In major surgery, ScvO2-based goal-directed therapy can be used to enhance fluid therapy and improve patient outcomes.Fluid loading-based goal-directed therapy is a cornerstone of anaesthesia management in major surgery. Its widespread application has contributed to a significant improvement in perioperative morbidity and mortality. In theory, only hypovolemic patients should receive fluid therapy. However, to achieve such a diagnosis, a surrogate marker of cardiac output adequacy must be used. Current methods of fluid loading-based goal-directed therapy do not assess cardiac output adequacy. Nowadays, new devices make it possible to continuously monitor central venous oxygen saturation . Patients in the control group will undergo standard fluid loading-based goal-directed therapy, as recommended by the guidelines. Patients in the intervention group will have ScvO2 to guide fluid therapy will result in a reduction of postoperative complications as compared with current goal-directed fluid therapy practices.This study is the first to address the issue of cardiac output adequacy in goal-directed therapy. Our hypothesis is that cardiac output optimisation during major surgery achieved by continuous monitoring of the ScvOClinicalTrials.gov. NCT03828565. Registered on February 4, 2019.The online version contains supplementary material available at 10.1186/s13063-023-07689-z. Fluid loading is a cornerstone of anaesthesia management in major surgery. Indeed, both fluid overload and hypovolaemia are deleterious for surgical patients , 2.Several studies have assessed fluid loading based on clinical variables  and against goal-directed variables . These studies found a reduction in postoperative morbidity and length of hospital stay in goal-directed fluid therapy patients, including in long-term follow-up –6.Since 2012, experts in the French Society of Anaesthesia and Intensive Care Medicine have recommended using goal-directed therapy for fluid loading in high-risk surgery patients . These g2).However, it is not known whether fluid responsiveness alone is sufficient to determine the need for fluid loading. Indeed, with regard to the Frank-Starling law, fluid responsiveness is a physiological status, which means that there is no need to provide fluid in all responders . Hypovol2 is an excellent surrogate for detecting cardiac output variations. Today, new devices make it possible to monitor ScvO2 continuously and therefore to assess the adequacy of perioperative cardiac output. This facility could be used to enhance fluid therapy during major surgery and improve patients’ outcomes.Serum lactate concentration is a late marker of cardiac output adequacy. In addition, it is not sufficiently accurate to detect slight changes in cardiac output. In contrast, ScvO2 to guide fluid therapy during major surgery will result in better cardiac output optimisation with a reduction in postoperative complications as compared with current goal-directed fluid therapy practices.Our hypothesis is that using the continuous monitoring of ScvOThis study will be a prospective, randomised, single-blinded, multicentre controlled superiority study with a 1:1 allocation ratio.Three French hospitals in which major surgery is performed: Hôpital Nord , Institut Paoli Calmette , Hôpital Caremeau .Adult patients over 50 years of age with an American Society of Anesthesiologists (ASA) score of between 2 and 4 who are scheduled for elective major surgery. Elective major surgery is defined as abdominal or thoracic surgery with a duration of at least 90 min and requiring the use of invasive blood pressure monitoring and a central venous line.Patients in the control group will be treated with standard care. Fluid loading will be performed in accordance with French guidelines . The car−1) infusion will be started  fluid loading of 250 mL of balanced crystalloid solution will be administered. If the stroke volume and/or cardiac output increase by at least 10%, additional fluid loading will be administered. When the stroke volume and/or cardiac output do not increase or increase by less than 10%, fluid loading will be stopped. If the patient is not fluid responsive with a mean arterial pressure below 10% of the value of his or her preoperative mean arterial pressure, a diluted norepinephrine .Patients in the intervention group will be treated via an algorithm based on continuous perioperative ScvO2 is above 70%, cardiac output will be defined as adequate. A diluted norepinephrine infusion will be used to maintain a mean arterial pressure within a 10% variation of the value of the preoperative mean arterial pressure, regardless of fluid responsiveness status.If ScvO2 is above 70%, but the need for norepinephrine is above 0.5 µg kg−1 min−1 to reach the blood pressure targets, a fluid loading of 250 mL of crystalloids will be administered. Administration will be repeated every 15 min until the norepinephrine dosage is below 0.5 µg kg−1 min−1.If ScvO2 is below 70%, cardiac output will be defined as inadequate. First, three out of the four determinants of ScvO2 will be assessed and corrected as needed . Then, fluid responsiveness will be assessed as described above. If the patient is fluid responsive, a fluid loading of 250 mL will be administered. If ScvO2 increases above 70%, fluid loading will be stopped. If not, the fluid loading will be administered as long as ScvO2 remains below 70%, with positive fluid responsiveness. If the patient is in the “grey zone” (when the fluid responsiveness assessment is not informative), the patient will be reassessed after 5 min  on day five after surgery . CCI is Regarding secondary outcomes, our hypothesis is that our experimental strategy will reduce the quantity of fluid administered during surgery. Therefore, the secondary outcomes will be the total amount of fluid administered perioperatively and the lung ultrasound score (LUS). LUS is a simple, fast and reproducible measure to assess the potential harm of excessive fluid administration . To assePatients will be recruited during their pre-anaesthesia consultation. The protocol will be explained, and written consent obtained. On the day of surgery, the patients will be randomly allocated to either the control group or the experimental group. During surgery, haemodynamic, ventilatory and pharmacologic data will be collected. In the post-anaesthesia care unit (PACU), an arterial and a venous blood gas analysis and an LUS will be performed in patients after tracheal extubation.The patients will be monitored daily during the 5 days following surgery. A phone call will be made on day 15 and on day 30 to collect information about potential postoperative complications. The duration of participation for each patient included in the study will be 30 days. Patients cannot be included in another study for 30 days after their randomisation.A sample size calculation was made from published data , 11. In Patients will be recruited during the anaesthesia consultation by a senior anaesthesiologist once the protocol has been explained and their written consent obtained.Randomisation will be performed with the block randomisation technique to reduce bias and achieve balance in patient allocation. Randomisation will be stratified by the treatment centre to reduce selection bias. Randomisation will be performed on surgery day by the attending anaesthesiologist with the nQuery Advisor software .Due to the study design, the attending anaesthesiologist can’t be blinded to the study intervention. Therefore, double blinding will not be feasible. However, the patient is blinded to his study group and postoperative data collection and statistical analysis will be performed by investigators blinded to the group allocation of each patient. Since the attending physicians is unblinded to the study intervention, no emergency unblinding strategy has been developed.Demographics .Comorbidities: Charlson comorbidities score and preoperative score to predict postoperative mortality (POSPOM).Surgical risk  and surgical procedure.2 for the patients in the experimental group.◦ Haemodynamic (continuous): mean, systolic and diastolic blood pressure, heart rate, cardiac output, stroke volume, stroke volume variation and ScvO2, positive end-expiratory pressure (PEEP).◦ Respiratory (every 30 min): tidal volume, respiratory rate, FiO◦ Neurologic (every 30 min): bispectral index (BIS), suppression ration (SR).Perioperative:At the end of surgery: total volume of fluids administered , type and total amount of anaesthesia drugs used, use of an epidural analgesic.In the PACU: arterial and venous blood gas analysis, LUS.Postoperative (daily): type and severity of postoperative complications until day 5 postoperative. CCI score calculation on day 5.At day 15 postoperative: collection of postoperative complications between day 5 and day 15 and CCI score calculation on day 15.At day 30 postoperative: collection of postoperative complications between day 15 and day 30 and CCI score calculation on day 30.All data will be collected in real-time by the clinical research team of the participating centres using a case report form. The variables to be collected are as follows:Data entry will be performed using REDCap software. Data analysis will be performed by a statistician from the clinical research team, using SPSS software .Table t-test to assess differences between parametric continuous variables, the Mann–Whitney test for non-parametric variables, the χ2 test for categorical variables and the Fisher exact test for 2 × 2 tables. No correction for multiple testing will be done. A two-sided p < 0.05 will be considered statistically significant. Stopping of the trial could also be based on interim data analysis if clearly one treatment is better than the other, but no interim analysis is planned.Statistical analysis will start only after the database has been validated . The statistical analysis will be blinded (no explicit identification of groups). The methodology and statistical analysis will be performed in accordance with the CONSORT statement . The popIn accordance with the Helsinki Declaration and French law, the research team will declare adverse events without delay to the study sponsor (Assistance Publique Hôpitaux de Marseille). Participation in the study will be clearly stated in the patients’ medical files. The data monitoring and the coordinating committees from the study sponsor will perform annual data-monitoring sessions for quality control and trial conduct auditions during the study period.This study was approved by an institutional review board (Comité de Protection des Personnes SUD-EST VI) on December 9, 2019. All participants will be able to consent to the study and written consent will be obtained from all participants. Minors and patients unable to understand the study or under guardianship will not be included.−1 min−1. All participating centres were notified without delay by the study sponsor of any protocol amendments. If a major deviation from the study protocol should happen, it will be documented by the investigators with a Breach Report Form.Two amendments have been made to the original protocol. The first amendment removed the diagnosis of heart arrhythmia from the exclusion criteria, because stroke volume variation was not used in the study to assess fluid responsiveness. Due to safety concerns, the second amendment added the 250 mL fluid loading in the experimental group should the norepinephrine dose exceed more than 0.5 µg kgAll participants will have consented to the study. Minors and patients unable to understand the study or under guardianship will not be included.The database will be anonymous and encrypted to ensure data safety and patient confidentiality.Professor Leone reports personal fees received from Aspen, AMOMED, LFB, Ambu and Gilead outside of the submitted work.Professor Zieleskiewicz reports personal fees received from General Electric Healthcare outside of the submitted work.Dr Pastene reports personal fees from Edwards Lifesciences outside of the submitted work.Dr Bernat, Dr Baumstark, Dr Bezulier, Dr Gricourt, Dr De Guibert, Dr Charvet and Dr Colin have nothing to disclose.The datasets analysed during the current study and statistical code will be available from the corresponding author, BP, on reasonable request, as is the full protocol.Patients enrolled in the study will be covered by indemnity for negligent harm through the standard indemnity arrangements of French national health insurance. Hôpitaux Universitaires de Marseille has insurance to cover non-negligent harm associated with the protocol.The authors aim to publish the present protocol in a high-impact, peer-reviewed anaesthesiology journal. The results will be published even in the event of non-significant results.Additional file 1.


