Fluid normally exchanges freely between the plasma and interstitial space and is returned primarily via the lymphatic system. This balance can be disturbed by diseases and medications. In inflammatory disease states, such as sepsis, the return flow of fluid from the interstitial space to the plasma seems to be very slow, which promotes the well-known triad of hypovolemia, hypoalbuminemia, and peripheral edema. Similarly, general anesthesia, for example, even without mechanical ventilation, increases accumulation of infused crystalloid fluid in a slowly equilibrating fraction of the extravascular compartment. Herein, we have combined data from fluid kinetic trials with previously unconnected mechanisms of inflammation, interstitial fluid physiology and lymphatic pathology to synthesize a novel explanation for common and clinically relevant examples of circulatory dysregulation. Experimental studies suggest that two key mechanisms contribute to the combination of hypovolemia, hypoalbuminemia and edema; (1) acute lowering of the interstitial pressure by inflammatory mediators such as TNFα, IL-1β, and IL-6 and, (2) nitric oxide-induced inhibition of intrinsic lymphatic pumping. The clinician is frequently confronted with the combination of hypovolemia and peripheral edema. In healthy humans, compensatory mechanisms including lymphatic return of extravascular fluid and transcapillary Starling forces act in concert to attenuate edema by increasing plasma volume. When this does not occur, treatment is a challenge, as administration of a diuretic will likely worsen hypovolemia and cause hypotension, while fluid administration worsens the edema. The most common explanation of this confusing condition is that capillary permeability has increased due to damage to the endothelial glycocalyx layer. However, increased capillary leakage normally accelerates lymphatic flow which normalizes the balance between the body fluid volumes, and might even increase the plasma albumin concentration  , 2.i) or failure of the intrinsic contractility of lymphatic smooth muscle cells. These mechanisms are distinctly different from lymphatic obstruction due to tumors, compression during compartment syndromes and resistance to thoracic duct outflow.This paper discusses how inflammatory insults and certain drugs may disturb the normal exchange of infused crystalloid fluid between the plasma and the interstitial space by mechanisms other than increased capillary leakage. Our review focuses on the possibility that decreased lymphatic return of fluid and protein alone can create hypovolemia, hypoalbuminemia, and peripheral edema. The pathophysiology responsible for poor lymphatic return might involve a reduction of the interstitial hydrostatic pressure  for urinary excretion by 50% in anesthetized but spontaneously breathing volunteers [k10 to low arterial pressure [Anesthesia also reduces the urinary output. Norberg et allunteers . A studypressure . Interesonstant k for urinInfused crystalloid fluid distributes between the plasma volume (3 L) and expandable parts of the interstitial space with a half-time of approximately 25–30 min. Volume kinetics identifies two fluid compartments in the interstitial space that equilibrate with the plasma but at different rates. The rapidly exchanging pool has a size similar to the plasma volume. Yet unpublished data from our group suggest that the expandable volume of the slowly exchanging pool is of negligible size when a few hundred mL are infused, while infusion of larger volumes of Ringer´s solution expand a progressively larger volume. During inflammatory states, the slowly exchanging pool expands even more and becomes larger than physiologically possible, which suggests that fluid can be bound or sequestered within the slow pool. Does this macroscopic view agree with anatomical and physiological studies of the interstitial fluid space?if, Fig. Recent advances in structural characterization of the interstitial space have revealed much complexity. Hyaluronan (HA) fills much of the interstitial space and forms a gel phase , 12. A rFluid in the interstitial space consists both of free fluid and fluid associated with the gel phase . The fibif [Surface charges on macromolecules attract electrolytes that contribute to the net osmotic force exerted by proteins and proteoglycans, a process called the Gibbs-Donnan equilibrium . The Donif . Howeverif  and, theif .General anesthesia creates a moderately severe maldistribution of fluid that is dominated by the strong inhibition of the diuretic response to volume expansion , 10. Thec) that forces fluid into the slow exchange pool. This is supported by the observations that small amounts of crystalloid fluid (300 mL during 15 min) expand mainly the plasma volume [Another finding during general anesthesia is that rate constant for fluid distribution to the slowly equilibrating interstitial pool is increased . The prea volume  while laa volume  which maif), and inhibition of lymphatic pumping. A recent finding that indirectly supports this hypothesis is that poor return flow from the interstitium to the plasma remains for several hours after awakening from general anesthesia, while the sympathetic-induced inhibition of the diuretic response to volume expansion resolves almost instantly [A complementary hypothesis is that general anesthesia initiates a mild form of the interstitial molecular events that occur during inflammation. These events are detailed below but include sequestration of fluid in the gel phase, resulting from a reduction of the interstitial hydrostatic pressure  determines lymph flow rates.. There is much evidence that inflammatory molecules play an important role in the maldistribution of fluid during acute inflammation, such as sepsis and burn injury, by reducing Pif. To discuss these findings in detail we first give a brief review of the factors that determine Pif.There are clinical conditions in which volume kinetic studies show that the return flow of fluid from the interstitium to the plasma (probably via the lymphatics) is severely reduced. These are the "transurethral resection syndrome" (studied in pigs) , sepsis if was assumed to be near zero or slightly positive relative to the atmosphere. Guyton and colleagues tested a wide range of experimental conditions and consistently measured a negative Pif in the range of − 3 to 7 mm Hg [if is maintained by an imbibition (suction) pressure created by colloids in the gel phase [Prior to the mid-1960’s, P 7 mm Hg , 31 whic 7 mm Hg , 33. Theel phase , 30, 31.el phase .The mechanisms cited above keep the interstitial space