Hypoperfusion or resultant hypoxia in solid tumours is a main reason for therapeutic resistance. Augmenting the blood perfusion of hypovascular tumours might improve both hypoxia and drug delivery. Cavitation is known to result in microstreaming and sonoporation and to enhance drug diffusion into tumours. Here, we report the ability to enhance both tumour blood perfusion and doxorubicin (Dox) delivery using a new sononeoperfusion effect causing a cavitation effect on tumour perfusion in subcutaneous Walker-256 tumours of rats using ultrasound stimulated microbubble (USMB).To induce the sononeoperfusion effect, USMB treatment was performed with a modified diagnostic ultrasound (DUS) system and SonoVue® microbubbles. The therapeutic pulse was operated with a peak negative pressure of 0.26 to 0.32 MPa and a pulse repetition frequency (PRF) of 50 Hz to 2 kHz. Contrast-enhanced ultrasound (CEUS) was used for tumour perfusion assessment.P < 0.05). The treatment also increased Dox delivery to tumours by approximately 3.12-fold more than that of the control (P < 0.05). Furthermore, ELISAs showed that vasodilators and inflammatory factors increased 4 hours after treatment (P < 0.05), suggesting that the inflammatory response plays an important role in the sononeoperfusion effect.The USMB treatment of 0.26 MPa and 1 kHz could significantly enhance tumour perfusion with a 20.29% increase in the CEUS peak intensity and a 21.42% increment in the perfusion area for more than 4 hours (The USMB-induced sononeoperfusion effect could significantly enhance the blood perfusion of Walker-256 tumours and promote drug delivery. It might be a novel physical method for overcoming the therapeutic resistance of hypoperfused or hypoxic tumours.The online version contains supplementary material available at 10.1186/s40644-023-00545-y. Due to the upregulation of angiogenesis in solid cancers, tumour cells often grow more rapidly than the cells that form blood capillaries , 2. ProlAnother important factor for the response to chemotherapy in tumours is the distance from tumour vessels to tumour cells. At approximately 40–50 μm away from the blood vessel, the doxorubicin (Dox) concentration drops to half of its perivascular concentration. Furthermore, the average distance from tumour blood vessels to hypoxic tissue is 90 to 140 μm .However, it is difficult to enhance tumour perfusion because vasoactive drugs may create vascular steal, leading to a decrease in tumour perfusion . PreviouConsidering the low intensity, low MI and tumour perfusion response features of the treatment, we named the effect “sononeoperfusion”, representing US, neoplasm and perfusion improvement. Sonoreperfusion usually refers to US-stimulated myocardial or muscular blood reperfusion effects. However, tumour vascular construction is chaotic, disorganized, immature, dysfunctional and mechanically vulnerable, which is quite different from normal developed vessels , 23.Therefore, the aim of this study was to investigate the proper parameters, Dox delivery and related mechanism for sononeperfusion. The parameters included the MI or acoustic pressure and the pulse repetition frequency (PRF). Cytokines such as vasodilators and inflammatory factors were tested to explain the mechanism.A commercial DUS system  connected to an X4-12 L linear array transducer was used for both therapeutic US exposure and US imaging. The system was equipped with contrast bubble imaging (CBI), integrated contrast-enhanced ultrasound (CEUS) imaging software and flash mode for microbubble destruction. The flash mode was specifically modified to deliver customized pulse sequences for regulating microbubble cavitation or so-called ultrasound stimulated microbubble (USMB), named Vflash. The Vflash pulses can be operated with adjustable frequency, MI, pulse length (PL), PRF and destruction (on)/replenish (off) time as described previously . In addiThe peak negative pressure (PNP) within a designated ROI of 1 × 1 cm was measured by a membrane hydrophone  positioned 2 cm away from the probe surface. The probe was placed above the hydrophone separated with degassed water in a sink .8 mL− 1 microbubbles with a mean diameter range from 2.0 to 4.0 μm after preparation in 4.0 mL of saline solution. The SonoVue® suspension was then used in both CEUS as an US contrast agent and in therapeutic US as cavitation nuclei. CEUS is a reliable method to assess tumour perfusion. According to the EFSUMB guidelines, the mean time-intensity curves within the tumours after bolus injection of a contrast agent were qualified to reflect microbubble wash-in and washout, thus representing the condition of tumour perfusion [SonoVue® microbubbles  are a commercially available US contrast agent that contains 10erfusion .7/mL) into the inner thigh of the rat. Then, the model was included in the study when the tumour size reached approximately 1 cm in diameter. All of the animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the university.A total of 81 Sprague‒Dawley (SD) rats bearing subcutaneous Walker-256 tumours were used. The tumour model was made by injecting 0.2 mL of Walker-256 cell suspension  according to different treatment parameter combinations and one control group. The treatment parameters of all groups are illustrated in Table In treatment plan 2 Fig. C, Dox seThe animals were anaesthetized by intraperitoneal injection of 2% pentobarbital sodium at 2 ml/kg, and the tumour surface was shaved and depilated. A catheter connected to a 22G needle was inserted into the caudal vein to establish the channel for intravenous injection. High-resolution two-dimensional (2-D) DUS was performed with the same VINNO70 system and the X4-12 L transducer to find the maximal dimension of the tumour section  was injected through the tail vein  of tumour contrast intensity, including the peak intensity (PI) and area under curve (AUC) data. The PI is the peak value of the TIC, and the AUC is integrated by the area under the TIC within 60 s starting from TIC elevation.For the calculation of the tumour perfusion area rate, the images of the largest tumour contrast perfusion area in the clip were intercepted. Then, the tumour perfusion area was manually delineated using Adobe Photoshop CC (Adobe), and the rate of tumour perfusion area was calculated by the perfusion area/entire tumour area ×100%. The increment of the tumour perfusion area rate was calculated by the percentage of perfusion rate after treatment minus the percentage of perfusion rate before treatment.2 at a filling rate of 20% cv/min. The tumours from Groups B and E and the control were harvested. The tumour tissues were minced into small pieces and homogenized. Then, the homogenates were centrifuged to obtain the supernatant for enzyme-linked immunosorbent assays (ELISAs).Immediately after the experimental procedures, 56 animals in the perfusion study were sacrificed by inhalation of carbon dioxide with exposure to 100% COThe contents of eNOS, PGE2, PGD2, PGF2, PGI2, C3a, C5a, LTC4 and TNF-α in tumour tissues were determined by the Rat eNOS-3 ELISA Kit, Rat PGE2 ELISA Kit, Rat PGD2 ELISA Kit, Rat PGF2α ELISA Kit, Rat PGI2 ELISA Kit, Rat C3a ELISA Kit, Rat C5a ELISA Kit, Rat LTC4 ELISA Kit and Rat TNF-α ELISA Kit, respectively . The absorbance optical density (OD) of each well was measured at 450 nm. The levels of ATP, NO and ROS in tumour tissues were determined by an ATP assay kit, NO assay kit and reactive oxygen species assay kit , and the OD values were measured by Microplate Reader according to the instructions.For the quantification of the Dox concentration, the rats in the treated group (n = 14) and the control group (n = 11) were sacrificed 40 minutes after Dox infusion. Approximately half of the tumour bulk tissues were taken, and the Dox content was determined by HPLC.Another half of the tumour sample was frozen and sliced, the nuclei were stained with DAPI, and the sections were examined under a fluorescence microscope . Dox can spontaneously emit red light, while the nuclei appeared blue under UV excitation.One tumour sample from the treated group or the control was stained with haematoxylin and eosin (H&E) for morphological observation. Under a light microscope, tumour cells are surrounded by connective tissue in a disordered arrangement.p value less than 0.05 was considered statistically significant.SPSS 25.0 software was used for statistical analysis. Multifactor repeated-measures ANOVA was used to determine the influence of different groups on the blood perfusion of Walker-256 tumours at different time points for the PI, AUC and tumour perfusion area of CEUS. If there was an interaction, it was necessary to test the separate effects, and the Bonferroni method was used for pairwise comparison. The contents of ATP, eNOS, PGF2, PGI2, LTC4, TNF-α and ROS in tumour tissues were analysed by one-way ANOVA with a completely random design, and the LSD method was used for further comparison between groups. The variance of NO, PGE2, PGD2, C3a and C5a in tumour tissues was uneven. The independent sample Kruskal‒Wallis rank sum test was used, and Bonferroni correction was used for further comparison between groups. The concentration of Dox in tumour tissues was determined by an independent sample T test. A P < 0.05) (Tables P < 0.05). The average incremental perfusion area rate was 11.84% in Group B and 10.53% in Group E (P < 0.05). However, for the other groups , the PI elevations ranged from 3.12 to 7.28%, the AUC from 2.81 to 7.33%, and the incremental perfusion area from 4.06 to 8.41%, and none of there were significant (P > 0.05). Four hours after treatment, the effect was further enhanced in Group B (PNP 0.26 MPa) with an increase of 20.29% in PI, 18.22% in AUC and 21.42% in incremental perfusion area when compared with the baseline (P < 0.05). There was no significant difference in Group E (PNP = 0.32 MPa) and the other groups 4 h later (P > 0.05) (Tables The results showed that the sononeoperfusion effects of Groups B and E (PRF 1.0 kHz) were significant immediately after treatment  Tables  and 3. IFour hours after treatment, we applied HPLC and fluorescence microscopy to evaluate the Dox concentration within the tumour tissues. HPLC showed that the Dox concentration in the control group was 1152.71 ± 369.83 ng/g and that in the treated group  was 3246.59 ± 1301.85 ng/g. The Dox concentration in the treated tumours was 2.82-fold higher than that of the control . There were no significant changes in PGD2, PGE2, or C3a among the groups (P > 0.05)  were higher than those of the control (P < 0.05), while there was no significant difference in the control (P > 0.05).Light microscopy revealed that microvascular hyperaemia and inflammatory cell infiltration were obvious Fig. C 4 hoursIn this study, we comprehensively investigated the proper acoustic parameters, duration time, perfusion area, Dox delivery, cytokines and related pathological changes associated with the sononeoperfusion effect. For the first time, we proposed the new term “sononeoperfusion” to represent this tumour perfusion enhancement effect induced by DUS and microbubbles. The effect had previously appeared in immunotherapy of MC38 colon cancer and chemotherapy of PANC-1 pancreatic cancer in mice , 21. ThiFirst, therapeutic US was generated from a modified DUS system (VINNO 70) as previously described . The sysSecond, the sononeoperfusion effect