Spontaneous tumour regression in patients after high dose therapy and autologous stem cell transplantation or patients with standard therapy is accompanied with the presence of high titers autoantibodies against carbonic anhydrase I (CA I). The concomitant presence of aplastic anaemia‐like syndrome in these patients points to parallel bone marrow suppression during this period. It seems that CA I, an ‘obscure’ enzyme, does not have any significant physiological role in humans. One possible explanation points to the fact that autoantibodies against CA I may target another antigen(s) which is(are) important in tumour growth as well as in normal haematopoiesis. One of the candidates for such a target is the DNA polymerase theta. The highest numbers of such cases have been reported in patients with lymphoma/leukaemia, malignant melanoma, neuroblastoma and renal cell carcinoma (‘hypernephroma’). The mechanism of spontaneous tumour regression is unknown. Some of the published data indicate that immune mediation, possible tumour growth inhibition by growth factors and/or cytokines, induction of tumour differentiation, hormonal action, tumour necrosis, psychological factors and epigenetic mechanisms may be involved in this process.2Spontaneous tumour regression after its relapse after HDT and autologous stem cell and AuSCT has been firstly described in 2003.3The anti‐CA I autoantibodies were found in patients with bona fide aplastic anaemia (AA).4The presence of anti‐CA I autoantibodies in the patient sera during the spontaneous regression of their tumours is striking. It is associated with the suppression of bone marrow, a phenomenon which got a classification ‘aplastic anemia‐like syndrome’ (‘AA‐like syndrome’). The anti‐CA I autoantibodies are present in up to 40% of patients in bona fide AA too.https://www.uniprot.org/uniprotkb/O75417/entry#sequences) can be found in three of them: SLKPI, DGLAV and SSEQL. The almost full alignment with SLKPI sequence is at the Polθ position 511‐515 (SLKPV), with the DGLAV sequence at the Polθ position 1811‐1815 (DGLQL) and finally with the SSEQL sequence at the Polθ position 1821‐1825 (SSESL) . From this point of view the pathophysiological explanation for bone marrow suppression (AA‐like syndrome and bona fide AA) and spontaneous tumour regression may have a common point: The suppression of the DNA polymerase Polθ which is vital for both bone marrow (haematopoiesis) and tumour cell growth. Clinically this resembles the picture of spontaneous tumour regression which is associated with AA‐like syndrome.Therefore, it is tempting to search for other models which could explain the observed data obtained in laboratory experiments. One of the candidates with strong possible cross reactivity could be the enzyme DNA polymerase theta (Polθ). The DNA polymerase Polθ has been described rather in detail.Ján Lakota: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; writing – original draft ; writing – review and editing .The author declares no conflict of interest.


The novelty and motivation of this research can be emphasized by examining how the heat transfer mechanism of a non-Newtonian Powell-Eyring fluid, which flows because of a stretched sheet, is affected by factors like viscous dissipation, the slip velocity phenomenon, and Joule heating. In addition, the investigation delves into the heat transfer behavior of the fluid flow when it comes into contact with a convectively heated stretched surface that is influenced by varying fluid properties. This analysis also takes into account the influence of changing fluid characteristics and the presence of magnetic field. The numerical solutions of modelled equations that governing the problem are detected using the shooting technique. Also, in order to confirm the validity of the present investigation, a proper comparison with certain published works as a particular case of the present model is presented, and a perfect agreement is noted. With the use of diagrams and tables, the flow problem’s effective parameters are thoroughly discussed. Likewise, through a tabular representation, the values of the local Nusselt number and the skin-friction coefficient are computed and analyzed. Many significant conclusions can be drawn from numerical results. Most importantly, the local Nusselt number rises monotonically with both the surface convection parameter and the slip velocity parameter, but the local skin-friction coefficient has the opposite trend. The results indicate that the nanofluid temperature is enhanced by factors such as the surface convection parameter, magnetic field, and viscous dissipation. On the other hand, the slip velocity phenomenon leads to the opposite effect. Beginning with the pioneer Sakiadis2 studied Blasius type flow over a stretching sheet where the speed was constant and the fluid at rest. Sequentially Crane3 investigated the flow of fluid which has common characteristics with boundary layer of tile Hiemenze flow and the velocity of stretching sheet is proportional to distance of the slit. The analytical solution of flow past stretching surface in the existence of magnetic field in two dimensions was studied by Kumari and Nath4. Abel et al.5 investigated the transfer of the heat in visco-elastic fluid through porous medium over surface where the viscosity was variable with temperature. Sanjayanand et al.6 obtained the analytical solution of zeroth order of stream function. The Mass and heat transfer equations are investigated past continuous stretching sheet to obtain the hypergeometric solutions. Chamkha and Mansour7 studied the transfer of heat past a stretching sheet in the presence of chemical reaction through porous medium and the unsteady free convection as well. The influence of temperature and space on flow which was generated over a stretching surface in the existance of normally applied magnetic field was studied by Ganga et al.8. The generation of entropy on steady incompressible Carreau liquid flow past a stretching sheet with curved shape in two dimensions was studied by Raza et al.9. Megahed et al.10 studied the MHD fluid flow with heat transfer past an unsteady stretching sheet under thermal radiation and heat flux effects. Tawade et al.11 obtained the solution of nonlinear ordinary differential equations using the approach of Runge-Kutta 4th-order with shooting for nanofluid flow which was laminar and unsteady past stretching surface in two dimensions. Some further problems related to the fluid flow under to a stretchable sheet with variables thickness for different conditions can be found in the literature14. Later, there are numerous researchers development about nanofluids flow subject to stretchable sheets are highlighted in Refs.17.Monitoring flow of fluids over stretching surfaces and investigation of their behavior recently occupied a significant regard between researchers in engineering and science aspects due to their major role in several of industrial applications such as plastic manufacturing and rubber as well, photographic films coating by liquids and manufacturing of lubricants etc.20. Powell-Eyring model is one of the non-Newtonian fluid models which is derived from kinetic theory of gases instead of empirical formula. Many researchers used the Powell-Eyring model under different physical conditions. Hayat et al.21 obtained exact solution of magnetohydrdynamic eyring powell fluid flow past a stretching surface and discussed radiative effect as well. The analytic solution for the Powell-Erying fluid flow past a nonlinear stretching sheet was investigated by Panigrahi et al.22. Gaffar et al.23 investigated the Eyring-Powell fluid flow over vertical surface through porous medium. Hayat et al.24 studied flow of Eyring-Powell fluid past an exponentially stretching surface in the Prescence of chemical reactions and heat flux. The influence of convective boundary conditions, viscous dissipation and activation energy on Eyring Powell nanofluid through porous medium was examined by Nazeer et al.25. Patil et al.26 studied the effect of chemical reaction and thermal radiation of unsteady Nano Powell-Eyring fluid flow behind stagnation point. Khader and Babatin27 carried out a numerical solution by using the spectral collocation method the flow of Powell-Eyring fluid in the existence of magnetic field and thermal radiation.Over the years, many researchers have aimed to study the properties of fluids, especially non-Newtonian fluids, which have become extremely popular in various industrial, bio-fluid, foods, and medical fieldsIt’s important to note that earlier research overlooked the heat transfer process of a Powell-Eyring fluid, which is impacted by elements such as viscous dissipation, slip velocity phenomenon, and Joule heating. Additionally, prior studies did not consider a convectively heated stretched surface that is influenced by changing fluid properties, all within the same investigation. Consequently, the novelty and impetus behind the present research are driven by insights from the literature review, urging an exploration into the impacts of some important phenomena like viscous dissipation, slip velocity, Joule heating, and magnetic field on the flow behavior of the convectively heated Powell-Eyring fluid. Notably, this fluid’s properties undergo thermal changes due to the stretching sheet.B are applied to fluid layers, causing them to impart in the transverse direction. In our research, we suppose that a hot fluid exists beneath the stretching sheet’s bottom surface.Consider a Powell-Eyring fluid moving as indicated in Fig. A and m are constants. The viscous dissipation phenomena is also considered for a better understanding of heat transfer mechanism. The energy equation takes into account the effect of a Joule heating. The rules for fluid and heat transport are determined by the foregoing axioms and the usual Boussinesq’s approximation 1:u, v), C are the Powell-Eyring fluid parameters. These parameters (C) are highly significant, as they control the distinct rheological characteristics and behavior of this non-Newtonian material. They directly influence how the fluid behaves and interacts in various situations, offering valuable insights into its response to external factors. Adding slip velocity to the velocity field’s condition adds to the originality. The continuity, momentum, and energy equations are all linked to the following boundary conditions 28:29:M is the magnetic parameter, Ec symbolize to the Eckert number and Pr is the Prandtl number. All of these parameters can be described as follows:This hot fluid is vital in heating the surface of the stretched sheet by convection with the temperature 29. The previous work of Hayat et al.29, which was performed with the homotopy analysis method, can be compared with our current work in the absence of To support the numerical calculations performed in this work, the results of this study were compared to those published in one of the famous journals, which is addressed further down by Hayat et al.mentclass2pt{minimThis portion of the discussion uses many variables that correspond to the flow under consideration to show physical descriptions of velocity and temperature profiles. Thus, Runge-Kutta of order four is used in conjunction with the shooting technique in Mathematica software. We found that the calculated results had significant impacts. Figure In Fig. Figure Figure M are directly related to the generation of increased resistance force, also known as Lorentz