dry and maintains a very low volume of free fluid, which has been suggested to occupy as little as 1% of the interstitial volume. The remaining interstitial fluid is bound in the gel phase . By contAnalysis of interstitial pressure and volume during lymphatic failure draws data primarily from studies of the skin, as it is among the largest organ system of the body and is estimated to hold approximately 3 L of fluid, second only to skeletal muscle that accounts for approximately 20 L . Peripheif from −2 mmHg to between −7.5 and −10 mmHg within minutes [if occurs in burn injury, where values of 40 mmHg and greater have been reported [Injecting lipopolysaccharide or inflammatory molecules intravenously and, in some cases, directly into the skin, reduces P minutes , 34. Thereported –41. Such. have shown that inflammatory mediators reduce Pif to more negative values. Numerous cytokines reverse integrin-collagen binding, including histamine (mast cell degranulation and Compound 48/80), PAF, TNFα, IL-1β, IL-6, LPS, and IFN-γ [The interstitial space is under tensile forces mediated by cell-collagen interactions and, specifically, by integrins. The basal tensile force sets the pressure–volume relationship. When tissue is excised and placed in isotonic saline it expands due to loss of these tensile forces , 42. In nd IFN-γ  1 β1, α2 β2, α10 β1, α11 β1 [if when injected intradermally, thus demonstrating the role of integrins in the Pif-lowering process [This mechanism has also been studied in vitro using fibroblasts mixed into collagen gel. Fibroblasts bind to collagen via cell surface integrins that serve as protein linkages between the cell’s cytoskeleton and the collagen. The integrins are composed of heterodimers of α- and β-subunits, with both subunits having distinct members. There are four collagen-binding integrins: α, α11 β1 –46. Fibr process .if. First, Pif becomes more negative due to the loss of tensile forces by cytokine-dependent interstitial relaxation. This expansion occurs rapidly and the ensuing more negative Pif increases fluid filtration and imbibes fluid from the capillary into the interstitial space. Pif starts at slightly negative pressures (− 2 to − 7 cm H2O) and might become deeply negative (− 10 to − 40 cm H2O). The shift can cause life-threatening hypovolemia during the first 6–8 h after burn injury [if are likely to be initially induced, to various degrees, in all conditions associated with acute inflammation. The presence of HA and glycosaminoglycans in plasma suggest that interstitial expansion is also associated with matrix fragmentation [if towards zero, where the compliance characteristics for interstitial volume expansion cease [During acute inflammation, there is a biphasic change in Pn injury  but negaentation . The secon cease .The interstitial edema and hypovolemia become aggravated if lymphatic pumping is impaired. During sepsis and states of sustained inflammation, increased levels of nitric oxide (NO), likely generated by inducible nitric oxide synthase (iNOS), inhibits lymphatic smooth muscle cell pumping and, thus, contributes to lymphatic failure –52. Slowif and poor lymphatic pumping greatly expands the interstitial space. As the interstitium fills with fluid, Pif shifts along its pressure–volume curve towards more positive values and eventually approaching zero, thus diminishing the forces for further fluid influx. During experimental thermal injury, the transition from largely negative Pif towards zero occurred over a time period of hours [The combined effects of low Pof hours . These cof hours . Lacune of hours .if increases the capillary leakage of blood plasma by a suction effect that does not necessarily involve alteration of the capillary permeability. This was illustrated by Arturson and Mellander in 1964 who studied the effects of second-grade burn injury on the cat paw; they found accelerated loss of intravascular fluid to the injured area but no change in the capillary filtration coefficient, which is a measure of capillary permeability [Reduction of Peability .. [Hypovolemia, hypoalbuminemia, and peripheral edema occurs in several medical diseases and is generally attributed to increased capillary permeability, as reviewed by Siddall et al. . Sepsis 2-receptor agonist) and theophylline [A special case is the rare Idiopathic Capillary Leak Syndrome (Clarksons´disease) which presents dramatically with attacks of hypovolemic shock and pitting edema , 59. Claphylline .if and the pressure–volume relationship is fairly accessible to interventions [kidney has an extensive network of specialized lymphatic vessels that are named for their intrarenal location [if rises quickly as a result of external capsular compression, lymphatic obstruction (metastasis), systemic venous hypertension , ureteral obstruction, and increased capillary permeability. Parenchymal edema has been suggested to collapse the capsular and hilar lymph vessels and prevents lymph outflow, contributing to further pathological increases in Pif [if, is a typical finding in acute kidney injury (AKI) and is commonly associated with elements of intra-renal lymphatic failure [Much of our analysis has drawn upon data from the skin where Pventions . The sitlocation . Due to s in Pif . Renal i failure , 65 likeSepsis is a leading cause of acute kidney injury  where thSkeletal muscle represents another encased organ system that develops compartment syndrome albeit more commonly due to cellular swelling rather than lymphatic failure. Skeletal muscles are composed of fibers arranged in bundles with each bundle surrounded by a connective tissue sheath [if. The lymphatics have no ability to respond to increases in interstitial fluid as they too are encased.e sheath . Their le sheath . Therefogut can develop both edema and compartment syndrome. The walls of the intestine are lined with an extensive population of leukocytes that, during acute inflammation, generate high levels of nitric oxide and cytokines [The ytokines . In gene2O [The mesenteric lymphatic vessels drain into the thoracic lymph duct and subsequently into the left subclavian vein. Increases in central venous pressure will reduce the pressure gradient for lymph flow and may worsen peripheral edema. Conditions commonly associated with elevated central venous pressure include heart failure, portal hypertension, volume overload, positive pressure ventilation, and positive end-expiratory pressure (PEEP). However, the outflow pressure which causes lymphangion stroke volume to begin to decrease is approximately 10 cm H2O  above wh2O . However2O . Sustain2O .kb. In conscious volunteers