was remarkable and repeatable under proper USMB treatment, i.e., only in Groups B and E. The best tumour perfusion improvement was observed in Group B (PNP 0.26 MPa and PRF 1.0 kHz) with a 20.29% increase in PI, an 18.22% increase in AUC and a 21.42% increment in the perfusion area rate Tables . This efThird, US-mediated drug delivery has been well documented in many studies . The USMIt is well known that solid tumours always develop hypoperfused and hypoxic areas, resulting in chemotherapy, radiotherapy and immunotherapy resistance. This hypoxic area lies between the perfused tumour and necrotic tumour , 36. We Finally, we tried to explain the mechanism of sononeoperfusion by detecting related cytokines within tumour tissues. The mechanical effect of stable cavitation under 0.3 MPa  may releSonoreperfusion effects have been discovered in skeletal muscle in recent years and may be a promising solution for peripheral vascular diseases or muscular ischaemia . Since sThis is a preliminary experimental study. We did not test more parameters or measure the cavitation magnitude for the sononeoperfusion effect, considering the complexity of cavitation. Proper acoustic parameter combinations, including the microbubble concentration, may greatly influence the effect. This study did not prove the improvement in the hypoxic microenvironment of solid tumours. In addition, the mechanistic study of the sononeoperfusion effect was only limited to the inflammatory response. Further signalling molecules and pathways related to the effect should be taken into consideration.In this work, we demonstrated that modified DUS combined with microbubbles enhances blood perfusion of rat Walker-256 tumours, which was named the sononeoperfusion effect, thus promoting chemotherapeutic drug (Dox) delivery by up to 3.12-fold. This study also demonstrated that the sononeoperfusion effect might be related to the inflammatory response by the release of vasodilators and inflammatory factors.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


The comparative analysis section compares the rheology of the optimal SBVE fluids and nanofluids in each category to the rheology of polymeric guar gum fluid for the entire range of shear rates and temperature conditions. The type 3 optimum fluid with high surfactant concentration of 0.2 M cetyltrimethylammonium bromide and 1.2 M sodium nitrate was the best of all the optimum fluids and nanofluids. This fluid shows comparative rheology to guar gum fluid even at elevated shear rate and temperature conditions. The comparison of average viscosity values under a different group of shear rate conditions suggests that the overall optimum SBVE fluid prepared in this study is a potential nonpolymeric viscoelastic fluid candidate for hydraulic fracturing operation that could replace polymeric guar gum fluids.Surfactant-based viscoelastic (SBVE) fluids have recently gained interest from many oil industry researchers due to their polymer-like viscoelastic behaviour and ability to mitigate problems of polymeric fluids by replacing them during various operations. This study investigates an alternative SBVE fluid system for hydraulic fracturing with comparable rheological characteristics to conventional polymeric guar gum fluid. In this study, low and high surfactant concentration SBVE fluid and nanofluid systems were synthesized, optimized, and compared. Cetyltrimethylammonium bromide and counterion inorganic sodium nitrate salt, with and without 1 wt% ZnO nano-dispersion additives, were used; these are entangled wormlike micellar solutions of cationic surfactant. The fluids were divided into the categories of type 1, type 2, type 3, and type 4, and were optimized by comparing the rheological characteristics of different concentration fluids in each category at 25 °C. The authors have reported recently that ZnO NPs can improve the rheological characteristics of fluids with a low surfactant concentration of 0.1 M cetyltrimethylammonium bromide by proposing fluids and nanofluids of type 1 and type 2. In addition, conventional polymeric guar gum gel fluid is prepared in this study and analyzed for its rheological characteristics. The rheology of all SBVE fluids and the guar gum fluid was analyzed using a rotational rheometer at varying shear rate conditions from 0.1 to 500 s Th. Th37]. −1 at 5 s−1 intervals and different temperatures of all SBVE fluids, nanofluids, and guar gum gel fluid. The fluid concentration was optimized based on the rheological behavior of the fluids when compared with fluids in the same category at 25 °C. It is important to study the factors affecting the key controlling properties of the fluid viscosity and flow to ensure its optimum application during fracturing operation. These are the shear rate due to the non-Newtonian nature of the fluid and the temperature of the surroundings Br), and the molar mass is 364.447 gm/mol. Its molecular structure is illustrated in 2O, was obtained from Sigma Aldrich. The guar gum was obtained from Swastik gum industries .Cetyltrimethylammonium bromide (CTAB), or cetrimonium bromide, is a cationic quaternary ammonium surfactant. The chemical formula for CTAB is . The Parallel Plate geometry (plate diameter: 40 mm) was implemented at a gap of 0.17 mm for all the fluid analyses see . The vis−1 at a difference of 5 s−1 under different temperature conditions of 25 °C to 75 °C by 10 °C intervals. Therefore, the viscosity data of the fluids was studied and compared for 100 different shear rate conditions under room temperature for optimization of the fluids in the type 1, type 2, type 3 and type 4 categories. The optimum fluids were compared with each other and with guar gum under all different temperature and shear rate conditions. The viscosity data were plotted and compared on clustered bar charts to visualize the differences in terms of the actual viscosity data of the different fluids on all 600 points for a wide range of shear rate and temperature conditions.The viscosity was observed by varying the shear rate from 0.1 to 500 s−1) and at different temperatures of 25 °C, 35 °C, 45 °C, 55 °C, 65 °C, and 75 °C.The viscosity plots show different values with the change in salt concentration. The values increase for the fluids with increased salt concentration from 0.2 M to 0.4 M and slightly decreases at 0.6 M. Further, the values reflect an increasing trend as the salt concentration increases to 1.2 M. The viscosity plot of 1.2 M counter ion concentration fluid reflect higher viscosity values maintained under low and high shear rate conditions.3. Thus, the fluid with 1.2 M NaNO3 concentration is the optimum fluid of the type 3 category of high surfactant concentration (0.2 M CTAB) fluids. However, a concentration of 1.0 M provides similar values under certain exceptional shear rate conditions.Further, when analyzing fluids in this category with increased salt concentration, the viscosity plot depicts reduced values for the fluid with 1.4 M NaNOIn this case, the viscosity behaviour seems to be different from other cases, as at 0.2 M salt concentration the plot resembles very smooth viscosity data. The values decrease with increasing shear rate without any ups or downs.−1. Then, the viscosities provide similar values to those obtained with a shear rate of 450  and nanofluid (with 1 wt%ZnO NPs additives) in the previous study were type 1 and type 2 SBVE fluids. The fluids had a CTAB concentration of 0.1 M, and the optimal counter-ion NaNOIt can be concluded that the type 1 and type 2 optimum fluids cannot replace guar gum, as they show poor rheology under elevated shear rate and temperature (ESRT) conditions. Therefore, it was necessary to develop more fluids with improved rheology. We prepared and optimized type 3 and type 4 fluids with increased surfactant concentration under the hypothesis that they would have comparable rheology to guar gum gel fluid.−1. The figures reflect that the optimum fluid and nanofluid show better viscosities than guar gum gel fluid up to 55 °C under all conditions. The guar gum shows similar viscosity values under initial shear rate conditions (up to 150 s−1 shear rate conditions) at 65 °C for both optimum SBVE fluid and nanofluid. In contrast, the optimum fluid and nanofluid show better viscosity at high shear rate conditions. Further, the guar gum fluid shows better viscosities at 75 °C temperature when compared to both optimum fluid and nanofluid at low shear rate conditions. The optimum type 4 nanofluid shows poor rheology compared to guar gum fluid at high shear rate conditions. However, the type 3 optimum fluid shows similar viscosities to guar gum at high shear rate conditions  . TherefoStatistical parameters are compared of viscosity data produced by optimum fluid (type 3) and nanofluid (type 4), and guar gum gel fluid, such as the standard deviation, mean, and standard error of the mean. The viscosity data of all three fluids was analyzed repeatedly at 100 same shear rate conditions under six different temperatures.The optimum SBVE fluid shows the highest mean viscosity value compared to the optimum SBVE nanofluid and guar gum. The standard deviation is higher for the optimum fluid than for the other fluids, meaning that the viscosity values are more dispersed for the optimum SBVE fluid around its mean value of 818.09 centipoise. In contrast, guar gum shows the lowest SD value. Therefore, the viscosity values of guar gum are clustered around its mean value of 688 centipoise see . The staThe low-surfactant optimum SBVE fluids of types 1 and 2 cannot replace guar gum, showing poor rheology under elevated shear rate and temperature (ESRT) conditions.The high-surfactant SBVE fluids (type 3) and nanofluids (type 4) of 0.2 M CTAB concentration show optimal rheology when prepared at 1.2 M and 0.4 M counter-ion sodium concentration, respectively.The optimal type 3 fluid and type 4 nanofluid show better viscosity than guar gum gel fluid up to 55 °C under all conditions.−1 shear rate conditions) at 65 °C to both optimum SBVE fluid and nanofluid. However, the optimum fluid and nanofluid show better viscosity under high shear rate conditions.The guar gum fluid shows similar viscosity values under initial shear rate conditions  shows better rheology than the optimum nanofluid prepared with the addition of 1 wt% ZnO nano dispersion (type 4).In our previous paper, we proposed ZnO nanoparticle-enhanced nonpolymeric SBVE fluids for hydraulic fracturing applications. However, the fluids were not compared with any polymeric fluids applied conventionally in fracturing operations. In this study, the rheology of the fluids was compared with traditional guar gum gel fluids to develop and optimize new high-surfactant SBVE fluids based on the hypothesis of obtaining fluids with highly improved rheology. The key findings of this study are as followings:−1 can be compared (Moreover, the type 3 optimum fluid seems to have comparable rheology to guar gum fluid under all conditions. For field implementation, the average viscosity values at shear rate groups of 1 to 100, 100 to 200, 200 to 300, 300 to 400, and 400 to 500 scompared . The oveTherefore, the presented novel optimal surfactant-based viscoelastic fluid of the type 3 category has the potential to replace polymeric guar gum fluid and can be implemented as a novel nonpolymeric fracturing fluid composition during hydraulic fracturing operations.