force, it acts in a manner perpendicular to the path of fluid flow; hence, as Lorentz force increases, impedance force also increases and attempts to oppose fluid flow, thinning the thickness of the boundary layer. It follows naturally that raising the magnetic parameter improves the temperature of the sheet 30–33. This lends further support to the understanding of how the magnetic field impacts these processes.Figure Photographs of the temperature and velocity patterns in relation to the thermal conductivity parameter Ec. The Eckert number Ec represents the transformation of kinetic energy into stored energy, which is then dispersed as heat, through work performed versus the stresses of the viscous fluid. Thus, an enhancement in the sheet temperature Figure The distributions of velocity and temperature are each subject to the influence of the surface-convection parameter As shown in Table The slip velocity phenomenon weakened the velocity, temperature distributions, and the skin-friction coefficient, in particular.As the magnetic number or the surface convection parameter rises, both the sheet temperature and the temperature distribution within the thermal boundary region rise as well.As the dimensionless material fluid parameters or the viscosity parameter increases, the rate of heat transfer at the wall decreases.Higher conductivity values result in a rise in the temperature profile, however a reversal of this pattern can be seen beside the sheet.By increasing the viscosity parameter’s value, the liquid’s velocity decreases while the temperature field increases slightly.In future research, we aim to build upon this study by exploring heat and mass fluxes. The goal is to regulate the cooling process utilizing nanofluids.A computational model that incorporates viscous dissipation and convective boundary conditions for a slippery, non-Newtonian Powell-Eyring fluid flowing toward a stretching surface has been interpreted. A set of nonlinear ordinary differential equations is produced by incorporating an appropriate dimensionless transformation into the governing model. Significant data are discussed here after hiring shooting approach to analyze the fluid velocity, fluid temperature, the local skin-friction, and Nusselt number. Following are some conclusions that can be derived from the findings of this study:


In July 2022, an outbreak of MPox was reported in the United States as part of a world-wide health emergency. Healthcare settings in the US started to see possible cases, and the public health and clinical diagnostics infrastructure was quickly overwhelmed. As the concept of diagnostic stewardship is becoming increasingly more important, we sought to identify clinical information obtainable at presentation that would predict a positive MPox PCR in order to streamline testing and optimize resource utilization.Table 1RR and diagnostic performance for single and combined clinical factors resulting in a positive MPox PCRThe charts of 146 patients presenting for MPox testing in a large healthcare system in Texas in July and August 2022 were reviewed to identify clinical factors upon presentation that would correlate with a positive lesion PCR.49.3% of patients tested positive. The mean (SD) age of patients was 31.9 (8.41) years. The mean (SD) time of symptoms was 5.47 (5.08) days. The mean (SD) time from last sexual encounter was 17.6 (11.01) days. Table 1 shows the RR and diagnostic performance for each clinical factor resulting in a positive MPox PCR as well as the RR for those with 2, 3, and 4 combined high risk clinical factors.Male gender, non-heterosexual sexual orientation, recent sexual encounter, painful lesions, contact with a known MPox case, systemic symptoms, and shorter symptom duration less than or equal to 6 days increased the risk of a positive Mpox PCR result and may be used to prioritize testing in a resource constrained environment. Patients presenting with a combination of 2, 3, and 4 risk factors further increased the likelihood of a positive test. Risk factors can be used for diagnostic stewardship in MPox testing. Further research will focus on creation and prospective validation of clinical prediction rules.Luis Ostrosky-Zeichner, MD, FACP, FIDSA, FSHEA, FECMM, CMQ, Astellas: Grant/Research Support|Cidara: Advisor/Consultant|Cidara: Advisor/Consultant|F2G: Advisor/Consultant|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|NIH: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: Honoraria|Pulmocide: Grant/Research Support|Scynexis: Grant/Research Support|T2 Biosystems: Grant/Research Support|Viracor: Advisor/Consultant


Fluorescent molecular probes are very powerful tools that have been generally applied in cell imaging in the research fields of biology, pathology, pharmacology, biochemistry, and medical science. In the last couple of decades, numerous molecular probes endowed with high specificity to particular organelles have been designed to illustrate intracellular images in more detail at the subcellular level. Nowadays, the development of cell biology has enabled the investigation process to go deeply into cells, even at the molecular level. Therefore, probes that can sketch a particular organelle’s location while responding to certain parameters to evaluate intracellular bioprocesses are under urgent demand. It is significant to understand the basic ideas of organelle properties, as well as the vital substances related to each unique organelle, for the design of probes with high specificity and efficiency. In this review, we summarize representative multifunctional fluorescent molecular probes developed in the last decade. We focus on probes that can specially target nuclei, mitochondria, endoplasmic reticulums, and lysosomes. In each section, we first briefly introduce the significance and properties of different organelles. We then discuss how probes are designed to make them highly organelle-specific. Finally, we also consider how probes are constructed to endow them with additional functions to recognize particular physical/chemical signals of targeted organelles. Moreover, a perspective on the challenges in future applications of highly specific molecular probes in cell imaging is also proposed. We hope that this review can provide researchers with additional conceptual information about developing probes for cell imaging, assisting scientists interested in molecular biology, cell biology, and biochemistry to accelerate their scientific studies. The cell is the fundamental constituent of living bodies and one of the basic units for biological investigation. At the subcellular level, various organelles, such as nuclei, mitochondria, endoplasmic reticulums (ERs), lysosomes, etc., are distributed inside cells and function synergistically. Thus far, the statuses of different organelles during distinct bioprocesses have attracted intense interest. It is important to provide visualized organelle signals in biological studies because the relative localizations between different organelles or between organelles and particular molecules significantly influence their activations. For example, the release of cytochrome C from mitochondria into the cytoplasm is a special marker of apoptosis ,2,3. A lMolecular probes with high specificity toward particular organelles are promising tools for biological studies at the subcellular level . Through2+ ions may cause a new kind of programmable cell death process called ferroptosis . Th. ThDAOTAy (FLIM) . Sun et P-2Bz 9,  by linki2+-chelating molecule to construct a new probe, called STDBT  ..2 has th2+ in the ER in different cell lines , lysosomes continuously pump protons into the lysosome through V-type ATPase, i.e., a proton pump, to maintain a stable acidic microenvironment and metabolic capacity . The aciLysosome-targeted molecular probes are usually derived from lipophilic amines, a lipophilic weak base group. Amine-containing molecules can accumulate in lysosomes through passive diffusion or active transport actions, including endocytosis and autophagy; however, compared to the accumulation mechanism mediated by passive diffusion, endocytosis or autophagy contributes less to the number of accumulation probes ,142. TheIn addition to weakly alkaline anchoring, scientists have also reported fluorescent probes based on an “AND” logic gate strategy. Probes that only release fluorescence signals in an acidic environment and after reacting with analytes are designed for the detection of lysosomal location matters. These kinds of molecules may not accumulate in lysosomes automatically, but would still exhibit an excellent signal-to-noise ratio for lysosome-targeted cell imaging.3+) in the lysosome of liver cells is one of the important biological indicators for diagnosing liver diseases such as cirrhosis, liver injury, and liver cancer [3+ fluorescence probes, using galactose as the liver-cell-targeting group, imidazole as the lysosome-targeting group, and naphthimide, with good photophysical and chemical properties, as the fluorescence response group to obtain monodentate, bidentate, and tridentate galactose probes—IM-Gal-1, IM-Gal-2, and IM-Gal-3  in hepatocytes, which enables the probes to exhibit good liver-targeting ability. Cu2+ in lysosomes is reported to relate to Munk’s syndrome, Wilson’s disease, Alzheimer’s disease, and other diseases [DHUCu-1  indiseases . Chen etCu-1 45, , with neLyso-NIR-HClO  level may cause or aggravate cardiovascular and Alzheimer’s disease ,153. MaoHClO 46, , for higysosomes . The fluYSNO 48, , with hiNino 49, , which iONOO 50, , that cao-RC 51, , for the  of the ER and mitochondria may cause the translocation of some transcription factors toward the nucleus to initiate feedback bioprocesses ,168. TheMNQI + channel on the ER surface may influence the bioactivity of the ER. Therefore, several ER localization signal sequences that guide intracellular protein distribution, such as ER retention signal sequences, KDEL (—Lys-Asp-Glu-Leu-COOH), and ER insertion signal sequences, Eriss , have been conjugated with molecular probes for ER-targeted imaging [Low cytotoxicity: The cytotoxicity of the fluorescent molecular probes includes two parts. The first is the phototoxicity generated when the probes were excited by irradiation sources. Under the excitation conditions during cell imaging, the generation of ROS is inevitable. The exceedingly exogenous ROS may break the intracellular redox balance and cause oxidative damage to intracellular biomacromolecules, including DNA, proteins, and lipids, thus influencing the statuses of organelles and living cells. This property of the molecules has been applied to photodynamic therapy for disease treatment; however, it is a limitation of probes in the application of cell imaging, especially in cases of super-resolution and longtime imaging, which need higher emission energy. The modification of the fluorophore is still necessary to obtain a probe that works better inside cells. Chen and co-workers have introduced a triplet-state engineering strategy with which to construct mitochondria-targeted probes with reduced phototoxicity, providing a potential strategy for this direction . The oth imaging . Some sp imaging .