this parameter attains a value being 10% of rate of entrance to the fast fluid pool, and approximately 25% of the urine flow rate [kb becomes approximately 50% larger, and the flow may even exceed the urinary excretion [The described microcirculatory and molecular events may be interpreted when having macroscopic analyses in mind. During the past decade, volume kinetic studies have quantified the entrance of isotonic electrolyte fluid to the slowly equilibrating interstitial pool by using the rate constant xcretion  Fig. 5)kb. In coPooling of many experiments suggests that fluid leaves the slowly equilibrating fluid pool and returns to the plasma with a half-time of approximately 2 h while the half-time is close to 10 min for the rapidly equilibrating pool . The lymphatic circuit operates like a “telephone line” and return flow of fluid to the plasma increases within minutes in response to intravenous fluid loading .if [The half-time for the persistence of fluid in the slow interstitial pool is much longer in inflammatory states. For example, re-analysis of a cohort of patients undergoing surgery for cholecystitis or appendicitis even yielded a negative return flow rate for the slowly equilibrating fluid phase . This reif ).. induced sepsis in 25 anesthetized sheep by injecting lipopolysaccharide and puncturing the cecum [Li et alhe cecum . One houPreeclampsia during pregnancy is characterized by hypovolemia, hypoalbuminemia, and peripheral edema, and hypertension . A compa. reported that the plasma concentrations of lumican  and metalloproteinase-2  increase with the degree of edema. By contrast, biomarkers of endothelial integrity  show an inverse relationship with the edema [Kwashiorkor is a nutritional disease which shares the pathological fluid distribution with sepsis. Hypoalbuminemia is an obligate finding but not enough to explain the peripheral edema. Recently, Gonzalez et alhe edema . These fA warning is often given when pharmacokinetic data is interpreted into physiological terms, which is frequently warranted, due to tissue binding issues and drug metabolism. Volume kinetics provide functional volumes and rates for fluids and have long given reasonable estimates of plasma volumes and descriptions of physiological events. Currently, it is the best available method by which dynamic whole-body events related to fluid infusions can be understood.Our data suggest that the rapidly equilibrating fluid pool correspond to the interstitial “free fluid phase” and likely to fluid in very close proximity to capillaries, and that the slowly equilibrating pool corresponds to the “gel phase” or interstitial space remote from capillaries or lymphatic. Our data further suggest that the interstitial free fluid phase normally occupies more volume than the 1% suggested by physiological studies, but the volume may also become very small, probably by compression, when the slowly equilibrating fluid phase swells.We have reviewed molecular and macroscopic evidence showing that the interstitial space plays an active role in the development of pathological fluid distributions characterized by hypovolemia, hypoalbuminemia, and peripheral edema. An important pathway consists of swelling of the gel phase due to loss of integrin-cellular interactions caused by inflammatory signals. This dramatically reduces the interstitial hydrostatic pressure which applies a suction effect on the plasma and prolongs the time period when distributed fluid resides in the interstitium. Associated actions of nitric oxide involves an inhibition of lymphatic pumping. These events are reflected in macroscopic studies using volume kinetics, which identifies a small interstitial pool of rapidly exchanging fluid and a large pool which equilibrates slowly. General anesthesia modestly increases of the absorption of fluid into the slowly exchanging pool. Inflammatory disorders (cholecystitis and sepsis) greatly prolong the time period when the infused fluid resides in the slowly exchanging fluid pool, and the resulting swelling occurs in part at the expense of the size of the rapidly equilibrating pool.


N-acetylglucosaminidase was a hallmark of several solid tumours. Having identified this glycosidase as a potential target for cancer therapy, we designed an enzyme-responsive albumin-binding prodrug of the potent monomethyl auristatin E programmed for the selective release of the drug in the tumour microenvironment. This tumour activated therapy produced a remarkable therapeutic efficacy on orthotopic triple-negative mammary xenografts in mice, leading to the disappearance of tumours in 66% of treated animals. Thus, this study shows the potential of induced-volatolomics for the exploration of biological processes as well as the discovery of novel therapeutic strategies.The discovery of tumour-associated markers is of major interest for the development of selective cancer chemotherapy. Within this framework, we introduced the concept of induced-volatolomics enabling to monitor simultaneously the dysregulation of several tumour-associated enzymes in living mice or biopsies. This approach relies on the use of a cocktail of volatile organic compound (VOC)-based probes that are activated enzymatically for releasing the corresponding VOCs. Exogenous VOCs can then be detected in the breath of mice or in the headspace above solid biopsies as specific tracers of enzyme activities. Our induced-volatolomics modality highlighted that the up-regulation of  via the detection of volatile tracers in animal breath, thereby allowing the discovery of a new tumour targeted therapy.We developed a volatile organic compound (VOC)-based probe cocktail for monitoring the dysregulation of tumour-associated enzymes  Thus, the design of novel strategies dedicated to the exploration of tumour-associated enzymes is of prime importance for cancer therapy and diagnosis.5,6 Within this framework, activable imaging probes have been developed to detect the dysregulation of some enzyme activities in tumours.7–17 However, most of these turn-on probes suffer from low signal penetration depth and background noise.18,19 Recently, we introduced the concept of induced-volatolomics for cancer diagnosis.20,21 We demonstrated that a volatile organic compound (VOC)-based probe can be activated in vivo by an enzyme overexpressed in solid tumours to trigger the release of an exogenous volatile tracer in the breath of individuals, thereby enabling cancer detection. Herein, we present an original approach allowing real time and simultaneous monitoring of the activity of several tumour-associated enzymes by means of a VOC-based