This communication briefings the roles of Lorentz force and nanoparticles aggregation on the characteristics of water subject to Titanium dioxide rotating nanofluid flow toward a stretched surface. Due to upgrade the thermal transportation, the nanoparticles are incorporated, which are play significance role in modern technology, electronics, and heat exchangers. The primary objective of this communication is to observe the significance of nanoparticles aggregation to enhance the host fluid thermal conductivity. In order to model our work and investigate how aggregation characteristics affect the system’s thermal conductivity, aggregation kinetics at the molecular level has been mathematically introduced. A dimensionless system of partial-differential equations is produced when the similarity transform is applied to a elaborated mathematical formulation. Thereafter, the numerical solution is obtained through a well-known computational finite element scheme via MATLAB environment. When the formulation of nanoparticle aggregation is taken into consideration, it is evident that although the magnitude of axial and transverse velocities is lower, the temperature distribution is enhanced by aggregation. The host fluid which contains the nanomaterials has a valuable influence on the dynamics of host fluid according to the experimental and numerical investigation3. The modern era researchers are fascinating due to wide range of nanofluids application in every field of engineering and science4. The host fluid temperature enhanced due to mixture nano-sized particles inside the fluid and it is happen because of effective thermal transport of tiny particles which are responsible for enhancing heat transfer rate5. The tiny solid particles different shapes influence the host fluid dynamics and significance influence on host fluid temperature6. The different particles features inside the nanofluid flows have been investigated numerically, analytically, and experimentally by the research community7. Recent, several researchers investigated the different nature of nanoparticles to improve the host fluid thermal enhancement subject to different types of techniques and geometeries10. The inclusion of nano-particles in the base fluid resulted in a rapid rise in temperature, whereas the mono fluid had a smaller impact on temperature than the hybrid nanofluids11. The combination of base fluids and small solid fragments can improve the thermal properties of several fluids13.Nanofluids are fluids that are created by the homogenous dispersion of tiny particles of metal or metal oxides at the nanoscale14. Titanium dioxide powders have assortments of applications, attributable to its ability to bestow whiteness furthermore, darkness to different things, including paints, papers,and beauty care products. The titanium dioxide has acquired incredible consideration as a promising photocatalyst because of its useful properties among the other photocatalysts, high compound strength, and minimal expense16. The numerous researchers presented titanium dioxide tiny particles used for the treatment of dangerous cancers types17 and many other aspect of human daily life18. Fujishima et al.19 specifically provided additional details regarding the outstanding titanium dioxide photocatalyst material for environmental purification.In our daily life, the titanium dioxide is key component which is studied in metal oxide surface science as a crystalline oxides because of its capacities as a photocatalyst with sensibly high proficiency for water breakdown20. Because of this, heat can be moved very quickly through the nanoparticles clusters. In addition to the greater influence volume of aggregations compared to that of nanoparticles, can increase the nanofluid’s heat conductivity21. Experiments have shown that particle aggregation can increase the efficient heat conductivity of nanofluids24. The nano-sized nanoparticles clustering have a remarkable influence to upgrade host fluid thermal properties and aggregated nanoparticles are more efficient heat transfer rate as compared to non-aggregated nanoparticles25. In light of practical applications like automobile radiators, printing inks, and polyester synthesis, as well as the significance of particles aggregation for enhancing heat transport, nanoparticles aggregation gain mush popularity now a days26. The aggregation inside the host fluid is influenced by both initial concentration and cluster size27. After that, the aggregated particles volume, number of nanoparticles per aggregated cluster, and the fractal dimensions of the size of the clusters were reported in references29. The significant applications of magnetohydrodynamic (MHD) engagement can be found in aerospace engineering, astrophysics, and medicine. Due to the connection between the electrically charged liquid and the magnetic field, numerous machines with MHD generators, boundary layer flow, bearings, and turbulent pumps are pretentious. Zeeshan et al.30 examined the significance of magnetic field subject to water contain titanium dioxide nanofluid and they found that the magnetic field has significance impact on the dynamics of nanofluid. The host fluid temperature enhanced due to higher strength of magnetic field and recede the fluid velocity31.The aggregation of nanoparticles have a significant impact on both thermal and rheological properties. In order to construct a passageway that has a lower thermal resistance, the aggregated particles typically form linear chains33, is used to solve MATLAB environment. When the influence of nanoparticle aggregation is taken into consideration, it is evident that although the velocity is lower, the temperature function is upgraded by aggregation. Thus, demonstrating aggregates’ significance and impact, as well as their value as a theoretical tool for future industrial and technological applications. The various types of boundary layer flow subject to different fluid related problems have been solved via finite element approach studied by34. This approach solved fluid problems rapidly, rapidly, and precisely. This report give the answers of below research questions: What is influence of magnetic parameter on the characteristics of water-titanium dioxide nanofluid subject to titanium dioxide particles aggregation and non-aggregation?What is impact of Coriolis force on the dynamic of water-titanium dioxide nanofluid subject to titanium dioxide particles aggregation and non-aggregation?What impact do the magnetic and rotating parameters have on Nusselt number and skin friction factors in the presence of titanium dioxide particles aggregation and non-aggregation?A review of the literature reveals that rotating nanofluid has received less attention subject to nanoparticle aggregation. As a result, the current manuscript concentrated on the transport phenomenon of Titanium dioxide nanoparticle aggregates in water-based fluids under the influence of an externally imposed magnetic field. Through aggregation, the thermal conductivity of nanoparticles can be significantly improved. Additionally, nanoparticles come with a number of difficulties. Trying to figure out how a system’s molecular interaction, like aggregation kinetics, affects the fluid’s thermophysical properties is one of engineering’s biggest challenges. Through this literature, we learn how aggregation kinetics can affect heat transfer, generated temperature, and thermal conductivity. In light of previous research, we therefore investigated the effect of aggregation kinetics on a rotating nanofluid subject to lorentz force. The prime objective of this communication is to observe the role of nanoparticles aggregation to enhance the host fluid thermal conductivity. The FEM, a well-known computational schemez direction, which is oriented transverse to xy plane, at an angular velocity of O has been chosen, with the x-axis representing the movement of the stretching surface, the y-axis representing the normal of the surface, and the z-axis representing transverse to the xy plane. In the axial (z-direction) direction, a static and uniform magnetic 35. The ambient temperature is represented by x, y, and z directions, respectively. Tables 37:T represents the fluid temperature. The formulated problem boundary conditions are39:Take into consideration time dependent three-dimensional rotating water-based nanofluid that flows across a stretching subject to magnetic field. On an elongated surface, a three-dimensional, non-transient flow of magnetized nano liquid TiO2) is considered, as depicted in Fig. mentclass2pt{minim is consi38):Similarity transformations \documententclass1pt{minimaw of Eq.  becomes By utilization of Eq. , we get 43. The first and second essential steps of this method are to assume the piecewise continuous function in order to obtain the solution and to locate the parameters that correspond to the functions in such a way as to minimize the error in the solution45. With the boundary conditions (12) for the solution of a system of simultaneous partial differential equations as shown in  red red12) rEquations –20) are are20) aIn above mentioned mathematical relation, The final equations model for finite element is shown below:46 and Ali et al.40. In addition, the 47 and Adnan et al.48, who present finite element results against rising inputs of Using a graphical representation, the physical effects of the various flow parameters, including the rotating, magnetic, and time dependent parameters, on the dimensionless fluid flow velocity and the temperature are shown. The effects of particle aggregation suspensions with microscopic particles and a fluid that is susceptible to Lorentz and Coriolis forces have been demonstrated by our findings. The each graph show two types of curves for nanoparticles aggregation (M), is depicted in Fig. M results in a significant decrease in the primary velocity 38, for transient Newtonian convection surface extending flow, this is due to a upgrade in unsteady convection currents, which enhances boundary layer thermal diffusion. In addition, the model and nanoparticle aggregation have a lesser distribution of . Figure M, a significant decrease in the axial friction is observed. Figure M is improved, the significance difference near the boundary surface is noted. Figure M(1:1:5) and M and Figure The nanoparticles aggregation causes a decline in magnitude of both primary and secondary velocities, and lesser the strength of The fact that the aggregation model has a temperature profile that is higher than that of typical model and a significant decline in improve thermal performance of base fluid and a negative influence on Increase the value of the skin friction factor, The magnitudes of axial momentum and transverse momentum decrease when the Coriolis and Lorentz strengths are exceeded, andA theoretical model for predicting the thermal conductivity of nanofluids has been developed by taking into account the structure of the aggregates and nanoparticles, as well as the physical properties of the base liquid and the nanoparticles. Effective nanofluid thermal conductivity and viscosity for homogeneous models and nanoparticle aggregation were investigated by the authors. It is reasonable to draw the following main findings based on present report analysis:


The majority of literature on cavitary pulmonary coccidioidomycosis is from four decades ago which was prior to the advent of triazoles and focused on surgical treatment. This observational study is a comprehensive retrospective study of pulmonary cavitary coccidioidomycosis from patients at Valley Fever Institute at Kern Medical over the last 12 years. This observational study aims to explore the spectrum of coccidioidal cavities and the evaluation and management of those cavities.IRB approved, retrospective review of electronic medical records of the Valley Fever Institute database was conducted. Demographics, comorbidities, types, and the number of cavities, complications, and medical and surgical treatment were gathered and compared to the literature. PubMed and Google Scholar were searched for cavitary pulmonary coccidioidomycosisOf the initially 276 identified patients, 137 met the inclusion criteria. This study found 52 (37.2%) patients with hemoptysis. One case (0.7%) required radiologic intervention to occlude the bleeding vessel, and one (0.7%) case of hemorrhage required right upper lobe lobectomy. Nine (6.6%) cases exhibited a ruptured cavity. Eight of those cases had initial chest tube placement, of which three  did not require surgical intervention. Seven of 137 (5.1%) cases presented with a pleural effusion not associated with a cavity rupture. Five (3.7%) were due to primary coccidioidomycosis. Three of the coccidioidal effusions required therapeutic thoracentesis, and none required a chest tube or surgery. The mean duration of the initial antifungal treatment was found to be 563 days (n=80). In 35% (28/ 80) of them, a triazole was switched to another triazole for variable reasons, including treatment failure or side effects.Coccidioidal pulmonary cavitation remains a complex disease to evaluate and treat. This study's ethnic demographic differed from other cohorts. It also contradicts the notion that pulmonary coccidioidal cavitary disease and dissemination infrequently manifest in the same patient. In the present age of triazole therapy, indications and the need for surgery continue to decline. Further investigation needs to be conducted to evaluate medical therapy’s efficacy and long-term outcomes.Rupam Sharma, PGY-1/MD, Astellas: Grant/Research Support George R. Thompson, III, MD, Astellas: Advisor/Consultant|Astellas: Grant/Research Support|Cidara: Advisor/Consultant|Cidara: Grant/Research Support|F2G: Advisor/Consultant|F2G: Grant/Research Support|Mayne: Advisor/Consultant|Mayne: Grant/Research Support|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Mundipharma: Advisor/Consultant|Mundipharma: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support


Candida palmioleophila is a rare human pathogenic fungus, which has been poorly characterized at the genome level. In this study, we reported the first fatal case of C. palmioleophila infection in China and investigate the microevolution of C. palmioleophila in the human host environment.C. palmioleophila stains were collected from the patient at different time points for routine microbial and drug sensitivity testing. The first C. palmioleophila isolate 07202534 was identified by de novo whole genome sequencing. The in vitro and in vivo genetic evolutionary characteristics of C. palmioleophila were discussed based on the analysis of bioinformatics data.A series of C. palmioleophila isolates displayed dose-dependent sensitivity to fluconazole. The C. palmioleophila genome contained homologous genes such as CDR1 and MDR1, which were recognized to be related to azole resistance. In addition, amino acid variation was detected at F105L and other important sites of ERG11. In addition, the mean divergence time between C. palmioleophila and Scheffersomyces stipites CBS 6054 was 406.04 million years, indicating that C. palmioleophila originated earlier than its closest relative. In addition, the six strains of C. palmioleophila isolated form the patient had higher homology and fewer mutation sites, which indicated the stability in C. palmioleophila genome. We also found that C. palmioleophila had a wide natural niche and may evolve slowly.The six C. palmioleophila and will aid in the prevention and control of its spread.We believe that this study will contribute to improve our understanding of the genetic evolution, pathogenicity, and drug resistance of  Candida palmioleophila is ascomycetous yeast, which was first isolated in 1988 from soil. In 1999, C. palmioleophila was reported as a human pathogen causing intravenous catheter-associated fungemia  (Candida guilliermondii (Meyerozyma guilliermondii)  perform de novo whole genome sequencing (WGS) of C. palmioleophila and compare its genetic and evolutionary characteristics with those of related species.In clinical practiceansenii)  or Candirmondii) . Sequencermondii . At pres in 2023 , the genC. palmioleophila-positive patient was collected via the Hospital Information System (HIS) system of the First Hospital of China Medical University.Clinical information  relating to the This study was reviewed and approved by the Ethics Review Committee (ERC) of the First Hospital of China Medical University (ERC number: 2019-53-2) and was conducted in accordance with the Declaration of Helsinki and its amendments.9 cells/L; neutrophil accounted for 85%; and a C-reactive protein level, 22.6 mg/L. Lung computed tomography (CT) indicated multiple spots under both lungs. Because aspiration pneumonia was suspected, the patient was administered antibiotics such as cefotaxime or meropenem. Aspiration pneumonia recurred several times.A 92-year-old man with aspiration pneumonia was admitted to the First Hospital of China Medical University on March 31, 2020. The patient’s chief complaint was fever for 4 days. He had acute onset of fever and respiratory symptoms. The patient was admitted for routine blood tests. His white cell count was 7.87 × 10The patient also had a 10-year history of Alzheimer’s disease, a 5-year history of cerebral infarction, and had been bedridden for a long time with nasogastric feeding. In addition, he had a history of prostatic hyperplasia for many years, a long-term indwelling catheter, and bullous pemphigus for 3 months. Several skin ulcerations could be seen on the patient’s limbs and trunk.C. palmioleophila strain (isolate 07202534) was isolated. The serum -β-D-glucan level was 227.86 pg./mL. Voriconazole 200 mg  was administered by nasal feeding.On June 9, 2020, a white floc was found in the patient’s indwelling catheter, which did not obstruct catheter drainage. The catheter was replaced and the bladder was rinsed with normal saline. The midstream clean-catch urine cultures were negative for bacteria, but the first The patient developed gastrointestinal bleeding on July 16, 2020. Diet and water were prohibited while parenteral, nutritional support was given via peripheral inserted central catheter (PICC).C. palmioleophila was isolated several times from a mid-stream urine sample ]. e sample . BecauseC. palmioleophila was isolated from the patient’s PICC catheter tip. The infection progressed, and the patient died of septic shock on October 16, 2020.On October 12, 2020, Candida sp. (isolate 07202534), with a count of 1 × 105 colony-forming units (CFU)/mL. This strain could not be identified using the VITEK 2, MALDI-TOF, or VITEK-MS systems, and was later confirmed as C. palmioleophila by internal transcribed spacer (ITS) sequencing , July 27 (isolate 08201684), August 6 (isolate 08201782), and August 18 (isolate 07204494); the colony counts ranged between 104 and 106 CFU/mL. On October 12, 2020, a strain of C. palmioleophila (isolate 07206383) was isolated from an intravenous catheter tip, with a colony count of 200 CFU/mL . The minimum inhibitory concentration (MIC) results were interpreted according to the breakpoints suggested for other Candida species by the Clinical Laboratory Standards Institute .C. palmioleophila cell were cultured on YPD medium at 37°C for 18 h and then used for genomic DNA extraction. The genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, with minor modifications . Paired-end libraries were prepared using the TruSeq DNA Sample Preparation Kit, followed by 2 × 150 bp sequencing using the Illumina Novaseq platform . Then 10-kb libraries were generated with the Template Prep Kit . Single-molecule real-time (SMRT) sequencing was performed by using the Pacific Biosciences platform . Raw data were processed after adapter contamination removal and data filtering using the Adapter Removal  was performed by Shanghai Personal Biotechnology Co., Ltd. (China). ications . The DNA Removal  and SOAP Removal . The fil Removal  and A5-m Removal  to const Removal . Subsequsoftware .Function genomic element prediction involved the prediction of coding genes, non-coding RNA, and repeated sequence. Genes prediction was performed using Augustus (version 3.03), glimmerHMM (version 3.0.1), and SNAP (version 2006-07-28). Homologous gene prediction was obtained from the protein sequences of related species using the “exonerate” tool (version 2.2.0). The predicted genes were integrated using EVidenceModeler software (version r2012-06-25) . The comFunctional annotation was completed by performing a Basic Local Alignment Search Tool (BLAST) search of the following databases: including Non-Redundant (NR) protein database, Gene Ontology (GO), Kyoto Encyclopedia of Gene and Genomes (KEGG), SwissProt, Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOg), Pathogen-Host Interactions (PHI), Transporter Classification Database (TCDB), Carbohydrate-Active Enzymes (CAZy), cytochrome P450, and Pfam.C. palmioleophila isolates, the extracted DNA of was randomly interrupted by ultrasound, and an insert fragment with an average length of about 400 bp was obtained according to different interrupted time and rotation speed, and then the fragment was constructed for subsequent genomic library. It is typically 6–8 cycles at the time of library construction. The sequencing data were then mapped to the above genome sequence of the C. palmioleophila isolate (07202534) to identify single nucleotide polymorphisms (SNPs), gene acquisition, recombination, loss as well as nucleotide insertions and deletions (indels). The filtered high quality data were aligned to the reference genome using the bwa-men program (version 0.7.12-r1039); the aligned parameters were based on the default parameters of bwa-men. The sam files were transformed to bam files using the bwa sampe and samtools  toolkit. Picard software (version 1.107)For the other five vs-all blastp analysis, and the threshold of series alignment was set as 1e-10. The orthomcl (version 2.0.8) software was used to process the result of sequence alignment, in which the length of sequence alignment was set to 70%, MCL was used to cluster the gene family, and I (Inflation) used for clustering was set to 1.5. Finally, a homemade Perl script is used to sort out and count the clustering results.First, the protein sequence of the reference genome  was downBased on the results of the homologous gene cluster analysis, single copy homologous genes were selected for multiple sequence alignment. An alignment quality control software system  of Paml software was used to estimate the divergence time between species based on amino acid sequence alignment information . AccordiThe expansion and contraction of gene families was analyzed using CAFÉ software (version 3.1), which used statistical analysis to obtain the size of each gene family. For a given phylogenetic tree, CAFÉ can estimate the total birth and death rates of each gene family, infer the most likely gene family size at each internal node, and identify branches where the gene family expands and contracts if the gene family size is known for each existing species. NCBI nr (Database release 2017.10.10), KEGG (KAAS Ver. 2.1) and GO annotation  were performed on genes related to expansion and contraction of the target gene family.C. palmioleophila grew as round, smooth, white, creamy colonies with neat edges at 35°C for 3 days. When grown on CHROM agar Candida medium , the colonies appeared white at 35°C for 24 h, gradually turning turquoise, with