(2)The capacity of in vivo imaging: Fluorescent imaging in vivo is always challenging but engaging because it provides the most straightforward biological information. Many of the probes developed at this point possess a relatively short wavelength of excitation/emission located in the UV–Vis wavelength region. This region overlaps with the excitation/emission spectra of biomolecules and biosystems. Therefore, the result might be cluttered by the high background signals. In addition, the absorbance of the short-wavelength light by the biological samples will also limit the application of the probes in deep sample penetration imaging. The satisfying wavelength for in vivo imaging is in the near-infrared range , which can avoid interference form the biological samples. A number of NIR fluorophores have been developed, suggesting the importance of considering the link design with particular moieties for in vivo imaging at the organelle level. Another encouraging method is the design of two-photon-excited fluorophores, which can be excited by two lower-energy NIR photons. Therefore, two-photon fluorescent microscopy can be applied for imaging in vivo by using fluorescent probes with an increased penetration depth and other advantages, such as a prolonged observation time.(3)2+ probes are promising tools for autophagy monitoring. Moreover, the development of novel probes may also encourage biologists to discover unknown bioprocesses. For example, high-resolution methods can assist in distinguishing the different modes of mitochondrial fission; this may explain how other organelles participate in modulating the fission process in cells [Biological guidance: Most molecular probes are developed by chemists, but the utilization of probes is most likely carried out by biologists. The gap between these two fields is obvious, implying that communication between scientists is integral to major breakthroughs and beyond. Our standpoint suggests that the development of biology should guide probe design. The studies on ferroptosis created an urgent demand for the detection of cellular iron and lipid peroxidase, as an example . It was in cells . Furtherin cells ,191,192.The development of fluorescent molecular probes provides powerful tools in the fields of cell-related research. At this point, elaborate cell images covering subcellular information, such as the patterns and statuses of organelles, can be obtained by utilizing specially designed probes. From these images, it is possible to investigate the relationship between particular organelles and different bioprocesses, revealing the signal molecules that play a key role in regulating bioactivity, explaining the molecular mechanisms of some diseases and bringing potential therapeutic targets to light. Hence, advanced fluorescent molecular probes facilitate the understanding of biology, pathology, and pharmacology. Here, we review the probes developed in the past decade for targeting organelles, such as nucleus, mitochondria, ERs, and lysosomes. We mostly focused on the probes proposed in the past five years and discussed the types of building blocks that can be selected for achieving organelle specificity according to the unique chemical/physical properties of different organelles. We also introduced the representative probes that can respond to special intracellular chemical/biological reagents at the subcellular level. The novel probes certainly showed advanced performances in comprehensive applications, but some drawbacks and challenges remain. We believe some of the following directions might receive more attention in the future in order to promote the capacity of the probes.Overall, the journey of organelle-targeted fluorescent molecular probes is just beginning. Deeper growth of chemical and biological knowledge will certainly lead to new probes as powerful tools for the relative fields.


A population pharmacokinetic (PK) model was constructed from nine Phase 1 studies and three Phase 3 studies in patients with acute bacterial skin and skin structure infections (ABSSSI) or hospital- or community-acquired bacterial pneumonia (HABP or CABP) and refined using Phase 3 data from patients with Staphylococcus aureus bacteremia (SAB).Ceftobiprole medocaril is an intravenously administered cephalosporin prodrug that is rapidly converted Structural PK models for systemic compartments and linear vs. non-linear elimination were considered. Covariate analyses were carried out. Model evaluation involved goodness-of-fit and prediction-corrected visual predictive check plots and a sampling-importance-resampling procedure. The model was externally qualified and refined using Phase 3 SAB PK data.Figure 1). Model-based simulations to predict exposure in subgroups  showed that renal function is the most clinically relevant covariate, consistent with the predominance of renal elimination for ceftobiprole . While model-based simulations showed that predicted exposure in subjects with extreme body weight (120 kg) fall below the bioequivalence reference range of healthy subjects weighing 75 kg, these weight-based differences are unlikely to be clinically significant. Neither sex nor infection type are expected to have a clinically relevant impact.The initial dataset included 4890 plasma concentrations from 773 healthy subjects or patients with ABSSSI, HABP, or CABP. This dataset was expanded to include 1134 plasma concentrations from 180 patients with SAB. External qualification showed that the model predicted data from patients with SAB well but the model was refined to best capture data from these patients. The refined model (three-compartment model with linear elimination) provided a robust fit to the pooled data (A population PK model that described the plasma PK of ceftobiprole well in healthy and infected patients was developed. The derived measures of plasma exposure are expected to be both accurate and precise.Anthony P. Cammarata, M.S., Adagio Therapeutics, Inc.: Grant/Research Support|Albany Medical Center: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|BioFire Diagnostics LLC: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Cidara Therapeutics Inc.: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Genentech: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Employee|Inotrem: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A.: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|Qpex Biopharma: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|Theravance: Grant/Research Support|tranScrip Partners: Grant/Research Support|University of Wisconsin: Grant/Research Support|Utility Therapeutics: Grant/Research Support|ValanBio Therapeutics Inc.: Grant/Research Support|VenatoRx: Grant/Research Support Karine Litherland, Ph.D., Basilea Pharmaceutica International Ltd, Allschwil, Switzerland: Full time employee|Basilea Pharmaceutica International Ltd, Allschwil, Switzerland: Stocks/Bonds M. Courtney Safir, PharmD, Adagio Therapeutics, Inc.: Grant/Research Support|Albany Medical Center: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Biofire Diagnostics LLC: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Cidara Therapeutics Inc.: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Genetech: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Employee|Inotrem: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A.: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|Qpex Biopharma: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|Theravance: Grant/Research Support|tranScrip Partners: Grant/Research Support|University of Wisconsin: Grant/Research Support|Utility Therapeutics: Grant/Research Support|ValanBio Therapeutics, Inc.: Grant/Research Support Sujata M. Bhavnani, PharmD; MS; FIDSA, Adagio Therapeutics, Inc.: Grant/Research Support|Albany Medical Center: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|BioFire Diagnostics LLC: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Cidara Therapeutics Inc.: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Genentech: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Ownership Interest|Inotrem: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A.: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|Qpex Biopharma: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|Theravance: Grant/Research Support|tranScrip Partners: Grant/Research Support|University of Wisconsin: Grant/Research Support|Utility Therapeutics: Grant/Research Support|ValanBio Therapeutics Inc.: Grant/Research Support|VenatoRx: Grant/Research Support Mikael Saulay, MSc, Basilea Pharmaceutica International Ltd, Allschwil, Switzerland: Full time employee|Basilea Pharmaceutica International Ltd, Allschwil, Switzerland: Stocks/Bonds Jennifer Smart, PhD, Basilea Pharmaceutica International Ltd, Allschwil, Switzerland: Stocks/Bonds Mark E. Jones, PhD, Astellas Pharma Global Development, Inc: Support for the present publication|Basilea Pharmaceutica International Ltd: Employee of Basilea Pharmaceutica International Ltd|Basilea Pharmaceutica International Ltd: Stocks/Bonds Marc Engelhardt, MD, Astellas Pharma Global Development, Inc.: Support for the present publication|Basilea Pharmaceutica International Ltd: Employee of Basilea Pharmaceutica International Ltd|Basilea Pharmaceutica International Ltd: Stocks/Bonds Christopher M. Rubino, PharmD, Adagio Therapeutics, Inc.: Grant/Research Support|Albany Medical Center: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|BioFire Diagnostics LLC: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Cidara Therapeutics Inc.: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Genentech: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Ownership Interest|Inotrem: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A.: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|Qpex Biopharma: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|Theravance: Grant/Research Support|tranScrip Partners: Grant/Research Support|University of Wisconsin: Grant/Research Support|Utility Therapeutics: Grant/Research Support|ValanBio Therapeutics Inc.: Grant/Research Support|VenatoRx: Grant/Research Support


