were placed in the thickest portion of the middle septum, excluding areas closer to the ventricular cavity to avoid blood contamination. Moreover, papillary muscles were avoided.In the pre-contrast scans, the ROIs were positioned at the same level chosen in the post-contrast scans (at 1 min and 5 min). ROIs were measured in the three phases  at time 0, within one year and after five years in order to obtain Hounsfield units (HUs) to include in the ECV calculation of the portal and delayed phase for each available examination .ECV was determined by the following equation, according to Miller et al. .ECV = , 8 patients were excluded because the hematocrit examinations were performed outside the indicated time interval (more than 4 weeks before or after the CT examination), 2 patients because the CT scans were incomplete, 4 patients because the CT images were compromised by motion artefacts, 2 patients who had already received radiotherapy for left breast cancer, 1 patient who had already received another cardiotoxic chemotherapy, 2 patients who had a previous heart disease (FE < 40%) and 1 patient because the chemotherapy dose had to be reduced to 75% during treatment due to febrile neutropenia. After applying the exclusion criteria, the population considered for the final analyses accounted for 82 patients , with 62 patients affected by ductal carcinoma, 4 patients by infiltrating lobular carcinoma, 14 patients by a poorly differentiated carcinoma and 2 patients by an undifferentiated carcinoma. About 44 patients reported a stage II tumor (following TNM 8th edition), 16 presented with a stage IV and 14 stage III and 8 stage Ic (24). Of the patients, 54 were treated with doxorubicin (DOX)-based treatment and 28 with a epirubicin–trastuzumab (EPI–TRAS) based treatment. All patients were healthy at Tectively . At the http://www.R-project.org (accessed on 3 October 2022)). Data are presented as mean ± standard deviation (SD) for normally distributed populations and median ± interquartile range (IQR) for not normally distributed ones. When not otherwise specified in the text, we referred as the mean ± SD. Shapiro–wilk tests were used in order to assess the normality of the curves. For multiple comparisons ANOVA test was chosen after carrying Shapiro–wilk and Mauchly’s test in order to assess normality and sphericity, respectively. Afterward, a pairwise test was conducted only for the most important post hoc analysis through t-student test for paired data. In the case of different, independent groups with different or not known variances, a Welch’s test was performed, whereas, for non-normal distributions, a Wilcoxon test for paired data was used. All the tests were performed with a two-sided α-value = 0.05 as significance level. Eventually, the inter-class correlation (ICC) function was used in order to assess inter-observer reproducibility [The statistical analysis and plots were performed with RStudio software  measured in the portal phase (PP) was 27% ± 6% vs. 29% ± 8% (median + IQR), as rated by the first and second reader, respectively, with a non-significant difference (p = 0.09). In the delayed phase (DP), ECV was detected by first and second reader to be 28.3% ± 5% vs. 27.9 ± 6% (mean + SD), respectively, with a non-significant difference (p = 0.64). Similar results were observed when the two raters were compared at the first year of FU (T1) both for portal phase  and delayed phase . At the fifth-year checkpoint (T5), no significant differences were found between the two readers, neither in the portal phase , nor in the delayed phase , respectively.The pre-treatment ECV  analysis was conducted with the aim to assess the agreement and reproducibility of the inter-observer tests. For this purpose, a random sample of 10 subjects was chosen from the two observer’s rating groups in order to limit the weight of systematic and random biases. An ICC coefficient of 0.514 (95% CI 0.201–0.733) and 0.518 (95% CI 0.201–0.737) for the portal phase and the delayed phase, respectively, revealing an agreement between the two observers.0), the first year after NACT termination (T1) and the fifth year thereafter (T5). The population analyzed was heterogeneously represented by DOX-based schemes (DOX) and the combination of epirubicin and trastuzumab (EPI–TRAS), in order to combine information and assessing ECV validity in a general population. Further analyses were performed in the two subgroups with the aim to highlight different trends between them.In order to establish the validity of ECV measurements in WB-CT, a total of 82 patients who underwent cardiotoxic NACT were evaluated. ECV was measured at