probe cocktail , α-l-fucosidase (α-Fuc), β-glucuronidase (β-Glu) and N-acetyl-β-d-glucosaminidase (β-GlcNAc) were selected as the target enzymes since their overexpression has been reported in several solid tumours.27–34 Thus, the probe cocktail was composed of four ethyl glycosides i.e. D2-ethyl-β-d-galactopyranoside , D4-ethyl-α-l-fucopyranoside, (D4-EtFuc), D5-ethyl-β-d-glucuronide (D5-EtGlu) and 13CD5-ethyl-N-acetyl-β-d-glucosamine (13CD5-EtGlcNAc), designed to release D2-, D4-, D5- and 13CD5-ethanol isotopes, respectively, in the presence of the corresponding glycosidase.In this proof-of-concept, we chose to target glycolytic activity of tumours since glycosidase dysregulation constitutes a hallmark of many cancers.After cocktail administration , step 1,2-, D4-, D5- and 13CD5-ethanol molecules, we first developed an analytical strategy involving the GC-MRM-MS methodology  for each VOC-based probe. For this purpose, we administered increasing probe concentrations to the animals that were then placed for over 1 h 30 in airtight cages equipped with an SPME fibre .2-, D4-, D5- and 13CD5-ethanol isotopes were trapped and subsequently analysed by GC-MRM-MS. Under these conditions, injection of 1, 100, 10 and 10 μg kg−1 doses of D2-EtGal, D4-EtFuc, D5-EtGlu and 13CD5-EtGlcNAc respectively, produced the LDS for the corresponding volatile tracers . Under such circumstances, the VOC-based probe cocktail seems to be a useful tool for monitoring disease progression in real time.21Thereafter, our experimental protocol was applied to highlight the potential upregulation of glycosidases in malignant tissues. The cocktail of probes was administered to athymic nude mice bearing subcutaneous human KB xenografts  on days 12 and 15 post-tumour implantation. A control experiment was also carried out with healthy animals. On days 12 and 15, the amounts of Dterparts . In contex vivo on tumour biopsies. The aim of this trial was to compare simultaneously the catalytic activity of the four glycosidases in tumours and healthy tissues such as kidneys. In these experiments, we used D2-EtGal, D4-EtFuc, D5-EtGlu and 13CD5-EtGlcNAc probes at 10−6, at 10−4, 10−6, and 10−7 mol L−1, respectively, since these concentrations produced the LDS for ethanol isotopes when used on healthy tissues .Thus, MDA-MB-231 human mammary tumours harvested from nude mice with orthotopic xenografts were incubated in acetate buffer (pH 5) in the presence of the cocktail of VOC-based probes . Volatil39–41 . The three last steps of the synthesis were undertaken without intermediate purification. Thus, alkyne 7 reacted with O-(2-aminoethyl)-O-(2-azidoethyl)-nonaethylene glycol and Cu(CH3CN)4PF6 to form the corresponding triazole through copper(i)-catalysed azide–alkyne 1,3-cycloaddition. Deprotection of the glycoside followed by the introduction of the maleimide moiety using the N-hydroxysuccinimide ester 8 finally led to the drug delivery system 1 (24% over 3 steps).The tumour-activated prodrug 1 was accessible from the commercially available β-samide 2 . First, samide 2  and 47 tN-acetylglucosamide trigger is the key step in the process of drug release, we first verified that the carbohydrate moiety was still accessible to β-GlcNAc once bound to albumin. For this purpose, prodrug 1 was incubated with human serum albumin (HSA) at 37 °C in order to form the corresponding bioconjugate through Michael addition. Under these conditions, nearly 90% of 1 was converted in two hours as a result of its rapid binding with the protein  were well tolerated without major body weight loss  of the drug.39 Furthermore, the therapeutic efficacy obtained with 1 confirmed the selective and efficient enzyme-mediated release of MMAE in the tumour mass. Thus, this study showed that the targeting of tumour-associated β-GlcNAc by means of the enzyme-responsive albumin-binding prodrug is a promising therapeutic strategy for the treatment of triple-negative breast cancer (TNBC), a clinically aggressive disease for which current drugs are ineffective.We next pursued our study by assessing the antitumour activity of the β-GlcNAc-sensitive prodrug 1 in Balb/c athymic mice bearing orthotopic MDA-MB-231 triple-negative mammary tumours . The ani animals . At the  animals . In contght loss  or detecIn this study, we demonstrated that a cocktail of VOC-based probes allowed the screening of several tumour-associated enzyme activities in both living mice and tumour biopsies. This induced-volatolomics modality led to the identification of an enzyme target, β-GlcNAc, which was exploited for the selective delivery of an anticancer drug that produced an impressive anticancer activity in mice bearing TNBC xenografts. Hence, our work highlights the potential of induced-volatolomics for the discovery of tumour-associated enzyme targets and the development of novel therapeutic strategies. Numerous VOC-based probe cocktails can be designed for the multimodal exploration of enzymatic dysregulation related to various diseases. Thus, induced-volatolomics modalities could become powerful tools to accelerate the process of drug discovery in the near future.Experimental procedures involving animals were conducted according to protocol no. 2017053011069010 approved by the national ethical committee and carried out in accordance with the guidelines of the French Agriculture and Forestry Ministry (decree 2013-118) and the European Communities Council Directive (2010/63/UE).All relevant data supporting this article have been included in the main text and the ESI.†in vivo studies to test the efficiency of the cocktail of probes for highlighting highly active enzymes in tumours. J. L., J. C., F. D. and E. B. performed the in vitro and in vivo experiments using VOC-based probes. R. E. completed the VOC-based probe sensitivity study. J. C. and E. B. performed cell viability assays. S. P., I. O., B. R., R. C. and W. T. designed and synthesized the anticancer agent and VOC-based probes. R. C. and R. E. performed biochemical assays to certify the formation of the albumin–prodrug conjugate. J. S. and A. L. P. designed and supervised the experiments in tumour bearing mice treated with the enzyme-responsive prodrug. J. L., E. B. and R. C. collected all the data and reconstructed the figures. J. L., E. B. and R. C. wrote the methods section. P. P. and S. P. wrote the paper with the contribution of all authors.P. P. and S. P. supervised the whole study. P. P., C. G. and M. T. supervised the induced-volatolomics approach and the development of analytical methods. P. P. and J. C. designed There are no conflicts to declare.SC-014-D2SC06797H-s001