some areas of light pink at 35°C for 5 days. C. palmioleophila can be grown at temperatures up to 40°C. C. palmioleophila did not form pseudohyphae, but budding was observed under the microscope , which accounted for 53.45% of the genome length; each gene had an average of 1 exon. Protein coding genes were annotated using the NR, EggNOg, KEGG, SwissProt, GO, P450, TCDB, Pfam, and PHI databases and yielded 4,343 (98.41%), 4,213 (95.47%), 2,978 (67.48%), 3,968 (89.92%), 3,605 (81.69%), 4,314 (97.76%), 980 (22.21%), 3,779 (85.63%), and 1, 249 (28.30%) genes, respectively , which is associated with azole resistance. Moreover, the similarity between the ERG6 gene of C. palmioleophila and Candida albicans was 89.5%. In addition, we found genes encoding the ATP binding cassette (ABC) transporter family CDR1 and major facilitator superfamily (MFS) transporter MDR1 in C. palmioleophila genome. However, the identity between these C. palmioleophila genes and their orthologs in related species such as Saccharomyces cerevisiae, C. albicans, and C. tropicalis was not overly high (33.7–89.5%).We next annotated the C. albicans, such as the secreted lipases (LIP1) and secreted aspartyl proteinases  were also enriched in C. palmioleophila. This suggests that C. palmioleophila may display similar pathogenesis as C. albicans . Furthermore, we downloaded the ERG11 sequence of 19 common Candida spp. from the NCBI website, combined them with the scaffold2.t669/OP691259 sequence generated in this study, and analyzed the amino acid mutation sites related to fluconazole resistance. By comparing the C. palmioleophila ERG11 with its homolog in C. albicans, we found that the amino acid mutations occurred in important sites such as F105L, S110N, D116E, E266K, R267T, N440K, and F487Y. Among these, the F105L, E266K, and F487Y amino acid mutation sites were consistent with the fluconazole resistance of C. auris and other closely related species  and human blood, tissue, urine, and oral specimens. Evolutionary analysis showed that the ITS sequences of these strains were highly homologous  sites between these strains, suggested that the genomes of C. palmioleophila were highly stable  protein. There were also eight SNPs in the upstream region of the gene encoding for the prenylated Rab acceptor 1 (YIP3) protein  had a 46-bp exonic deletion in the gene encoding for the transcriptional repressor TUP1. The other 51 indels occurred in the upstream, downstream, and intergenic gene regions, including those of SGT1, ERG7, and LIP1  were observed in the five  strains .C. palmioleophila isolate 07202534 genome tested in this study and 23 genomes of related fungal species downloaded from the NCBI database . The average divergence time between C. palmioleophila 07202534 and S. stipitis CBS 6054 and S. Tezawaensis NRRL Y-17324 was 406.04 million years (range: 310.57–472.54 million years) (The mean divergence time between n years) .C. palmioleophila belongs to the order Saccharomycetales, the family Debaryomycetaceae, and the clade Candida glaebosa. C. palmioleophila are rare fungi that have been reported to cause an increasing number of human infections . The patient later developed Candida catheter-associated infection and eventually died of septic shock. Two recent studies have shown that C. palmioleophila can cause fatal infections in animals. An example was the death of a green sea turtle from disseminated candidiasis and candidemia  and secreted aspartyl proteinases (SAPs). Surprisingly, we observed a 46-bp indel in the TUP1 gene in the genome of the catheter-associated C. palmioleophila 07205383 isolate. The TUP1 gene encodes a transcriptional repressor and contributes to the transcriptional control of virulence-associated genes in C. albicans   . Thus, tC. palmioleophila isolates identified in this study that exhibited a dose-dependent sensitivity to fluconazole. Besides above mentioned strains, we also isolated several fluconazole-resistant strains from the same patient; however, these strains were not retained and they were not included in this analysis. Thus, these findings collectively suggested that the resistance of C. palmioleophila to antifungal medication should not be ignored. Another study confirmed the presence of fluconazole-resistant C. palmioleophila infection  and S. tanzawaensis   . The fiv an exon . This inC. palmioleophila strains were compared and analyzed. The results showed that C. palmioleophila isolated from different regions at different times had high level of homology. This information also suggests that C. palmioleophila evolved slowly.Thirdly, by screening ITS gene sequences published on NCBI, more than 30 C. palmioleophila has a wide ecological niche, which included marine and soil environments, plants , and animals   . These f sources . As the diseases . More woleophila  was isolC. palmioleophila might be underestimated because C. palmioleophila can be easily misidentified as C. famata  can be found in the article/The studies involving human participants were reviewed and approved by the Ethics Review Committee (ERC) of the First Hospital of China Medical University (ERC number: 2019-53-2). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.NW, YW, YC, ZR, CR, MY, NJ, YJ, and HL made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. JZ and JC involved in drafting the manuscript. ST have given final approval of the version to be published. All authors contributed to the article and approved the submitted version.This work was supported by the National Key Research and Development Program of China (2021YFC2300400). This work was supported by the National Key Specialist Construction Project for Clinical Laboratory Medicine, Laboratory medicine innovation unit (2019RU017), Chinese Academy of Medical Sciences.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Clostridioides difficile infection (CDI) following clinical treatment guideline updates placing it as first-line therapy. This puts a selection pressure on the development of fidaxomicin resistance, which has been associated with in vitro fitness costs compared to wildtype isolates. Here we aimed to assess the epidemiology of reduced susceptibility (RS) to fidaxomicin and prevalence of rpoB mutations in a clinical cohort.Fidaxomicin use is increasing to treat rpoB single nucleotide polymorphisms (SNPs).Stool samples from patients with CDI were collected from two different healthcare systems in Houston, TX between 2016-21. Fidaxomicin minimum inhibitory concentrations (MIC) were determined via agar dilution susceptibility testing following CLSI standards. RS was defined as MIC > 0.5 mg/L based on the EUCAST [T]ECOFF. A subgroup of 40 isolates with RS underwent Sanger sequencing to detect rpoB SNP mutations were detected in 12.5% (5/40) of isolates at 7 novel base positions; no SNPs were detected at previously reported position 3428.Of 535 patients included, 114 (21.3%) had infecting strains with fidaxomicin RS . Fidaxomicin RS was more common in RT 027  compared to non-027 strains . There were no differences in the proportions demonstrating RS across collection years (P=0.18) or based on location in or out of the Texas Medical Center (P=0.29). Mean MICs were similar between isolates collected before and after the IDSA/SHEA guideline update in 2018 (P =0.1). rpoB SNPs were observed in isolates with RS. Studies are needed to determine potential association(s) between fidaxomicin exposure and MIC changes.RS to fidaxomicin occurred in one fifth of clinical isolates with no changes in incidence between 2016-21. Several new Anne J. Gonzales-Luna, PharmD, BCIDP, Cidara Therapeutics: Grant/Research Support|Ferring Pharmaceuticals: Personal Fees|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support Kevin Garey, PharmD, MS, BCIDP, Acurx Pharmaceuticals: Grant/Research Support|Cidara Therapeutics: Grant/Research Support|Ferring Pharmaceuticals: Advisor/Consultant|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support|Vedanta Therapeutics: Grant/Research Support


Little is known about the prevalence of respiratory symptoms and associated respiratory virus detection in students/ staff in primary and secondary schools.School KIDS is a prospective respiratory viral surveillance program in a large Missouri school district. Participating students/ staff complete a monthly electronic symptom survey that asks about respiratory symptoms (sx) in the preceding 7 days; participants are categorized based on the presence of any respiratory sx: ongoing (current), resolved, and no sx. Within 36 hours of survey completion, participants undergo surveillance respiratory viral testing at school. Self-administered nasal swabs are tested via multipathogen PCR assay (Table). Prevalence of respiratory viruses in participants with and without sx were calculated. A mixed-effect multivariable logistic regression model was used to compare odds of a sample testing positive based on surveillance sx and enrollee age.A total of 544 students (30 pre-kindergarten (preK), 320 elementary, 117 middle, and 77 high) and 224 staff enrolled. A total of 2,537 sx surveys with corresponding respiratory samples were collected from November 2, 2022–April 15, 2023 (Table). Of the samples provided, 1,138 were preK-elementary (45%), 368 middle (14%), 276 high (11%), and 755 staff (30%). Participants reported ongoing (20%), resolved (14%), and no sx (66%). Prevalence of commonly reported sx were congestion (21%), runny nose (17%), cough (16%), and fever (3%). Overall respiratory virus test positivity was 26%; 50% of positive samples came from participants without sx. Flu A, PIV, and RSV were more frequently detected in those with ongoing sx. AdV, HMPV, RV/EV, and SARS-CoV-2 were more frequently detected in those with no sx. Increased odds of a positive test were observed among enrollees who reported runny nose , congestion , cough , or sore throat  but not fever .More research is needed to understand if students with respiratory sx are more likely than asymptomatic students to shed transmissible respiratory viruses.Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support