three different checkpoint times: at baseline  and between T0–T5 with a relative increase (RI) of 17.5%  as shown in  was found the ANOVA test. Afterward, a one-to-one subgroup analysis was performed analyzing the differences between the T0–T1 and T0–T5. The ECV values were compared between T0–T1 showing an RI of 20.6%  and a significant increase of 15.6% between T0–T5   .0 = 27.2% ± 10% (median ± IQR) and at the first year of FU, T1 = 34% ± 6% (Median ± IQR), with a relative difference (RD) of 22.4%, a value strongly significant . Data were confirmed also at five years of FU (T5 = 31.4% ± 4%) compared to baseline with a relative reduction (RD) highly significant   A.0 = 28.4% ± 5%), at the first year of FU (T1 = 32.9% ± 7%), and at five years of FU (T5 = 33.3% ± 7%). Among these three groups, compared with each other, a statistically significant different was found  and the post hoc subgroup analyses revealed a significant increased cardiac ECV between T0–T1  and between T0–T5 , respectively  setting, at the baseline (Tectively B.0 (26.7% ± 4%), at T1 (32.7% ± 4%) and at T5 (29.7% ± 4%), in PP setting. The global comparison with ANOVA test revealed a statistically significance among groups .A total of 28 WB-CT examinations of EPI–TRAS treated BC women were included in the analyses. All the women completed successfully all the chemotherapeutic cycles. Similarly to DOX, cardiac ECV was measured also for EPI–TRAS treated women at T0–T1 of 18.5% , but the result was not confirmed at the fifth year of FU (T0–T5), with an RD of 10.3% not reaching the significance . However, a significant reduction of ECV values was observed between T1–T5 with a significant RD of −10%   A.0 (27.4% ± 5%), at T1 (32.5% ± 6%) and at T5 (30.4% ± 6%). The comparison among the three temporal groups was statistically significant , so we could proceed with the post hoc subgroup analyses, which evidenced a significant increase of 28.7% between the T0–T1  and a RD of −13.6% between T1–T5 , respectively .The same 28 patients were examined after 5 min in DP setting, acquiring the images at Tectively B. Moreov0, no difference in the measured ECV values was observed between DOX- and EPI–TRAS-treated patients neither in PP (p = 0.51) nor in DP setting (p = 0.39), confirming the absence of cardiac damage before starting the NACT. However, in the PP, no difference was observed between the two different treated groups at T1 , and a similar result was achieved even at T5 . On the other hand, also in the DP setting between the two schemes, not statistically significant difference was observed neither at T1 (p = 0.48) nor at T5 (p = 0.10), respectively , T1 (54% ± 6%) and T5 (53% ± 8%).In our 82 patients undergoing cardiotoxic chemotherapy, echocardiography was performed at T1 and T5 was increased if compared to the baseline values .Among the selected 82 patients, we recorded 4 cases of cancer therapy-related cardiac dysfunction (CTRCD) during the period of follow-up from the end of the treatment (EOT). Indeed, we observed 2 patients suffering of LVEF reduction more than 15% after 16 and 23 months from EOT, respectively, 1 patient reported incidence of atrial fibrillation and pericardial effusion 14 months after EOT, and 1 patient reported an asymptomatic decrease of LVEF under the value of 50% (LVEF 40%) 16 months after EOT. In this subgroup, ECV measured in TOur investigation is the first study, to our knowledge, comparing DOX with the EPI–TRAS combination using ECV values as an imaging marker measured from a WB-CT scan as source. This marker was already proven to be effective in the particular context of cardiac MRI and CT scans ,13,16 inClassical biochemical markers such as troponin I (cTnI), troponin T (cTnT) and Natriurec Peptides (NP) have important limitations such as assay selection, different laboratory methods and cut-off values used, the variable follow-up and the lack of standardized cardiac endpoints ,28 resul1 and T5, whereas LVEF showed a slight decrease, without falling below the threshold value (LVEF < 50%).Our results showed ECV values increasing more than 30% in T1) and for chronic settings (T5) and between PP an DP. In these settings, we observed in a court of 82 patients a general increase in ECV values, significantly different between pre- and post-NACT, within the first year (T0–T1) and the fifth year (T0–T5). An accepted explanation is the subclinical damage caused from the antiblastic toxicants generating a tissue oedema which is eventually evolved in fibrosis, as also described by other authors [Cardiac ECV measured from WB-CT scans can be used as early marker of myocardial damage in BC patients undergoing