Immune checkpoint therapies (ICT) can reinvigorate the effector functions of anti-tumour T cells, improving cancer patient outcomes. Anti-tumour T cells are initially formed during their first contact (priming) with tumour antigens by antigen-presenting cells (APCs). Unfortunately, many patients are refractory to ICT because their tumours are considered to be ‘cold’ tumours—i.e., they do not allow the generation of T cells  or the infiltration of existing anti-tumour T cells (T-cell-excluded tumours). Desert tumours disturb antigen processing and priming of T cells by targeting APCs with suppressive tumour factors derived from their genetic instabilities. In contrast, T-cell-excluded tumours are characterised by blocking effective anti-tumour T lymphocytes infiltrating cancer masses by obstacles, such as fibrosis and tumour-cell-induced immunosuppression. This review delves into critical mechanisms by which cancer cells induce T-cell ‘desertification’ and ‘exclusion’ in ICT refractory tumours. Filling the gaps in our knowledge regarding these pro-tumoral mechanisms will aid researchers in developing novel class immunotherapies that aim at restoring T-cell generation with more efficient priming by APCs and leukocyte tumour trafficking. Such developments are expected to unleash the clinical benefit of ICT in refractory patients. EM), which has been recently associated with durable responses following tumour-antigen re-exposure . An. An27]. Blocking specific mechanisms of immunogenicity suppression, such as MIF/CD74 in metastatic melanoma, as opposed to a ‘shotgun’ approach of promoting more GM-CSF expression to increase overall immune activity, could offer a more precise, efficient, and potentially less toxic therapeutic route. The utilisation of GM-CSF tumour vaccines, while successful to an extent, essentially coerces the immune system into a heightened state of immunogenicity. But this does not specifically target the underlying issue of tumour-induced immune subversion.Regarding the potential of targeting immunosuppressive scavenger receptors in the TME, such as Clever-1, exciting pre-clinical combinatorial results opened the potential for investigating this rational in clinical settings . ResultsNew therapeutic approaches are specifically needed to overcome the T-cell desertification nature of UM. A new generation of immunotherapies to overcome this deficiency is based on developing tebentafusp in cancer types with severe T-cell desertification, such as mUM . This coWhile the initial findings with tebentafusp are promising, its optimal utilisation still needs further research to understand better its efficacy and safety profile in a broader population of mUM patients. The current study does suggest the potential of tebentafusp to enhance survival in mUM patients; however, a more in-depth analysis is necessary to understand the underlying mechanisms. Unexplained discrepancies, such as the disconnect between survival improvements and traditional radiological indicators, indicate that we might need to look beyond conventional metrics for evaluating this treatment. Personalising the use of tebentafusp based on individual patient responses and genetic diversity within the UM population could lead to more effective therapeutic strategies. Long-term safety studies, large-scale longitudinal studies, and exploring patient-specific factors influencing the treatment response will be crucial to validate and expand on these initial findings.Moreover, the therapeutic landscape of mUM is not confined to the potential of tebentafusp alone. Advancements in targeted therapies present additional pathways to disrupt the proliferation of UM cells and counteract their metabolic mechanisms to suppress local immune responses. For instance, darovasertib, a protein kinase C (PKC) inhibitor, has demonstrated potential in hampering UM cell growth, thereby potentially enabling immune responses to act more effectively against the disease . The impIn light of these critical areas requiring further innovation, significant strides have been made in personalised cell therapy. In a pioneering example, scientists have developed a clinical-grade method that not only deactivates endogenous TCR genes but also introduces two chains of a neoantigen-specific TCR (neo-TCR) into the locus encoding TCRα. Employing non-viral precision genome-editing tools, scientists have developed a clinical-grade method that deactivates endogenous TCR genes and inserts two chains of a neo-TCR into the locus encoding TCRα. In a first-in-human phase I clinical trial, 16 patients with treatment-resistant solid cancers received up to three neo-TCR transgenic cell products. Despite anticipated side effects, this therapy proved safe Fig. . The traFinally, other advances have underscored the potential of combinatorial approaches to improve ICT responses in pre-clinical and clinical settings. Integrating VEGF-targeting therapies with ICT has exhibited a noteworthy anti-tumour synergy , 134. SiIt is now clearly established that the ever-evolving landscape of cancer immunotherapy unveils the pivotal role of DCs dynamics in antigen processing and priming of T lymphocytes as an immunosurveillance step of supreme importance, shaping ICT efficacy within cold tumours. Accordingly, there is an increasing effort to develop combinatorial approaches to control these dynamics. The current combinatorial approaches are based on pharmacological strategies that target specific innate checkpoint regulators induced or secreted by tumour cells in cold tumours, suppressing immunogenic responses from local DCs. Furthermore, novel autologous DCs vaccination strategies and gene-therapy approach using mRNA vaccines offer the potential for enhancing T-cell priming and co-stimulation. The continuous growth of the immuno-oncology field highlights the importance of understanding the molecular mechanisms underlying T-cell desertification and exclusion, particularly related to tumours’ genetic instabilities driving cold tumours. Further research exploring the diversity of tumour’s secretomes and utilising epigenetic evidence from refractory biopsies holds the potential to uncover new insights. These findings will likely contribute to the development of next generation of innate immunotherapies aimed at unleashing immunogenicity in refractory cold tumours, complementing ICT and improving overall responses in cancer patients. With ongoing scientific advancements and a comprehensive understanding of tumour-immune interactions, the future holds promising prospects for more personalised and effective combinatorial immunotherapies.