Hepatocellular carcinoma (HCC) ranks as the third most lethal cancer with 15% 5‐year survival.β = 7  regression was employed to build a novel classifier. Ten‐fold cross‐validation was applied to avoid over‐fitting, selecting an optimal λ value of .0938 Figure . A totalGiven the apparent differences in prognoses between LPRS‐stratified subgroups, we subsequently probed the underlying mechanisms. Differential gene expression analysis identified 1637 prominently DEGs Figure . MetascaIntratumoural immune cells are crucial for HCC development and prognosis.Drawing on canonical gene insights,Collectively, compelling evidence demonstrated that LPRS scoring system exhibited a robust value for survival prediction, offered promising capabilities for comprehensive characterization of oncogenic features, and prospectively stratified patients into different subgroups to determine the administration of adjuvant therapies, especially anti‐cancer immunotherapy. Using it to characterize the heterogeneity of HCC may provide valuable clues for treatment decision‐making and eventually facilitate personalized management.The authors have declared that no competing interest exists.The National Natural Science Foundation of China, Grant Numbers: 82100664 and 82270646; The Fundamental Research Funds for the Central Universities, Grant Number: 0214‐14380510; The Nanjing Health Science and Technology Development Project for Distinguished Young Scholars, Grant Number: JQX19002; The Natural Science Foundation of Jiangsu Province, Grant Number: BK20190114; Jiangsu Province Postdoctoral Research Funding Program, Grant Number: 2021K116B; Medical Science and Technology Development Foundation, Nanjing Department of Health, Grant Number: YKK19070; Project of Modern Hospital Management and Development Institute, Nanjing University and Aid project of Nanjing Drum Tower Hospital Health, Education and Research Foundation, Grant Number: NDYG2020047; The Affiliated Drum Tower Hospital, Medical School of Nanjing University, Grant Numbers: 2021‐LCYJ‐PY‐46 and 2022‐LCYJ‐PY‐35; The Chen Xiao‐ping Foundation for the Development of Science and Technology of Hubei Province, China, Grant Numbers: CXPJJH121001‐2021073 and CXPJJH122002‐019Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Despite the intensive use of radiotherapy in clinical practice, its effectiveness depends on several factors. Several studies showed that the tumour response to radiation differs from one patient to another. The non-uniform response of the tumour is mainly caused by multiple interactions between the tumour microenvironment and healthy cells. To understand these interactions, five major biologic concepts called the “5 Rs” have emerged. These concepts include reoxygenation, DNA damage repair, cell cycle redistribution, cellular radiosensitivity and cellular repopulation. In this study, we used a multi-scale model, which included the five Rs of radiotherapy, to predict the effects of radiation on tumour growth. In this model, the oxygen level was varied in both time and space. When radiotherapy was given, the sensitivity of cells depending on their location in the cell cycle was taken in account. This model also considered the repair of cells by giving a different probability of survival after radiation for tumour and normal cells. Here, we developed four fractionation protocol schemes. We used simulated and positron emission tomography (PET) imaging with the hypoxia tracer 18F-flortanidazole (18F-HX4) images as input data of our model. In addition, tumour control probability curves were simulated. The result showed the evolution of tumours and normal cells. The increase in the cell number after radiation was seen in both normal and malignant cells, which proves that repopulation was included in this model. The proposed model predicts the tumour response to radiation and forms the basis for a more patient-specific clinical tool where related biological data will be included. Cancer is considered the leading cause of death in many developed countries , and it The effectiveness of radiotherapy depends on several factors and especially on the total dose delivered. However, the response of the tumour to a given treatment can vary from one patient to another. Several underlying biological effects that could explain the variation in the biological response of tissues occurring during fractionated radiotherapy have been identified. Such biological principles can be summarized by a simple viewpoint called the five Rs of radiobiology: repair, repopulation, reoxygenation, redistribution in the cell cycle and radiosensitivity .The heterogeneity of the tumour response in patients with the same type of cancer and the non-uniformity of tumour cells within the same individual tumour remain a key challenge for the cure of cancer . UnderstIn recent years, there has been a significant amount of interest in the search for an optimal and personalized treatment plan ,11,12. MThe effects of repair and repopulation have been modelled . In thisFakir et al.  presente2)  HX4 PET images as input data. This tracer is a hydrophilic variant of the 2-nitroimidazole class of radiotracers, which is used as a marker of hypoxia for the preclinical evaluation and validation of PET imaging ,37.The proposed model was developed to describe the evolution and tumour response at both macroscopic and microscopic scales. In this work, the macroscopic scale at the tumour level is represented by PET images. These patient-dependent images are used as the input data, and at this scale, the model consists of a series of µ (vf (relative volume of capillary cells within a voxel). Since the amount of oxygen available may be heterogeneous in the cell microenvironment, a specific oxygen histogram was assigned to each voxel. The shape of the histogram depended on the vascular fraction of the voxel and was build according to the method described by Espinoza et al.  HX4 PET images from an image database generated by our previous study , which aThe model input data are PET images and a distribution of the partial pressure of oxygen. The model returns simulated PET images. Throughout this study, we hypothesized that the number of tumour cells is proportional to glucose consumption as described elsewhere ,41.maxi). Then, we divided the values of each voxel by the max. Then, tµ = 80% of the tumour cells were assigned to each voxel, and in the remaining 20%, nµ = 96.4% were normal cells and cµ = 3.6% capillary cells.To calculate the initial distribution of cells in each voxel, we searched for the voxel with the maximum intensity  is a model that predicts and quantifies the biologic radiation response of tumours, the survival. The most used model is based on Poisson statistics describing the distribution of the surviving tumour cells. In general, the LQ model is used for calculating cell survival and it allows for the quantification of TCP for tumours.Here, a TCP curve was generated by simulating the tumour response to radiotherapy. We consider a tumour controlled if all tumour cells were killed.The presented model allows us to simulate the tumour response under radiotherapy. In In In In the case of mouse number 3, the effect of radiotherapy is not visible. In the case of mouse 4, we can see a slight change. This result is also visible in the case of mouse 4, despite treatment with TH-302, an anti-tumour agent that is activated in a hypoxic environment.In our study, we modelled the response of normal cells to radiotherapy with the linear quadratic model. In During the first 72 h, the decrease in the total number of normal cells is due to the absence of radiotherapy. In fact, when radiotherapy is not administrated, the number of tumour cells per voxel increased. Since there is a fixed cell density in a voxel, tumour cells continued to proliferate at the expense of other cell types. Then, when radiotherapy was administered, the number of dead tumour cells following irradiation gave way to other cell types. Therefore, the number of normal cells increased after 72 h. Then, a slight change in this number was noted between two irradiations caused by cellular sensitivity to irradiation and cellular repopulation.The distribution of cells in the cell cycle affects the outcome of radiation therapy. A weighting coefficient was added to manage the sensitivity of the cells according to their location before irradiation , the percentage in G1 and S increases and that in G2 decreases.In Tp will be equal to 1200 h and then change to 120 h until the end of the treatment. In During the first two weeks of radiotherapy, Here, we considered the spatiotemporal evolution of oxygen during radiotherapy treatment.In α = 0.339 and β = 0.067, which gave a α/β ratio equal to 5.06 Gy  HX4 PET images of mice with rhabdomyosarcoma as input for our simulation model. In In the context of the numerical simulation of tumour growth and response to radiotherapy, we proposed to combine two series of biological processes. The first set includes the main processes often studied independently so far, such as tumour cell proliferation, angiogenesis, cell survival after irradiation, resorption of dead tumour cells and cell replacement. The second set includes the main concepts of what is referred to as the five Rs of radiobiology. These five Rs are cell cycle redistribution, reoxygenation, intrinsic radiosensitivity, cell repopulation and repair. This is the first approach where these concepts are integrated into a unified environment, using both the microscopic cell cycle scale and the macroscopic population scale. To our knowledge, no other study on the modelling of the tumour response to radiotherapy considered these processes altogether. Most previous studies indeed simulated these processes separately , for exaMore specifically, we have shown that our approach responds correctly to the modification of input parameters, through application to a virtual cell population. We have also shown that it is possible to quantify the impact of fractionation on the response, by studying a theoretical population. An example of a practical evaluation is also proposed, using, as input data, a set of preclinical [18F] HX4 PET images acquired under different oxygen conditions. Images obtained after simulation were consistent with the real post-irradiation images. Moreover, our model has been used to test [18F] FDG PET images (non-hypoxia tracer) of patients with rectal cancer, and the results are promising (data not shown).In this study, we investigated the impact of four different fractionation schemes on the evolution of a cell population. The objective was to find the protocol that allowed for better control of the tumour while limiting healthy tissue damage. The results showed that the reduction in treatment time has a considerable effect on local tumour control. In the cases of simulated and real tumours, the CHART protocol showed better tumour control, especially when compared with the conventional protocol. In the case of rectal cancer, studies have shown that the CHART protocol, as a preoperative treatment for rectal cancer, is feasible and appears to be associated with low acute and late toxicity. In the study of S. Brooks et al. , the autImages used in this work were images of mice treated with NaCl or TH-302. TH-302 is an investigational hypoxia-activated prodrug used to treat cancer. TH-302 is activated only in hypoxic regions where the oxygen level is very low. It was developed to target the hypoxic zones existing in tumours. Generally, it is used combination with chemotherapy and shows rather satisfactory results. Recently, in the study of Peeters et al. , the autAs future work, it will be interesting to simulate different dose-painting strategies targeting hypoxic regions in tumours. Indeed, a non-uniform response can be observed within the tumour due to the heterogeneity of oxygenation. This point is consistent with the fact that hypoxic cells are more radioresistant than well-oxygenated ones, which can negatively affect the radiotherapy result. Simulation could provide insights about the non-uniform dose distribution required to destroy an equal proportion of cells within a heterogeneous tumour. In recent work, research demonstrated that radiotherapy induces an immune response. During radiotherapy, some immunosuppressive barriers might increase. This process can lead to tumour resistance. Boustani et al.  reviewedFor radiotherapy, the treatment outcome is determined by several factors. Some factors diminish the effect of radiation therapy, for example, the repopulation of tumour cells and repair from sublethal damage. Some factors increase the local control of the cancer, such as processes of reoxygenation and the redistribution of tumour cell into more sensitive phases of the cell cycle. The impact of the individual factor varies across different tissues, but it is important to consider all of these factors when developing a successful radiotherapy model. In the presented work, we tried to develop a model considering all five Rs of radiobiology. The model developed could be expanded in the future to include patient biological data for a truly specific clinical tool.