cardiotoxic chemotherapies, showing different kinetics for acute  can be partially explained by different contrast permeability over time, inter-individual physiological or paraphysiological differences. Nevertheless, methodological issues should be taken into account; we conducted a non-randomized and non-matched retrospective study.2 and reaches an unacceptable threshold at 550 mg/m2, whereas epirubicin showed comparable cumulative thresholds at 360 mg/m2 and 900 mg/m2, respectively [The AC induced CTX is widely described as cumulative and dose-related cardiotoxicity causing phenomena such as arrhythmias, ischemic disease, prolonged QT interval, valve dysfunction and systolic dysfuction . Trastuzectively ,38. Contectively ,40.0, T1, T5) and at different phases  in BC patients. Indeed, in the DOX group, we observed concordant increasing ECV values at the first year for PP  and for DP , respectively. Noteworthy, the values remained stably high at T5 with 31.4% ± 4% with an RD from baseline of 14% (p < 0.0001) and 33.3% ± 7% with a RD of 17% (p = 0.005) for PP and DP settings, respectively (1 (32.7% ± 4%) vs. baseline (T0 = 26.7% ± 4%) detecting a RD of 18% for PP (p = 0.001), whereas a RD of 29%  was found in DP. However, in both phases, a drop was observed almost to baseline values at T5 . The decreasing ECV values in T1–T5 lapse were statistically significant both in PP  and in DP , arrhytmias (Taxanes), LVEF depression (5-Fluoruracil) used in polychemotherapeutic schemes, in agreement with current guidelines.Another important limitation was that the chosen hematocrits were performed in a different moment from the WB-CT, but still no more than 4 weeks before or after the imaging acquisition. Little influence on intra-individual variability was expected, as is analogous to other authors as described in the relevant literature .The main limitation of this study remains the low diagnostic accuracy, due to the lack of ECG gating scans, leading to possible motion artifacts and intra-individual variability. Nevertheless, this is also a strength due to the attempt to simplify a cumbersome investigation in order to use it as a possible clinical routine method. In the attempt to raise accuracy, an interesting perspective could be performing an additional arterial-phase scan with a restricted field of view (FOV) over the heart in follow-up studies of selected patients at high risk of developing CTX. Although a minimal increase in radiation dose is expected in this way a greater accuracy in CTX diagnosis would be warranted.However, after a possible validation, WB-CT could be used for CTX screening, while ECG gated CT scans, additional FOV heart-centered arterial phase or cardiac MRI could be reserved for a further selected investigation.We determined that BC women of age 55 ± 9 years who underwent different schemes of NACT expressed different ECV values at CT scans during FU. Our study has confirmed that myocardial ECV can be effectively calculated from CT scans ,21,22, b


Scientific Reportshttps://doi.org/10.1038/s41598-022-12943-6, published online 03 June 2022Correction to: 1H‑NMR.’The original version of this Article contained errors in the niacin content units. In the Results and discussion section, under the sub-heading ‘Persistent metabolome and ravages of time revealed by 34), no niacin signal above the detection limit could be found in modern equivalent . Niacin is stable throughout the brewing process and storage35, directly correlates with the gravity of the beer and is not produced during fermentation in considerable amounts33,36. Its low content in nowadays beers (5 mg/L37) cannot be attributed to higher concentrations in historical barley cultivars with levels being consistent between 80 and 120 µg/g33,34,38,39.” “While the found content of 6.2 g/L niacin in the historical beer is plausible for a lager beer of the time (compare 10.3 g/L in a strong beer of 1872now reads:34), no niacin signal above the detection limit could be found in modern equivalent . Niacin is stable throughout the brewing process and storage35, directly correlates with the gravity of the beer and is not produced during fermentation in considerable amounts33,36. Its low content in nowadays beers cannot be attributed to higher concentrations in historical barley cultivars with levels being consistent between 80 and 120 μg/g33,34,37,38.”“While the found content of 6.2 mg/L niacin in the historical beer is plausible for a lager beer of the time .37. Punčochářová, L., Pořízka, J., Diviš, P. & Štursa, V. Study of the influence of brewing water on selected analytes in beer. The original Article has been corrected.