Spontaneous bacterial peritonitis (SBP) is a severe complication that occurs in 8-27% of hospitalized patients with liver cirrhosis and ascites, with high mortality rates. This study aimed to identify the clinical characteristics associated with SBP.  Cross-sectional study, conducted in a public university.  The study consecutively included individuals with liver cirrhosis and ascites between September 2009 and March 2012. Forty-five patients were included: mean age 53.2 ± 12.3 years, 82.2% male, 73.8% Caucasian, mean Model of End-stage Liver Disease (MELD) score of 19.5 ± 7.2, and 33.3% with SBP. The subjects were divided into two groups: SBP and controls.  Comparison between individuals with SBP and controls showed that those with SBP had lower mean prothrombin activity time  and lower median serum-ascites albumin gradient (SAAG) . There was a tendency towards higher mean MELD in the SBP group, not significant . There was a strong positive correlation between the neutrophil count in ascitic fluid and serum leukocyte count  and a negative correlation between the neutrophil count in ascitic fluid with prothrombin activity time .  A few characteristics are associated with the presence of SBP, especially liver dysfunction, SAAG and peripheral leukocytosis. Extra-intestinal bacteria, such as those from the respiratory and urogenital tracts or the skin, are much less frequent. Catheters and other equipment used during invasive procedures represent another possible source of infection. Currently, the most accepted hypothesis regarding the pathogenesis of SBP consists of a bacteremia episode during fluid exchange between the peritoneal and intravascular cavities, with consequent infection of the ascitic fluid.,In fact, only a few patients with SBP present typical symptoms suggestive of peritoneal infection, such as fever, abdominal pain and peripheral leukocytosis. SBP is most frequently suspected when the patient develops signs of hepatic encephalopathy, increased abdominal volume or renal dysfunction, without any apparent precipitating factor. In addition to this, in a significant proportion of the cases, SBP may be completely asymptomatic and the diagnosis can only be made by analyzing the paracentesis results.The diagnosis for SBP consists of polymorphonuclear (PMN) counts ≥ 250 cells/mm³ and a positive ascitic fluid culture, without any evidence of external or intra-abdominal infectious source. Neutrophilic ascites is defined by negative cultures and also by PMN counts in the ascitic fluid higher than 250 cells/mm³. Presence of a positive ascitic fluid culture with neutrophil count less than 250 cells/mm³ is diagnosed as bacterascites, and management of this condition varies from patient to patient.Given the scarcity of studies assessing characteristics of patients with SBP in Brazil, and also the regional differences that exist, there is a need to evaluate this infection in our setting. Through observing its behavior, new strategies aiming towards diagnostic improvement can be sought. The main objective of this study was to identify the characteristics associated with presence of SBP in individuals with decompensated liver cirrhosis with ascites. We also aimed to describe the clinical profile of individuals with SBP.This cross-sectional analytical study was conducted by reviewing the medical records of individuals with decompensated liver cirrhosis with ascites who were admitted to the gastroenterology ward of the "Polydoro Ernani de São Thiago" university hospital of the Federal University of Santa Catarina , between September 2009 and March 2012. Over the same period, we evaluated the results from ascitic fluid cultures made in the university hospital laboratory of UFSC, for inclusion in the study. Among these, after analysis of the medical files, we excluded the patients who did not have cirrhosis (patients with ascites from other causes) and also those with insufficient clinical and laboratory data registered in their medical records. The study protocol conformed to the ethical guidelines of the 1975 Helsinki Declaration and had been approved by our institutional review board under the number 885/10.Patients were interviewed regarding their demographic and clinical characteristics as stated below. Additional clinical and laboratory variables relating to all the individuals from medical charts. The following clinical variables were studied: age; gender; skin color; SBP (which was defined as a neutrophil count in ascitic fluid higher than 250 cells/mm³ and/or a positive culture); jaundice; hepatic encephalopathy; upper gastrointestinal bleeding (UGIB) during hospitalization; ascitic fluid cultures; maximum axillary temperature; abdominal pain; diarrhea; comorbidities: diabetes mellitus (DM), systemic arterial hypertension (SAH), dyslipidemia and HIV; etiology of the cirrhosis: alcohol, hepatitis C or hepatitis B; and duration of prophylactic antibiotics for paracentesis. Among the laboratory variables, the following were evaluated: neutrophil count in ascitic fluid; prothrombin activity (PA); serum-ascites albumin gradient (SAAG); hemoglobin; leukocytes; platelet count; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT); albumin; total and direct bilirubin; creatinine; sodium; glucose; total protein in the ascitic fluid; and albumin in the ascitic fluid. The results from the liver biochemical tests  were expressed as the number of times the upper limit of normal (xULN). The other laboratory variables were expressed as absolute values. The bilirubin tests, international normalized ratio (INR) and creatinine were used for calculating MELD (Model of End-stage Liver Disease).Continuous variables were compared using Student's t test, or the Mann-Whitney test when appropriate. Categorical variables were compared using the chi-square test or Fisher's exact test. P values less than 0.05 were considered to be statistically significant. The correlation between the neutrophil count in the ascitic fluid and laboratory variables was assessed using Pearson's correlation coefficient. All tests were performed using the Statistical Package for the Social Science , version 17.0. From September 2009 to March 2012, 86 patients were evaluated for inclusion in the study because they presented decompensated cirrhosis with ascites and/or positive results from ascitic fluid cultures in the laboratory. Three individuals were excluded from the study because no neutrophil count in the ascitic fluid was available, along with another 38 patients who did not present cirrhosis . We included 45 patients with decompensated cirrhosis with ascites in the study, and 15 of these (33.3%) presented SBP . The meaWith regard to the etiology of the cirrhosis, it was observed that 46.7% showed alcoholism alone , 20% HCV and alcoholism, 20% hepatitis C alone, 6.7% hepatitis B alone and 2.2% HBV and alcoholism. 3, and three patients (21.4%) presented positive ascitic fluid cultures: two with Klebsiella pneumoniae and one with Streptococcus sp. The diagnostic paracentesis was performed, on average, after 3.6 ± 3.5 days of hospitalization.In the SBP group, the mean neutrophil count in ascitic fluid was 1,393.5 ± 1,115.1 cells/mmIn comparing the patient with SBP and the controls  and 2, iWe observed a strong positive correlation between the neutrophil count in ascitic fluid and the serum leukocyte count . A negative correlation was also noted between the neutrophils count in ascitic fluid and PA . Significant correlations with hemoglobin, platelets, AST, ALT, ALP, GGT, serum albumin, INR, total bilirubin, creatinine, sodium, glucose, total proteins in the ascitic fluid, albumin in the ascitic fluid, SAAG and maximum axillary temperature were observed. --,-,,SBP has previously been described more frequently among males, at percentages ranging from 72.8% to 83.7%, which was similar to what was found in this study.-With regard to the etiology of cirrhosis, Heo et al. reported that the greatest prevalence of cirrhosis was due to HBV (71.3%), followed by alcoholic cirrhosis (19.7%) and cirrhosis by HCV (6.4%),3, cellularities of up to 8,000 neutrophils per mm³ have been described.,,,,,,Even though SBP is diagnosed as an ascitic liquid neutrophil count ≥ 250 cells/mm,From reviewing the clinical and laboratory characteristics in relation to the presence of SBP, it was seen that there is controversy regarding the findings in the literature. The heterogeneity of the findings that have been correlated with the presence of SBP justifies indication of diagnostic paracentesis for all patients with decompensated cirrhosis with ascites who are admitted to hospital.Figueiredo et al.Such et al.From comparing the factors associated with SBP in the present study with what has already been described in the literature, we perceived that these studies are discordant. Thus, each study found different variables to predict the appearance of SBP. Nonetheless, the present study, as well as the others, identified some variables as factors for the existence of SBP. These characteristics confirm that there is higher prevalence of SBP among individuals with advanced liver disease. The present study reinforces the recommendation that diagnostic paracentesis should be performed on hospital admission for all cirrhotic patients with ascites, in order to investigate the presence of SBP, even for patients admitted for reasons other than ascites, since no clinical characteristics other than the severity of liver disease can predict ascitic fluid infection.A few characteristics are associated with the presence of SBP, especially liver dysfunction (prothrombin activity), SAAG and peripheral blood leukocytes.