The importance of fluid resuscitation therapy during the early stages of sepsis management is a well-established principle. Current Surviving Sepsis Campaign (SSC) guidelines recommend the early administration of intravenous crystalloid fluids for sepsis-related hypotension or hyperlactatemia due to tissue hypoperfusion, within the first 3 h of resuscitation and suggest using balanced solutions (BSs) instead of normal saline (NS) for the management of patients with sepsis or septic shock. Studies comparing BS versus NS administration in septic patients have demonstrated that BSs are associated with better outcomes including decreased mortality. After initial resuscitation, fluid administration has to be judicious in order to avoid fluid overload, which has been associated with increased mortality, prolonged mechanical ventilation, and worsening of acute kidney injury. The “one size fits all” approach may be “convenient” but it should be avoided. Personalized fluid management, based on patient-specific hemodynamic indices, provides the foundations for better patient outcomes in the future. Although there is a consensus on the need for adequate fluid therapy in sepsis, the type, the amount of administered fluids, and the ideal fluid resuscitation strategy remain elusive. Well-designed large randomized controlled trials are certainly needed to compare fluid choices specifically in the septic patient, as there is currently limited evidence of low quality. This review aims to summarize the physiologic principles and current scientific evidence regarding fluid management in patients with sepsis, as well as to provide a comprehensive overview of the latest data on the optimal fluid administration strategy in sepsis. Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection, whereas septic shock is a subset of sepsis in which profound circulatory, cellular, and metabolic abnormalities are associated with a higher risk of mortality than with sepsis alone . AlthougThe management of sepsis has not significantly changed over the past 40 years. Current guidelines recommend the early administration of antibiotics and intravenous (IV) fluids, in addition to source control and the judicious use of vasopressors . Fluid rAlthough there is a consensus on the need for adequate fluid therapy in sepsis and despite the multiple recent clinical trials examining fluid management in sepsis, the ideal fluid management strategy is still controversial and elusive, as there are no clear guidelines about the optimal fluid resuscitation in critically ill patients with sepsis. The purpose of this narrative review is to summarize the physiologic principles and current scientific evidence regarding fluid management in patients with sepsis, as well as to provide a comprehensive overview of the latest data on the optimal type  and volume  of fluids in sepsis and septic shock patients.A literature search was performed to identify all published research, such as original articles, reviews, and systematic reviews/metanalyses, using the key words “fluid resuscitation”, “sepsis”, “septic shock”, “critically-ill”, balanced crystalloids”, “normal saline”, “liberal fluid administration”, and “restricted fluid administration”. Records were retrieved from PubMed/Medline and Scopus, without prior application of language or other restrictions for the database search. Reference lists of included articles were also screened to identify potential studies missed by the initial literature search.The physiology of fluid administration in sepsis is a complex syndrome with multiple underlying mechanisms contributing to its pathogenesis. The initial step involves the recognition and the binding of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) on the surface of host immune cells ,5. Toll-Sepsis is characterized by a dysregulated inflammatory response with derangement of both macro- and microcirculation, leading to a status of actual and relative hypovolemia, resulting in tissue hypoperfusion and imbalance between oxygen delivery and demand ,8. The aFluid administration is considered the cornerstone of initial hemodynamic resuscitation in sepsis in order to restore circulating fluid volume and increase cardiac output and eventually, oxygen delivery ,14. FluiThis reasonable and well-established mechanism is excessively simplistic and it is increasingly recognized that, apart from hemodynamics, oxygen delivery and organ perfusion are also affected by the bioenergetic failure and impaired oxidative metabolism ,20. ThisExcessive fluid resuscitation may exacerbate shock, as a significant increase in filling pressures can overcome cardiac compensation mechanisms when the patient reaches the plateau of the Frank–Starling curve ,30,31. TMoreover, sepsis produces alterations in vascular permeability and only 5% of fluid bolus volume remains intravascular after 90 min in the critically ill patient, due to rapid fluid redistribution . It is wThe volume and the type of fluid used for initial resuscitation and the maintenance of fluid therapy have an impact on salt and water retention and by extension to fluid overload, which affects all the major organ systems, causing potential unfavorable outcomes on end-organ function ,42. MoreDespite the scientific advances of the last 20 years, sepsis management has not changed drastically, apart from the introduction of the bundles, which designate multiple interventions that should be completed within a specific time frame. After initial airway and respiratory stabilization, sepsis bundle should be performed within the first 3 h of presentation. The SSC 2021 bundle includes fluid resuscitation, antibiotic administration, lactate measurement and obtainment of cultures . In the CVO2) of at least 70% [Fluid resuscitation remains an integral part of sepsis management, since it was first employed during the European cholera epidemic as early as 1830 . The foleast 70% . The eraeast 70% .There is a growing scepticism regarding aggressive fluid resuscitation, since this approach may lead to massive fluid overload and, inevitably, to adverse outcomes . An incrThe 2021 SSC guidelines suggest the use of crystalloid fluids . HoweverCurrent SSC guidelines suggest using albumin in septic patients who received large volumes of crystalloids over using crystalloids alone . AlbuminIn septic patients, human albumin solution can be given for two indications: to restore or expand intravascular volume and to supplement serum albumin in the septic patients with hypoalbuminemia . In addiThe ideal fluid for septic patients, which would have similar osmolarity to plasma, increase intravascular volume and cardiac output, and improve tissue perfusion without causing tissue edema, while at the same time be cost effective, has yet to be discovered .Fluids are classified according to their composition in two major categories: crystalloid and colloid solutions. Crystalloids are recommended as first-line resuscitation fluids in patients with sepsis as they are inexpensive, widely available, and lead to fewer serious adverse effects ,68. IsotThe most commonly used unbalanced solution is 0.9% normal saline (NS). It is an isotonic solution and contains equal concentrations of sodium and chloride (154 mmol/L) and no organic anion to act as acid buffer. As a result, it has a strong ion difference equal to zero. Notably, its chloride concentration is almost 40% higher than that of plasma. This high chloride concentration has been associated with hyperchloremic metabolic acidosis, impaired tissue and renal perfusion, acute kidney injury, coagulopathy, and altered inflammatory response ,71,73,74Balanced solutions (BSs), apart from sodium and chloride, include other ions, such as potassium, calcium, and magnesium, and may also contain buffers such as bicarbonate, lactate, acetate, or gluconate, leading to electroneutrality  . MoreoveNumerous studies have been carried out in order to investigate and compare the effectiveness of BSs and NS on septic patients’ outcomes and identify the optimal fluid solution. However, which type of crystalloid solution should be administered during the management of patients with sepsis and septic shock remains unanswered, due to the low quality of existing evidence. Current SSC guidelines strongly recommend crystalloids as first-line fluid resuscitation, and further suggest using a BS instead of NS for the management of patients with sepsis or septic shock  .A double-blind, cluster randomized, double-crossover trial was conducted in four ICUs in New Zealand during a 7-month period  trial) aiming to determine the effect of BS in comparison with NS on acute kidney injury . The resIn 2018, the single-center cluster-randomized isotonic Solutions and Major Adverse Renal Events Trial (SMART) was conducted in five ICUs and compared the effect of BS and NS on mortality and renal outcomes . This waConversely, the large RCT Balanced Solution in Intensive Care Study (BaSICS), which aimed to determine the effect of BS (Plasma-Lyte 148) or NS on 90-day survival in ICU patients, did not reveal any significant reduction in mortality between the two groups . InteresRecently, another RCT, the Plasma-Lyte 148 versus Saline Study (PLUS) trial examined the relationship between the administration multi-electrolyte BS (Plasma-Lyte 148) versus NS, and the outcomes of 90-day mortality and renal complications in critically ill adults . NotablyMultiple observational, retrospective, and cohort studies have also been published, comparing the use of BS and NS. Specifically, a retrospective cohort study was designed to determine the effect of a BS (Normosol) compared with NS on the outcomes of patients with sepsis, defined as acute kidney injury and the need for renal replacement therapy . This stVarious systematic reviews and meta-analyses have been conducted comparing the two fluid treatments. Of note, the vast majority of these studies did not reveal any significant difference in the effect of BS or NS on the outcomes of patients with sepsis ,92,93,94Despite the abundance of data regarding the effectiveness and safety of BS and NS use on adults with sepsis, few studies have been carried out in children. Currently, NS remains the preferred resuscitation fluid for children with sepsis, although the SSC guidelines suggest the use of BS rather than NS for the initial resuscitation  . Due to CVO2 > 70% did not improve mortality, but instead resulted in a worse sequential organ failure assessment (SOFA) score, more days on cardiovascular support, and a longer ICU stay. However, in these trials the pre-randomization administered fluids were close to the 30 mL/kg goal, underscoring the wide adoption of SSC guidelines in the clinical practice [Fluid resuscitation is of paramount importance during the early stages of sepsis and septic shock management in order to address the deficit in effective vascular volume and the resulting tissue hypoperfusion. Several RCTs have been designed to investigate the timing and amount of fluid administration, as well as to identify appropriate target goals to guide fluid resuscitation . Since tpractice .+ counts, and were admitted in regular medical wards without access to MV, conditions that prevent the generalization of these results in higher resource settings.On the other hand, trials conducted in resource-limited settings point towards an excess mortality in patients that received bolus fluid therapy. The Fluid Expansion as Supportive Therapy (FEAST) study, conducted in Africa, found an increased risk of death among children with sepsis who received early treatment with bolus 5% albumin or 0.9% saline, in comparison with the control group . SimilarIncreased attention has recently been drawn to the optimal fluid management after the initial resuscitation. It has been shown that a higher volume of fluid during the first 3 h, but lower volume in the first 24 h, reduces mortality in severe sepsis and septic shock patients, and that positive total fluid balance increases mortality by 1.7 times . In ordeEvaluating the heart’s flow response to fluid administration has been proposed to differentiate between fluid responsive and fluid refractory septic states. As static measures, such as CVP, are poor indices of fluid status ,117, dynIn intubated patients, changes in intrathoracic pressure during MV can be used for the dynamic assessment of fluid responsiveness. Pulse pressure and stroke volume changes between inspiration and expiration, assessed by means of pressure/pulse waveform analysis, have been associated with intravascular volume status and the probability that a volume challenge will increase the SV ,126. HowSince findings suggest that fluid restriction and negative fluid balance benefits patients with ARDS, whereas liberal fluids are associated with prolonged MV, extravascular lung water (EVLW) measurement has been suggested to guide fluid administration in addition to dynamic measures . EVLW, aDynamic assessment of fluid responsiveness in a meta-analysis has been associated with a decrease in mortality, ICU length of stay, and duration of MV, but only one study with septic patients was included . In thisUntil recently, efforts have been mainly focused on the assessment of macrocircula-tion, such as cardiac output and blood pressure, to guide fluid resuscitation in patients with sepsis, with the exception of CRT. However, the assessment