Pseudomonas aeruginosa (PSA) pose a challenge clinically as therapeutic alternatives are limited, and clinical data is scarce. Murine models provide translational data that can provide insight into therapeutic alternatives, and assist in clinical guidance. Herein, we evaluated the efficacy of human-simulated regimens (HSRs) of ceftazidime/avibactam (CZA), imipenem/relebactam (I/R), and meropenem/vaborbactam (MVB) in a murine thigh infection model to evaluate in vivo efficacy against serine-carbapenemase-producing PSA.GES and KPC harboring 10 kill at 24h relative to 0h. The percent of isolates meeting the 1-log10 kill endpoint were assessed.9 PSA clinical isolates harboring GES 5 (n=1), GES 20 (n=1), GES 5/20 (n=1), GES 19/20 (n=3), and KPC (n=3) were evaluated. Groups of 6 mice were administered HSRs of CZA 2.5 g q8h as a 2h infusion (inf), I/R 1.25 g q6h as 0.5h inf, and MVB 4 g q8h as a 3h inf. Groups of 6 mice were sacrificed at 0h to determine initial bacterial burden (0h control) or treated with sham control and sacrificed at 24 hours to assess in vivo growth (24h control). Change in CFU/thigh relative to 0h was assessed and efficacy was defined as achieving ≥ 1-log10 CFU/thigh. Adequate growth was observed in control mice with ≥ 2 log10 CFU/thigh. CZA and I/R HSRs produced ≥ 1-log10 of kill against 100% and 83% of GES positive isolates, respectively . Activity was consistent against GES and KPC harboring isolates despite elevated in vitro MICs in some isolates. MVB activity was variable with only 44% of isolates reaching 1-log10 kill (MIC range 16 - >64). MVB failed in 67% of GES and 33% of KPC producing isolates all with elevated MICs. I/R and CZA in vivo/ in vitro discordance may be due to higher relebactam and avibactam concentrations achieved in the humanized exposure relative to the in vitro fixed concentrations.Baseline bacterial burden was 5.2 ± 0.4 login vivo. The activity of MVB was variable suggesting this may be an inferior treatment option. Further studies to evaluate clinical outcomes in GES and KPC producing PSA using CZA and I/R are warranted given their increasing prevalence worldwide.I/R and CZA were active against all KPC and GES harboring PSA isolates tested Christian Gill, PharmD, Cepheid: Grant/Research Support|Entasis: Grant/Research Support|Everest Medicines: Grant/Research Support|Shionogi: Grant/Research Support David P. Nicolau, PharmD, Allergan: Advisor/Consultant|Allergan: Grant/Research Support|Cepheid: Advisor/Consultant|Cepheid: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Tetraphase: Advisor/Consultant|Tetraphase: Grant/Research Support|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant|Wockhardt: Grant/Research Support


Maintaining the proper fluid balance is a fundamental step in the management of hospitalized patients. The current study evaluated the impact of negative fluid balance on outcomes of patients with confirmed COVID-19.  We considered the negative fluid balance as a higher output fluid compared to the input fluid. The fluid balance was categorized into four groups  and included ordinally in the model. The outcomes were all-cause mortality, length of hospitalization, and improvement in oxygen saturation. p < 0.001). After adjusting for potential confounders, there was a significantly lower frequency of mortality in patients with negative fluid balance compared to the controls . Similarly, the length of hospitalization was significantly shorter in the negative fluid balance group in comparison to the control group .  The fluid balance differed significantly among nonsurvivors and survivors (MD: −317.93, 95% CI: −410.21, −225.69, and  We determined that the negative fluid balance was associated with favorable outcomes in COVID-19 patients. The negative fluid balance was associated with the reduced mortality rate and length of hospitalization as well as improvement in oxygen saturation. Moreover, the NT-proBNP >781 pg/mL and fluid balance >−430 mL might be the predictors for positive fluid balance and mortality, respectively. In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, and spread rapidly worldwide. Death through COVID-19 could result from acute respiratory distress syndrome (ARDS) and respiratory failure, cytokine storm and subsequent multiorgan failure, and cardiovascular events. Although respiratory failure is one of the major causes of death among COVID-19 patients, the high mortality rates among the intensive care unit (ICU)-admitted patients suggest the multifactorial nature of COVID-19 [Increased production of inflammatory cytokines and bradykinin contributes to the increased permeability of pulmonary vessels and fluid accumulation in lung interstitial and alveolar space. This process leads to pulmonary hypertension and exerts an additional overload on the right ventricle . It is wStudies have showed that early resuscitation with intravenous fluids is essential for the improvement of hemodynamic stability, tissue perfusion, and mortality reduction in ICU admitted patients. Moreover, studies showed that negative fluid balance might be associated with promising outcomes in ARF, heart failure, and ARDS in non-COVID-19 patients. However, the negative fluid balance efficacy in COVID-19 patients is not well known. Additionally, it has been suggested that positive fluid balance could deteriorate the patients' general condition by affecting the renal, cardiac, and pulmonary functions. The fluid overload is associated with pulmonary and peripheral edema, increased chance of respiratory failure, delayed weaning from mechanical ventilation, and poor prognosis . We aimeThis retrospective cohort study was conducted in two university hospitals in the Babol city, north of Iran, from July 2020 to May 2021. This study has been approved by the Ethics Committee of Babol University of Medical Sciences . Before enrolment, written informed consent was obtained from study participants.All adult patients  in the two university hospitals were screened for inclusion and exclusion criteria. We defined the cohort as hospitalized patients with positive reverse transcriptase-polymerase chain reaction (RT-PCR) for SARS-CoV-2. An infectious disease specialist verified the positive RT-PCR tests and confirmed the infection with clinical symptoms and computed tomography (CT) scan finding (ground glass opacification). Inclusion criteria included (1) age ≥18 years old and (2) normal and acceptable range of serum electrolytes. The exclusion criteria were (1) age <18 years old; (2) pregnancy; (3) development of major arrhythmic events, including sudden cardiac death (SCD), sudden cardiac arrest (SCA), ventricular fibrillation (VF), and supraventricular tachycardia (sVT); and (4) changing the treatment protocol such as the application of plasmapheresis or adding a new antiviral agent. We defined critical illness as the presence of respiratory failure, septic shock, and/or multiorgan failure. The treatment protocol was remdesivir, dexamethasone, and heparin. Moreover, 166 patients received invasive ventilation and six patients needed dialysis.We retrieved patients' characteristics such as demographics, clinical features, comorbidities, and laboratory findings on admission from medical records. A trained team of researchers reviewed and retrieved data for each patient.The daily net fluid balance was calculated by subtracting daily fluid output from daily fluid input. Fluid input included oral, enteral, and intravenous fluid. We used calibrated containers for measuring the oral and enteral fluid volume. Fluid output included urine and fluid loss from drains and tubes. Urine volumes were collected and measured via Foley catheter and urine bag. Fluid input and output were calculated (milliliters per day) and charted every 24 hours. We considered the negative fluid balance as a higher output fluid compared to the input fluid. We applied oral and intravenous fluid restriction and administered furosemide (loop diuretic). We adjusted the fluid balance based on the presence of congestion, vital signs, clinical findings, and laboratory findings such as the creatinine level. After determining the proper fluid balance in each patient, we maintained the established amount during the hospitalization. However, we overlooked minor changes in fluid balance during hospitalization.2 saturation were selected as the key secondary endpoints.The primary outcome was all-cause mortality. The length of hospitalization and improvement in Ot-test for continuous data and a chi-square test for categorical data. Prior to statistical comparisons, all data were tested for normal distribution by the Kolmogorov–Smirnov test and visually by the Q-Q plot. We used receiver operating characteristic (ROC) curves to identify the classification threshold of NT-proBNP and fluid balance. We reported diagnostic indices such as area under the curve (AUC), sensitivity, specificity, and likelihood ratio (LR). We estimated the risk ratios (RRs) and 95% confidence intervals (CIs) for mortality using a negative log-binomial regression model. The linear mixed effects models were fit to assess changes from the baseline within the groups and differences of those changes between groups with respect to oxygen saturation during hospitalization and the length of hospital stay. Fluid balance was categorized into four groups and included ordinally in the model  and GraphPad Prism 9 software . The baseline characteristics were compared between the two groups using the independent sample l Tables . The magp=0.064). Furthermore, deceased patients had almost the same frequency of pre-existing medical problems  compared with the control group  (p=0.886). The laboratory results showed that interleukin-6 (IL-6)  and c-reactive protein (CRP)  were significantly higher in deceased patients compared to the survivors. No statistically significant differences existed in the creatinine levels on the day of hospital admission and before hospital discharge or death . The mean ± (SD) of NT-proBNP in all patients at the time of admission was 8254 ± (10061.2) pg/mL. Moreover, we measured the NT-proBNP before discharging survived