Hepatocellular carcinoma (HCC) is a highly lethal disease with an increasing incidence. Despite the advancements in diagnosis and recent therapeutic options, improving the prognosis of HCC patients remains challenging. One of the reasons of the unsatisfactory outcome of patients with HCC is the complex tumour microenvironment (TME), which is composed of immune and stromal cells, limiting effective treatments. Recent research has highlighted the importance of macrophages in the development and progression of HCC, opening new possibilities for therapy. This review focuses on the heterogeneity of tumour-associated macrophages (TAMs) in HCC, the mechanisms through which HCC tumour cells polarize macrophages, and the therapeutic targets that are currently being tested to explore novel therapies that can improve the prognosis and quality of life of HCC patients.Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer that comprises a complex tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are one of the most abundant immune cells present in the TME, and play a key role both in the development and in the progression of HCC. Thus, TAM-based immunotherapy has been presented as a promising strategy to complement the currently available therapies for HCC treatment. Among the novel approaches focusing on TAMs, reprogramming their functional state has emerged as a promising option for targeting TAMs as an immunotherapy in combination with the currently available treatment options. Nevertheless, a further understanding of the immunobiology of TAMs is still required. This review synthesizes current insights into the heterogeneous nature of TAMs in HCC and describes the mechanisms behind their pro-tumoural polarization focusing the attention on their interaction with HCC cells. Furthermore, this review underscores the potential involvement of TAMs’ reprogramming in HCC therapy and highlights the urgency of advancing our understanding of these cells within the dynamic landscape of HCC. Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ~80% of cases . CurrentA significant proportion of HCC cases are still diagnosed at advanced stages, compromising the therapeutic options. In this regard, the advancements in the understanding of the molecular biology of the HCC has allowed us to develop molecular targeted agents (MTAs), including immune-checkpoint inhibitors (ICIs) ,5, tyrosThe tumour microenvironment (TME) includes innate and adaptive immune cells, stromal cells, endothelial cells, cancer-associated fibroblasts and the extracellular matrix (ECM). Together, these components create a niche where tumour cells can grow and disseminate . Tumour-TAMs are considered a diverse and heterogeneous cell population originating from multiple sources and displaying various phenotypes and functions. Macrophages can be either embryonically seeded in the liver, where they continue to renew themselves, or derived from monocyte precursors that infiltrate tissues and undergo differentiation in response to specific microenvironmental conditions . TAMs ar+ macrophage cluster in patients with HBV/HCV-related HCC that might be involved in anti-tumour responses, although further experiments need to be conducted to confirm this statement [Traditionally, macrophages have been categorized into two different activation states based on the expression of cell surface polarization markers as classically activated (M1) and alternatively activated (M2). TAMs are generally classified as pro-tumourigenic M2-like macrophages and their presence has been associated with a worse clinical outcome . In thistatement . Multiple single-cell atlases of the HCC TME have been conducted using sc-RNA-seq technology and revealed the distinct transcriptional landscapes of each subpopulation within the TME ,28,29,30+ macrophages newly recruited into tumours, which shared similarities with the FOLR2+ TAM1 cluster from Sharma’s HCC dataset [+ cluster [+ LYZ+ VCAN+ macrophages, VEGFA+ macrophages associated with oxidative stress, and MMP9+ SSP1+ macrophages, which were considered terminally differentiated TAMs. The latter TAMs were believed to originate from monocyte-derived FCN1+ LYZ+ VCAN+ or TREM2+ macrophages and promote HCC cell migration, invasion, and tumour angiogenesis. These MMP9+ SSP1+ macrophages shared some similarities with SPP1+ TAM2s expressing TREM2 in Sharma’s HCC dataset [+ SSP1+ TAM and HSPA1B+ TAM subpopulations were increased in tumour tissue. However, it remains uncertain and requires further confirmation whether these APOC1+ SPP1+ TAMs identified by Gao et al. represent the same population previously defined by Sharma et al. and Lu et al. [+ macrophages interact with cancer-associated fibroblasts (CAFs) to form a spatial structure named the tumour immune barrier (TIB) that limits the infiltration of immune cells into the tumour core [In a study by Lu et al. , five ma dataset  and Liu  cluster . Additio dataset ,27. Furt dataset  observedu et al. ,27. Inteour core . + macrophages, which exhibited a signature similar to myeloid-derived suppressor cells (MDSCs) and were thus referred to as MDSC-like macrophages, and C1QA+ macrophages, which simultaneously presented signatures for TAMs, M1, and M2 macrophages [+ macrophages highly expressed other genes such as APOE and TREM2. Additionally, Song et al. found a macrophage population that was absent in previous sc-RNA-seq studies and was mostly infiltrated in advanced HCC patients, and characterized by a higher expression of CCL18 [In addition, Zhang et al. found two distinct macrophage clusters enriched in tumour tissues. These clusters were THBS1rophages . Interesrophages ,27, and of CCL18 . These mBesides the heterogeneity observed at the individual cell level, it has been demonstrated that immune cell infiltrates exhibit notable differences between intrahepatic metastatic lesions in multifocal HCC and those in cases of multicentric occurrence. Indeed, in metastases, a higher presence of M2 macrophages and a lower abundance of T cells have been observed . HoweverThe TME, and especially TAMs, orchestrate complex and dynamic interactions with HCC tumour cells by cell-to-cell contact and/or soluble messengers . In thisHCC tumour cells interact with TAMs through the secretion of diverse signalling molecules and exosomes that contain proteins, metabolites and nucleic acids . Cytokines represent a group of signalling proteins that play key roles in the immunomodulation of cancer . In HCC,Regarding specific signalling pathways, it has been demonstrated that HCC tumour cell-derived WNT