of microcirculation parameters, in relation to microvascular flow and density, tissue perfusion and oxygenation, and glycocalyx integrity has been on the epicenter of intensive research with promising results. Hand-held vital microscopes (HVM) have been investigated for the visualization of the sublingual capillary network, providing a wealth of information in relation to blood flow characteristics, vascular density, and glycocalyx ,137. ConAlthough fluid management in the septic patient has been extensively studied during the last decades, the “which”, “when”, and “how much” questions are still to be addressed. Evidently, fluid resuscitation has been associated with both benefits and harm; however, there is still a paucity of high-quality evidence to guide the clinical practice regarding fluid management in sepsis. Therefore, controversies remain in the scientific community, and more high-quality, robust studies are warranted to guide future developments. The “one size fits all” approach may be convenient, but it seems that personalized fluid management and taking patient-specific hemodynamic indices into account will provide the basis for better patient outcomes in the future. In this respect, cutting-edge tools for the assessment of perfusion on the tissue level are being investigated and their implementation in real-world settings, however challenging, is expected to revolutionize the management of these patients. Well-designed RCTs are required to compare fluid choices, specifically in septic patients, and they have to be powered enough to evaluate patient subgroups that may respond differently to different types or volume of fluids administered. Existing diagnostic tools have been proven to be valuable in assessing fluid responsiveness, although further trials are yet to determine improvement in patient-important outcomes.Data on the optimal type  and volume  of fluids in sepsis and septic shock patients are still controversial and elusive.Current SSC guidelines recommend the early administration of 30 mL/kg of IV crystalloid fluids for sepsis-related hypotension or a lactate ≥ 4 mmol/L, within the first 3 h of resuscitation. This is a weak recommendation and is based on low-quality evidence.Regarding the type of fluid administered during resuscitation, the majority of clinical trials demonstrated no significant difference between the balanced crystalloids and normal saline in the acute kidney injury and mortality.Excessive fluid administration during resuscitation can lead to worse outcomes in the septic patient.Fluid administration after initial resuscitation should be preferably guided by dynamic measures of fluid responsiveness.Fluid management in the critical patient can be optimized by the personalized, bedside, and dynamic assessment of macro- and microcirculation indices.The take-home messages are as follows:


For a transparent well with a known volume capacity, changes in fluid level result in predictable changes in magnification of an overhead light source. For a given well size and fluid, the relationship between volume and magnification can be calculated if the fluid’s index of refraction is known or in a naive fashion with a calibration procedure. Light source magnification can be measured through a camera and processed using computer vision contour analysis with OpenCV. This principle was applied in the design of a 3D printable sensing device using a raspberry pi zero and a camera. The automation of cell culture procedures has the potential to greatly increase the throughput and consistency of cell culture based experiments . ManipulSurface level measurement within a container of known volume is a simple way to determine fluiod volume. This can be done with electrically active probes dipped into a fluid being measured . These pHere we propose a method for non-contact optical detection of fluid level using a CMOS camera sensor and an LED. We detail the principles at work and show a 3D printed device that can be used to perform measurements in 24 well culture plates. Similar to the approach shown in , this meThe measurement principle behind this system relies on the lensing effects of fluids with different indices of refraction. In θ1 can be chosen so long as it results in a ray that intersects the fluid, and from this chosen angle θ2 can be derived via Snell’s Law. In terms of these constants the resulting transfer function is:I to the fluid level L and is a first degree polynomial. The full derivation can be found in section of the supplemental materials. This function is a useful approximation that ignores several factors including refraction caused by the plate material and refraction from the meniscus geometry of the fluid surface, our data show that accurate results can be obtained with this approximation in most scenarios. Meniscus has a substantial effect on measurement in two extremes, an almost empty well, and an almost full well. These meniscus effects are further explored in section.To derive a function relating the fluid depth to the apparent distance of the light source, we define the following constants.The polynomial can be characterized analytically so long as we can determine how the image distance relates to size on the camera sensor. This can be determined by taking a picture of a ruled measurement calibration slide at several known distances. It can also be characterized by a simple calibration step, being that the relationship is linear, a 2 point calibration would theoretically be sufficient, but for greater accuracy a 5 point calibration would be more prudent. Once this polynomial is characterized, image data is sufficient for capturing fluid level.To test this approach, we designed a 3D printable rig that holds a raspberry pi camera and a white LED. Using openCV , a free A Jupyter notebook that implements the described pipeline can be found in the supplemental material and any future updates will be accessible through the github repository linked in the supplemental materials. Through the use of Jupyter widgets, live changes to various parameters can be adjusted on the fly to optimize the detection process for any given setup.The plot shown in The region between points A and B are where this sensing process will be most generalizable, as the relationship between the spotlight image contour size and the fluid level is highly linear.μl within the linear range. Outside this range, the estimation error increases substantially.Deriving an effective transfer function for this system can be done by fitting a curve to a set of calibration points. Deciding on the best fit curve is a question of trade offs. The phenomenon we are measuring is linear within the range of volumes that have a stable meniscus geometry. A simple 2 point linear fit with points from the linear region, generates outputs with average error below 100In order to represent the entire curve, we need a high order polynomial fit. Using a least squares approximation method, we can fit polynomials to any subset of points we choose. Considering the phenomena shown in To evaluate the consistency of this measurement principle, data was captured in 2 different wells during 2 different runs. Measurement error along these runs are shown in The percentage error values of each fit are examined in In summation, we find that a 5th order least squares polynomial fit applied to 6 points  results in a transfer function that giving us a mean error of 5.51% and a standard deviation of 6.21%. This is a reasonable error for a low cost, low complexity system. The error is also consistent across multiple runs, indicating that the transfer function is generalizable to different wells and different runs. When constrained to the central linear region, this transfer function has a mean error of 4.62% and a standard deviation of 3.10%.The data presented so far have been captured under ideal conditions using distilled water on a level surface as our fluid of interest. To demonstrate the generalizability of the measurement principle. We also tested using food grade canola oil. The results are shown in The theoretical analysis shown in When a well is completely dry, droplets hold their shape rather than forming a layer along the bottom. Since this approach relies on the lens-like properties of a fluid layer, it does not work when the fluid in question has not formed a complete layer across the base of the well. The optical consequences of an incomplete fluid layer are shown in One of the most common ways to sense the height of a fluid’s surface is to use optical or ultrasonic time of flight sensors. These sensors interpret the time delay between signal emission and the return of the reflected signal to determine the distance to the surface. This method is highly effective. What is advantagous about the method presented here is that it uses hardware that may be already present in the system. For instance some lab-on-a-chip microfluidic systems contain imaging modules as well as the materials to move fluids . In systWhile the results presented here rely on calibration to numerically determine a suitable transer function, system characterization could theoretically be done through detailed modeling. Compared to the calibration approach, this would require detailed information about system parameters like camera lens optics, fluid properties, well geometry, and the optical properties of the cell plate material. However, if such a model were developed with sufficient accuracy it could offer additional insights and error checking. For instance, if the response of a fluid in the well is not as the model predicts, that may indicate mixups or mistakes in the fluid handling process. However, the additional complexity of the modeling approach vs pure calibration based fitting would make this feasible only for applications at large scale using the same system and fluids. The calibration based approach is more generalizable and can be applied to any system with a camera, led, and a well plate.By leveraging the availability of low cost camera hardware and open source machine vision tools, devices that use computer vision for sensing tasks are likely to become more ubiquitous. The proliferation of cheap mass-produced camera sensors is an opportunity for designers to consider many new methods of measurement.The device shown here serves as a simple proof of concept for the use of this fluid level measurement principle. For practical purposes, this should be deployed with many cameras in parallel, each monitoring a single well. Previous work has been published laying out the design and use of a 24 well parallel microscope system using similar camera hardware and LEDs, this system is called the “Picroscope” , 12. TheIt would also be possible to use this principle without the overhead LED being permanently affixed above the culture plate. In applications using pipette robots, LEDs could be attached to the end effector, if the arm positions the LED at a known position, spot measurements can be taken and used to detect potential dosing errors or losses due to evaporation.This proposed fluid level sensing method is a simple, reliable, and accurate approach that has been validated for cell culture plates and has potential for uses in other applications.S1 AppendixDetailed derivation of image distance transfer function.(PDF)Click here for additional data file.S1 FileData from sensing pipeline used to generate plots shown in the paper.(ZIP)Click here for additional data file.S2 Filehttps://github.com/pvbaudin/Optical-fluid-level-measurement-for-culture-plates.Notebook used for live image capture and processing. Allows for calibration and testing of the sensing pipeline. Current version can be found at (IPYNB)Click here for additional data file.