patients. The NT-proBNP was significantly lower in survived patients before discharge compared to the time of admission .The recruitment of patients was commenced in July 2020 and ended in May 2021. During eleven months, a total number of 315 patients were evaluated and 294 patients were included (according to inclusion and exclusion criteria). The demographic and baseline characteristics of the patients are summarized in p < 0.001) (One hundred ninety-six patients (66.66%) achieved negative fluid balance. In the two hundred ninety-four patients included in this study, the mean ± (SD) of fluid balance was −268.61 ± (408.99) mL  and differed significantly among nonsurvivors (−58.80 ± 414.57)  and survivors (−376.75 ± 362.24) ,  . The are< 0.001) . A fluid< 0.001) . The med< 0.001) . An NT-pp < 0.001). Moreover, after adjusting for potential confounders, there was a significantly lower frequency of mortality in patients with negative fluid balance compared to the controls .The unadjusted result showed a significantly lower frequency of mortality in patients with negative fluid balance compared to the controls . Similarly, after adjusting for potential confounders, the length of hospitalization was significantly shorter in the negative fluid balance group in comparison to the controls   .2 saturation for patients with negative and positive fluid balance at each time-point. Both groups showed a significant increment in oxygen saturation over time. However, during the hospitalization, the negative fluid balance group showed a significantly greater increment compared to the positive fluid balance group .We showed that negative fluid balance was associated with a decreased mortality rate, even after adjusting for the potential confounders such as the presence of critical illness. We categorized the fluid balance into four groups and included them ordinally in the model. For every one level increase in the fluid balance category, there is a decrease of ∼30% in the mortality rate . The same model was applied for the length of hospitalization, and as is shown in The ventricle pressure-volume relationship is curvilinear, and the atrial pressure increases rapidly as the ventricle reaches the plateau of the Frank–Starling curve. Elevated atrial pressure contributes to pulmonary and venous hydrostatic pressure overload, associated with the intravascular fluid shift to interstitial space and disturbed capillary blood flow and oxygen saturation . The aboBoth negative and positive fluid balance groups had low oxygen saturation at the beginning of admission. However, at the time of admission, the negative fluid balance had lower oxygen saturation compared to the positive fluid balance group. Pulmonary edema secondary to ARDS or heart failure could be the possible reason for low oxygen saturation in patients , 13. MorThe present study showed that males were associated with higher mortality rates compared to females. This finding is consistent with other studies. The studies suggested that COVID-19 has higher severity and mortality in males compared to females, especially in ICU-admitted patients , 16. ThiHypoxemic respiratory failure (ARDS) is the most common cause of admission to the ICU and is responsible for more than 80% of deaths . It has It has been shown that AKI is a common 5.1% to 29%) complication in critically ill COVID-19 patients, associated with high mortality, especially in ICU-admitted patients . Moreove% complicSeveral studies evaluated the impact of loop diuretic administration and negative fluid balance on ICU-admitted patients and conditions such as AKI. Moreover, there is no consistency between these studies. Some suggested beneficial effects , 34, whiIn addition to respiratory failure and shock, critically ill patients with COVID-19 might manifest heart failure . StudiesPhysicians should be vigilant during the aggressive fluid administration in COVID-19 patients who have declined cardiac output secondary to cytokine storm, heart failure, or other causes that has no beneficial effects, and overload might lead to adverse results due to pulmonary infiltration that contributes to diminished respiratory function.As our study showed, the negative fluid balance in critically ill patients was associated with better outcomes. Murphy et al. noted that the administration of adequate and early resuscitation fluid therapy besides conservative late fluid therapy, in patients with acute lung injury, is associated with better outcomes compared to the patients with only one of these approaches or none of the two . TherefoWe eliminated an important confounder, critical illness, which was not adjusted in similar studies. Additionally, we used a large database with high granularity. However, the current study has several limitations. First, this is a retrospective study, and we cannot eliminate the residual confounding due to unobserved factors. Furthermore, we did not include insensible perspiration in the fluid balance calculation. In addition, we did not evaluate the NT-proBNP levels in deceased patients. Moreover, we are unable to derive a definite mechanism for the beneficial effects of diuretics and negative fluid balance. Accordingly, we highly recommend prospective studies in this context. It is essential to explore whether more precise fluid management through invasive methods such as central venous pressure measurement or pulmonary arterial catheterization or noninvasive methods such as bioimpedance spectroscopy or echocardiography is necessary or not in a particular subset of patients, including those with underlying heart or renal failure.In summary, we determined that negative fluid balance was associated with favorable outcomes in COVID-19 patients. The increase in oxygen saturation and decrease in mortality and length of hospitalization may be due to the improvement in the cardiovascular and pulmonary function and tissue perfusion. Although we cannot securely determine that negative fluid balance is beneficial, it is highly likely that negative fluid balance affords COVID-19 patients' material benefit. In the absence of compelling contradictory data from a randomized, blinded clinical trial, we should encourage maintaining negative fluid balance in COVID-19 patients, especially critically ill patients and higher NT-proBNP levels.


The immunotherapies based on ICIs in CRC are nowadays limited to microsatellite unstable tumours which are approximately 15% of all CRC cases. There are a few new immune checkpoints belonging to the B7 family, including B7H4. B7H4 expression is associated with so-called “cold tumours”, and its function is linked to the downregulation of various immune cell populations. Our study aimed to investigate whether B7H4 expression is dependent on microsatellite status in CRC and on elucidating the immunological context in which the expression of B7H4 occurs. We enrolled 167 patients in the study. We prepared the homogenates from tumour tissues and healthy adjacent tissue to assess the B7H4 levels and the Bio-Plex Pro Human 48-cytokine panel. We assessed the microsatellite status of the tumour, B7H4 expression, CD8+ T cell population, and the TILs and budding in H + E stained slides by the IHC method. We used an online available database for further exploring the biological characteristics of B7H4. The expression of B7H4 was more frequent in microsatellite stable tumours, and was negatively associated with TILs. B7H4 is positively correlated with antitumour immunosuppressive iTME, thus contributing to the immunosuppressive environment in CRC. The B7H4  protein belongs to the B7 family . The genThe members of the B7 family include, among others, PD-L1, PD-L2, PD-1, and CTLA-4. These molecules have a highly important place in oncological clinical practice as targets for immunotherapy. An immune checkpoint blockade against them is used in a broad range of human malignancies . HoweverThe selective upregulation of PD-1, CTLA-4, LAG-3, Tim-3, and killer immunoglobulin-like receptors is often present in MSI-H tumours. The presence of these co-inhibitory receptors may explain the phenomenon.MSI-H tumours are not eliminated naturally, despite high immune activation in this type of cancer. Furthermore, tumour-infiltrating cells (TILs) express high levels of PD-1 in MSI colorectal cancer, which is absent in microsatellite stable tumours (MSS); this explains why a checkpoint blockade is effective in MSI status tumours ,15. For The tumour microenvironment (TME) consists of stromal, extracellular components, and immune cells. The immune part creates only immunological tumour microenvironment (iTME). The iTME can vary across the cancer subtypes and the disease stage . The iTMThis study aimed to evaluate the expression of the B7H4 to MSI/MSS status and other clinicopathological features of CRC. Furthermore, another goal was to explain the immunological context in which the expression of B7H4 occurs, through the 48-cytokine screening panel of cancer tissues homogenates and immunogenic features, immune composition, and functional annotations analysis of online available datasets. The samples from 167 patients obtained during surgery due to CRC were used in the study. Patients were treated in the 1st Specialist Hospital in Bytom, Poland . The collected specimens included colorectal tumour tissues and adjacent normal tissue. Patients were enrolled based on the inclusion and exclusion criteria described in our previous paper . ResearcFragments of the tumour tissue and surgical tissue margin were weighted and homogenized according to the standard homogenization protocol already described in our previous paper . To asseFor B7H4 expression, the B7H4 immunostaining was performed in 76 cases in which the MSI/MSS statuses were also established. Tissue samples were obtained from formalin-fixed paraffin-embedded tissue blocks with primary CRC and tumour-free margin samples. Then, the samples were deparaffinized and rehydrated. In the next step, antigen retrieval was performed by incubating slices in EnVision Flex Target Retrieval Solution High pH  for 20 min at 95 °C. Prepared samples were incubated with Peroxidase-Blocked