The duration of the tumour-penetrating effect of CEND-1 was evaluated by assessing tumour accumulation of Evans blue and gadolinium-based contrast agents in hepatocellular carcinoma (HCC) mouse models. The plasma half-life was approximately 25 min in mice and 2 h in patients following intravenous administration of CEND-1. [3H]-CEND-1 localised to the tumour and several healthy tissues shortly after administration but was cleared from most healthy tissues by 3 h. Despite the rapid systemic clearance, tumours retained significant [3H]-CEND-1 several hours post-administration. In mice with HCC, the tumour penetration activity remained elevated for at least 24 h after the injection of a single dose of CEND-1. These results indicate a favourable in vivo PK profile of CEND-1 and a specific and sustained tumour homing and tumour penetrability. Taken together, these data suggest that even single injections of CEND-1 may elicit long-lasting tumour PK improvements for co-administered anti-cancer agents.CEND-1 (iRGD) is a bifunctional cyclic peptide that can modulate the solid tumour microenvironment, enhancing the delivery and therapeutic index of co-administered anti-cancer agents. This study explored CEND-1’s pharmacokinetic (PK) properties pre-clinically and clinically, and assessed CEND-1 distribution, tumour selectivity and duration of action in pre-clinical tumour models. Its PK properties were assessed after intravenous infusion of CEND-1 at various doses in animals  and patients with metastatic pancreatic cancer. To assess tissue disposition, [ There is a substantial volume of literature reporting that poor penetration of anti-cancer agents into solid tumours is an important factor limiting their efficacy. Adequate drug penetration into the tumour can be compromised by several factors, including the dense connective tissue stroma of the tumour microenvironment . This phThe tumour-penetrating peptide CEND-1  has been shown to improve the tumour penetration of various simply co-administered chemotherapeutics, leading to an improved drug dose efficacy ,5,6 thatThis study examined the pharmacokinetic (PK) properties of CEND-1 in mouse, rat, dog and monkey models for translational purposes. The clinical PK properties were investigated in patients with metastatic pancreatic ductal adenocarcinoma before and during combination therapy with nab-paclitaxel and gemcitabine. Moreover, in vivo distribution and tumour selectivity of CEND-1 was assessed in mice bearing orthotopic 4T1 mammary carcinoma. Finally, we examined the duration of the tumour-penetrating effect of CEND-1 in a mouse model of hepatocellular carcinoma (HCC).max and AUClast, generally increased with dose in a more-than-dose-proportional manner, with rapid elimination. The average half-lives were approximately 20–25 min in mice, 30 min in rats and 40 min in dogs. The in vivo pharmacokinetic assessment of CEND-1 in mice, rats, dogs and monkeys after a single intravenous dose showed pharmacokinetic profiles comparable in all species . The sys0-inf) of CEND-1 at 5 mg/kg and 50 mg/kg were 28,230 hr*ng/mL and 421,119 hr*ng/mL, respectively  and combination therapy with nab-paclitaxel and gemcitabine (cycle 1 day 1 and cycle 6 day 1) at 1.6 and 3.2 mg/kg CEND-1 are summarised in Administration of subsequent CEND-1 doses at Cycle 1 Day 1 and Cycle 6 Day 1 again resulted in plasma concentrations rapidly increasing with peak concentrations occurring at the first post-dose PK sampling timepoint, before decreasing in an exponential manner B,C. Mean0-t, AUC0-6h and AUC0-inf) followed the same pattern described for Cmax with a linear increase with an increased dose. The median half-life values of CEND-1 (T1/2) were between 1.6 and 1.8 h over PK sampling timepoints. Mean clearance (CL) values were between 106.8 mL/h/kg and 266.5 mL/h/kg. The terminal volume of distribution (Vz) mean values were between 220.9 mL/kg and 277.4 mL/kg over all days of PK sampling.Assessment of plasma CEND-1 parameters demonstrated that exposure (AUCThe PK profile of CEND-1 at the beginning of the study (run-in and C1D1) and after five completed cycles of therapy (C6D1) were similar.3H]-CEND-1 in female BALB/c mice with 4T1 tumours was assessed at 30 min and 3 h after intravenous administration  of [e tumour . Radioace tumour . 3H]-CEND-1 in female BALB/c mice with 4T1 tumours was assessed at 30 min, 3 h and 8 h after intravenous administration  (The quantitative tissue distribution (QRA) of -CEND-1 administration in mice bearing 4T1 mammary carcinoma tumours were assessed using QWBA and QRA. Initially, [3H]-CEND-1-related radioactivity was detectable in most organs at 30 min post-administration but was subsequently largely cleared in non-tumour tissues within 3 h post-administration, with the exception of the tumour, and tissues known to be related to the metabolism and secretion of CEND-1 . The renal excretion is similar to other tumour-targeting RGD peptides such as cilengitide -CEND-1 became apparent by 3 h post-administration. Although at 3 h the [3H]-CEND-1-related signal was mostly eliminated systemically from tumours and healthy tissues, the signal was still detectable in tumours, demonstrating specific, potent and prolonged retention in tumours. Furthermore, the tumour concentrations of radioactivity remained at least seven-fold higher than any other tissue at the 12 mg/kg dose. These results are supportive of previous non-clinical studies that demonstrated the ability of CEND-1/iRGD in enhancing drug delivery and efficacy in both primary and metastatic tumours in a variety of mouse and human tumour types [The pronounced tumour specificity of [3H]-CEND-1-related radioactivity was not detected in the tissues of the central nervous system including the brain, suggesting that CEND-1 does not cross the blood-brain barrier. Nevertheless, since CEND-1/iRGD has shown efficacy in multiple pre-clinical models of glioblastoma, there is a possibility that the disrupted blood-brain barrier (BBB) may allow peptides such as CEND-1 to penetrate the brain in certain pathological conditions  and RGD control peptide (CRGDDGPKC) for pre-clinical use was sourced either from GenScript  or CPC Scientific . It was manufactured using solid phase peptide synthetic techniques with high chemical purity. It has a cyclic structure (S-S bonded through the cysteine side chains). The purity of all materials was >95%.The CEND-1 drug product for clinical use was provided by Cend Therapeutics, Inc. . CEND-1 for injection was a sterile, white, lyophilised powder supplied as 100 mg per vial of active ingredient dose strength for intravenous administration. CEND-1 injection consisted of the CEND-1 drug substance with sodium acetate trihydrate and mannitol as excipients.TGFα/c-myc bitransgenic mice and tumour-induced NMRI Foxn1 nude mouse models were approved by the local animal care committee in agreement with German legal requirements . The clinical pharmacokinetic study protocol and all amendments were approved by Central Adelaide Local Health Network Human Research Ethics Committee (HREC) (HREC/18/CALHN/225), St John of God Hospital Ethics Office (Ref 1397) and Sydney Local Health District HREC, Concord Repatriation General Hospital on behalf of Alfred Hospital (HREC/18/CRGH/72). All human participants provided written informed consent prior to enrolment. The human study is registered with ClinicalTrials.gov (NCT03517176) and the Australian New Zealand Clinical Trials Registry (ACTRN12618000804280).The animal pharmacokinetic studies were conducted under approvals from Pharmaron Institutional Animal Care and Use Committee. The murine orthotopic 4T1 tumour model animal experimentation was conducted under approvals from the Animal Welfare and Ethical Review Body (AWERB)  and in accordance with the Animal Welfare Act 2006, with UK Home Office Guidance on the implementation of the Act and with all applicable Codes of Practice for the care and housing of laboratory animals. Experiments involving the The in vivo pharmacokinetic profile of CEND-1 after a single dose was evaluated in four non-GLP animal studies including Sprague-Dawley rats, Beagle dogs, cynomolgus monkeys and BALB/c mice following the intravenous injection of CEND-1 at various doses  . Blood sPlasma CEND-1 was quantified by validated liquid chromatography coupled with a tandem mass spectrometry (LC/MS/MS) assay. Tolbutamide or leuprorelin acetate were used as internal standards. AB Sciex Triple Quad API 5500 , operated in the positive electrospray ionization (ESI) mode, was used to monitor the precursor to production ion transitions of m/z 495.4→69.9.n = 14) and 3.2 mg/kg (n = 14) in the expansion cohort were analysed. Details of this phase I clinical study, conducted in patients with metastatic pancreatic ductal adenocarcinoma receiving CEND-1 in combination with nab-paclitaxel and gemcitabine, are described in detail by Dean et al. -NSP and re-dissolved in PBS pH 7.4, followed by gentle mixing for approximately 2 h, or overnight at 2–8 °C, until protein tagging and hydrolysis of unbound NSP was complete. The NSP-peptide solution was then purified by reverse phase chromatography and the resulting solution was concentrated using a centrifugal evaporator. The resulting material was dissolved in 1:1 ethanol:water and stored at –80 °C prior to formulation for dose preparation. Radiochemical purity and specific activity of the [3H]-labelled test peptide was determined using reverse phase HPLC and quantitative radioactivity analysis (QRA). However, the pharmacologic activity of the labelled peptide was not determined. CEND-1 A, manufa3H]-NSP) B. Briefl7 cells suspended in PBS. The 4T1 triple negative breast tumour cell line was obtained from Crown Bioscience UK . To produce 4T1 tumours, female BALB/c nude mice , obtained from Charles River Ltd. , were orthotopically injected in the mammary fat pad with 2 × 106 4T1 cells into the mammary fat pads to induce tumour development. [3H]-CEND-1 was administered when the tumours were approximately 2 weeks old (approximately 80–250 mm3). Radiation was quantified in tissues using QWBA . The mice were deeply anesthetised to a surgical plane with isoflurane and a blood sample collected by cardiac puncture at 0.5-, 3- or 8-h post-dose administration. The mice were then perfused by using ~20 mL of PBS containing 1% BSA via a 25G 5/8 fixed needle into the left ventricle and making a cut to the right atrium. Immediately following perfusion, the mice were killed by cervical dislocation and the tissues were dissected for QRA analysis. Female 4T1 tumour-bearing BALB/c mice (as above) each received a single intravenous administration of [TGFα and albumin/c-myc single transgenic mice in CD13B6CBA background were crossed to generate the male TGFα/c-myc bitransgenic mice, as previously described [2 (via the drinking water) to induce the expression of TGFα. The animals were inspected every 2 to 3 days. To detect and monitor the endogenously formed HCCs in the TGFα/c-myc mice, gadoxetic acid (Gd-EOB-DTPA)-enhanced MRI was performed on a 3T MRI scanner  as described recently [Homozygous metallothionein/escribed ,21. Heparecently ,21,22. MHepG2 cells were obtained from American Type Culture Collection (ATCC) and were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin . The ceTGFα/c-myc mice with confirmed liver tumours according to Gd-EOB-DTPA (Dotarem)-enhanced MRI or nude mice with HepG2 tumours received iRGD/CEND-1 or RGD control peptide (4 μmol/kg each), or PBS by tail vein injection. Evans blue  was injected intravenously 15 min later. Another 30 min later, the mice were terminally perfused with Ringer solution by cannulation of the left heart ventricle. Organs were inspected macroscopically following laparotomy, and the liver, tumour and other organs were excised. Evans blue was extracted from tissues in N,N-dimethylformamide for 24 h at 37 °C and quantified spectrophotometrically  at 600 nm. TGFα/c-myc mice with confirmed liver tumours or nude mice with HepG2 xenografts were anaesthetised by intraperitoneal injection of ketamine (70 mg/kg body weight) and xylazine (10 mg/kg body weight), followed by a basal T1–weighted MRI and immediately thereafter a Gd-DTPA-enhanced MRI [The nced MRI . In the nced MRI . For quanced MRI . ROIs weTM WinNonlin, version 6.1). The linear log trapezoidal algorithm, with a weighting of 1/Y*Y was used for parameter calculations. Mean PK parameters were calculated from individual animals in each treatment group. Concentrations below the LLOQ (if any) were excluded for the calculation of PK parameters. The mean values of PK parameters were calculated using Microsoft Excel version 2010. No formal sample size calculation was performed. Samples of 2–12 animals and 31 pancreatic cancer patients were considered acceptable to perform initial pharmacokinetic evaluations. The concentrations of CEND-1 were used to calculate PK parameters by employing a non-compartmental analysis (Phoenix