Reagent (Dako) and then incubated with antibody: B7-H4 Polyclonal Antibody, Invitrogen, incubation time: 40 min.; dilution: 1:1500. After this process, they were put in EnVision FLEX HRP (Dako). Then, antigen–antibody complexes were stained using 3,3′-diaminobenzidine. Finally, tissue sections were counterstained with hematoxylin, dehydrated, and covered with coverslips for further analysis. For MSI/MSS status evaluation in 101 cases, the IHC staining for MSH2, MSH6, PMS2, and MLH1 was performed on 4 µm thick sections of a representative formalin-fixed, paraffin-embedded (FFPE) tumour tissue block on a Dako Autostainer Link 48. Samples underwent deparaffinization and rehydratation. In the next step, antigen retrieval was performed by incubating slices in EnVision Flex Target Retrieval Solution High pH  for 20 min at 95 °C. Prepared samples were incubated with Peroxidase-Blocked Reagent (Dako) and then incubated with one of the following antibodies: Mouse Monoclonal antibody MSH2 (G219-1129), Cell Marque, incubation time: 30 min.; dilution: 1:400; Mouse Monoclonal antibody MSH6 (44), Cell Marque, incubation time: 45 min.; dilution: 1:100; Mouse Monoclonal antibody PMS2 (MRQ-28), Cell Marque, incubation time: 40 min.; dilution: 1:50; Mouse Monoclonal antibody MLH1 (G168-728), Cell Marque, incubation time: 40 min.; dilution: 1:100. After this process, they were put in EnVision FLEX HRP (Dako). Then, antigen–antibody complexes were stained using 3,3′-diaminobenzidine. Finally, tissue sections were counterstained with hematoxylin, dehydrated, and covered with coverslips for further analysis.Tumours were assessed as to whether nuclear staining of invasive tumour cells for MSH2, MSH6, PMS2, and MLH1 was seen in the presence of positive internal control . Tumours with nuclear staining for markers in at least 1% of invasive tumour cells were considered to have positive marker staining. The algorithm on which the interpretation of immunohistochemistry testing was based is presented in the article of Olave and Graham . The MSIBriefly, 4 µm thick tissue sections were used for immunohistochemical (IHC) analysis. They were deparaffinized with xylene, rehydrated in graded alcohol, and washed in deionized water. In the next step, antigen retrieval was performed by incubating slices in EnVision Flex Target Retrieval Solution High pH  for 20 min at 95 °C. Prepared samples were incubated with Peroxidase-Blocked Reagent (Dako) and then incubated with antibody: (CD8+/144B) Mouse Monoclonal Antibody diluent, incubation time: 40 min.; dilution: 1:100. After this process, they were put in EnVision FLEX HRP (Dako). Then, antigen–antibody complexes were stained using 3,3′-diaminobenzidine. Finally, tissue sections were counterstained with hematoxylin, dehydrated, and covered with coverslips for further analysis.Tumour-infiltrating lymphocytes (TILs) assessment was performed in 102 specimens. The percentage of tumour-associated lymphatic infiltration was estimated semi-quantitatively on a five-grade scale on the same H&E-stained slides by the two pathologists, according to the criteria defined by Salgado et al. in breast cancer . These iTumour budding was assessed in the same 101 specimens. Tumour buds were estimated in one FOV at a hotspot area in the invasive front under ×20 magnification. The number of buds was adjusted by the normalization factor (1.210). Budding was reported in the following manner: low budding: 0–4 buds; intermediate budding: 5–9 buds; high budding: >10 buds. The mean number of buds per FOV was also used in the statistical analysis.Homogenates’ supernatants were collected after they were stored at −80 °C. The concentrations of cytokines/chemokines/growth factors were measured in 77 homogenates by the Bio-Plex Pro Human cytokines screening panel 48 cytokines assay  according to the manufacturer’s instructions. In brief, 50 µL aliquot of the sample was diluted 1:2 with sample diluent, incubated with antibody-coupled beads and biotinylated secondary antibodies, and followed by streptavidin-phycoerythrin. Standard curves for each studied parameter were performed using respective cytokine standard solutions. The beads were analysed in the Bio-Plex Array Reader (Bio-Plex Manager 6.2 software from the Bio-Plex 200 System). The intra-assay %CV varied up to 15%, and the inter-assay %CV varied up to 25%, depending on the analysed parameter. The obtained results were then normalised to the corresponding total protein level. This method has been previously used in analysing cytokines in lymphocyte cell culture supernatants and in blood serum samples ,25. http://220.189.241.246:13838/#shiny-tab-188 home (accessed on 15 December 2022)).We performed functional annotations analysis based on mRNA expression profiles in the CRC online dataset from the FieldEffectCrc Package among cohort A, consistent with 311 CRC samples . We normt-test  and Mann–Whitney U test  were used. Independent variables were also compared using the Student’s t-test and Mann–Whitney U test. Pearson’s coefficient or Spearman coefficient were used for assessing the relationships between the examined variables . Tau-Kendalls’ Tau Rank Correlation Coefficient was used to determine the association between the levels of the examined proteins, T, and N parameters. p values < 0.05 were considered significant. Hierarchical clustering and principal component analysis were performed to reduce the number of variables and clarify the influence of B7H4 expression on the cytokines profile. The factors obtained in PCA were used as new variables named Dim 1 and Dim 2 according to their proportions of the explained variance. Next, the Dim 1 and Dim 2 values were used in further analyses. p values ≤ 0.05 were considered significant. Statistical analysis was performed using STATISTICA 13 software (Statsoft) and the R Studio .Data distribution was determined using the Shapiro–Wilk test. The log transformation of the concentrations of the studied proteins provided a better fit for the Gaussian distribution. The data are presented as mean ± SD for the variables with normal distribution, and as median with interquartile range for the variables with non-normal distribution. To compare the tumour and margin concentrations, the paired Student’s p < 0.0001, In this study, to elucidate the link between B7H4 expression, tumour microsatellite status, and the immunological background, the B7H4 expression level was first compared between tumour tissues and normal tissues adjacent to CRC. A total of 159 pairs of cancer tissues and normal adjacent tissues’ homogenates were analysed for B7H4 level, measured by ELISA. B7H4 was elevated in tumour tissues’ homogenates compared to normal adjacent tissues . Subsequently, we further explored the effect of B7H4 on tumour immunity with online available tools. We juxtaposed the B7H4 immune landscape with the landscape of a known immune checkpoint in MSI type CRC PD-1  in the enrichment of the Molecular Signature Database Collection for Hallmark gene sets are shown in For further investigation of the B7H4 relation with transcriptome activity in CRC, we conducted GSEA between low and high B7H4 expression datasets to identify the B7H4-related pathways activated in CRC. Significant differences , as we had expected. Tumour-Infiltrating Lymphocytes and CD8+ T lymphocyte density has been reported to be inversely correlated with B7H4 expression in many solid tumours ,35,36. HUnderstanding the tumour type-specific cytokine profile could provide insights into iTME targeted therapy using the plasticity of pro-tumour or anti-tumour polarised immune cells. The iTME targeted drugs could change the immunosuppressive environment of “cold tumours” to “hot” tumours and restore proper antitumour response . To elucAfter exploring the effect of B7H4 on tumour immunity, we noticed that the mutational landscape of B7H4 high tumours was significantly different compared to the PD-1 related gene mutation load. Moreover, B7H4 expression exhibits a relation to the high mutation frequencies of the APC and TP53 genes, which were not observed in the PD-1 group. A highly similar mutational phenotype was observed by Lin et al. in the MSS/MSI-L CRC group . ImmunogIntratumour Heterogeneity, Number of Segments, Fraction Altered, and Aneuploidy Score, were significantly lower in the B7H4 high expression group. Intratumour Heterogeneity (ITH) reversely association with B7H4 could be explained by results that link ITH levels with MSI status and, conversely, its lower score with CMS2 CRC subtype, which corresponds to the canonical epithelial tumour harbouring high chromosomal instability in a microsatellite stable (MSS) context ,51. AlonFinally, we used GSEA to survey the transcriptome of CRC cases to compare B7H4 high-expressed CRC versus low-expressed. The results showed that among the upregulated immune-related pathways, there were genes upregulated by STAT5 in response to IL-2, genes encoding components of the complement system, which are part of the innate immune system, and genes upregulated by IL-6 via STAT3, and IFN-γ response. IL-6/STAT3 as well as the IL-2/STAT5 pathway activates downstream target genes to protect tumour cells from apoptosis, increase tumour cell proliferation, cell cycle progression, invasion, and metastasis, and are involved in drug resistance ,58. SimiIn conclusion, the findings of this study revealed that B7H4 expression is upregulated in CRC and is associated with the MSS status of tumours. Furthermore, B7H4 may play a pivotal role in the shaping of iTME in the microsatellite stable consistent immune landscape. B7H4 is positively correlated with antitumour immunosuppressive iTME. The iTME in the presence of B7H4 positive tumours might be mainly shaped by Th2 cells and their cytokine network. Additionally, the B7H4 positive tumour’s genomic landscape is similar to the one of MSS type CRC, which supports our finding that B7H4 expression is associated with microsatellite stable tumour types with a preserved mismatch repair gene mechanism. Taken together, the data suggest that B7H4 might be a potentially promising therapeutic target in MSS type CRC.