ligands interact with macrophages, inducing polarization into an immunosuppressive M2-like phenotype, which in turn leads to tumour growth, migration and metastasis . LikewisOther signalling molecules that are secreted by HCC tumour cells include the protein high mobility group box 1 (HMGB1), which promotes autophagy-regulated M2 polarization of TAMs via the production of ROS in these cells through the TLR2/NOX2 axis . In vivoOn the other hand, the secretion of reactive nitrogen species (RNS) such as nitric oxide (NO), which were shown to be regulated by the transcription factor forkhead box O-1 (FOXO1) in HCC cells, modulates TAM infiltration and polarization towards an antitumour phenotype by reducing IL-6 and CD206 expression in these macrophages . + T cell suppression in HCC [+ T cell dysfunction and anti-PD-1 resistance. Furthermore, a recent study shows that tumour-derived exosomal hsa_circ_0074854 participates in the differentiation of M2-like TAMs and its suppression inhibits HCC tumour growth [In the context of HCC, tumour cell-derived exosomes are known to modulate macrophage polarization and promote immune escape and tumour progression ,55. Amonn in HCC . Addition in HCC . Moreoven in HCC . Besidesn in HCC . An examn in HCC . Mechanir growth . In recent years, many studies have aimed to elucidate the molecular mechanisms that hide behind the complex interactions of M2-like TAMs with tumour cells favouring HCC progression . In respect of signalling molecule-mediated crosstalk, it has been shown that in hypoxic and inflammatory conditions, M2-like TAMs secrete high amounts of IL-1β, inducing an overexpression of hypoxia inducible factor (HIF)-1α in HCC cells, the latter being responsible of an increased EMT of HCC cells and HCC metastasis in mouse models . MoreoveSimilarly, IL-8 has been shown to promote EMT of HCC tumour cells via the JAK2/STAT3/Snail signalling pathway, and hence induce their migration and invasion . In addiBesides cytokines, the secretion of TGF-β by TAMs promotes EMT and confers stem-like properties to HCC tumour cells . LikewisExosome-mediated crosstalk is also involved in the interplay between M2 TAMs and HCC tumour cells. Exosomal miR-17-92 clusters from M2-like TAMs were shown to induce a mismatch in the TGF-β1/BMP-7 pathway of HCC tumour cells by modulating post-transcriptional and post-translational modifications of various proteins. As a consequence, miR-17-92 increased HCC tumour cells’ stem-like properties and their invasive capacity . AnotherDuring the last few years, several preclinical and clinical studies have started to evaluate TAM-centered therapeutic strategies in HCC. In this regard, several approaches for targeting TAMs have been proposed for limiting liver cancer progression, including targeting TAM recruitment and accumulation in the tumour and the functional reprogramming of TAMs . Although the inhibition of monocyte/macrophage recruitment by chemokine targeting approaches such as blocking the CCL2/CCR2 axis with a CCR2 antagonist suppresses tumour growth  and poteAlthough strategies directed to target TAMs have initially been focused on macrophage depletion, there is growing evidence that reprogramming of TAMs to overcome an immunosuppressive environment may constitute a more effective therapeutic strategy in cancer . One of Metabolic reprogramming of TAMs is also linked to the plasticity of these cells. In this respect, it has been shown that receptor-interacting protein kinase 3 (RIPK3) is downregulated in HCC-associated TAMs, its deficiency being associated with a reduction in reactive oxygen species (ROS) production and fatty acid oxidation (FAO) via PPAR pathway activation and an induction of M2 polarization of TAMs . In line+ T cell proliferation and differentiation to achieve a long-term immune surveillance to boost anti-PD-1 therapy in HCC [Other strategies of promoting reprogramming of TAMs towards an M1 phenotype includes the use of TLR agonists, such as resiquimod (R848), a potent agonist of toll-like receptors TLR7 and TLR8, to enhance cancer immunotherapy . In liney in HCC . In thisy in HCC .On the other hand, other strategies to enhance the anti-tumour responses of TAMs include restoring their phagocytic capacity by blocking the “don’t eat me” signal of CD47-SIRPα. CD47 expression in HCC cells is correlated with the poor overall survival of patients with HCC  and antiRecent genetic engineering technologies, such as chimeric antigen receptor macrophages (CAR-M), have started to pave the way for using macrophages in adoptive cell therapies to treat solid tumours . In vitrRegarding their potential applicability in HCC, a liver macrophage-targeting mRNA-laden lipid nanoparticle (LNP) was recently generated to produce CAR-Ms coexpressing glypican-3 (GPC3)-specific CAR, a glycoprotein attached and extensively upregulated in HCC tissues, and Siglec-G lacking immunoreceptor tyrosine-based inhibition motifs (Siglec- GΔITIMs). Siglec-G is expressed on the surface of macrophages and interacts with the anti-phagocytic signal protein CD24 upregulated in the tumour tissues of HCC patients. This interaction activates the ITIM motif in macrophages, which is known to restrict the phagocytic function of macrophages. Thus, this novel approach may help macrophages to identify and competitively bind to CD24 of HCC cells, augmenting the cellular phagocytosis by evading the activation of ITIMs, subsequently favouring the antigen cross-presentation of CAR-Ms . In factIn cancer, the existence of a distinct microenvironment of each tumour contributes to the heterogeneity of macrophages . AlthougTAMs can dynamically interact and crosstalk with HCC tumour cells modulating the development, growth and invasiveness of HCC cells by a great variety of mechanisms. In this regard, HCC tumour cells are known to secrete signalling molecules such as cytokines, chemokines and growth factors that are able to promote the transition of macrophages into TAMs acquiring a predominant M2-like phenotype. Most of the studies characterizing the secretome of HCC cells and the influence of the secreted factors on macrophages are based on in vitro models that do not recapitulate the great complexity and diversity of macrophages that are present in the TME of HCC tumours.Recently, TAMs have emerged as a relevant cell type for targeted therapy in liver cancer and several preclinical and clinical therapeutic approaches have been developed aiming to deplete or reduce this population in HCC tumours. Nevertheless, pan-macrophage therapeutic strategies targeting all macrophages are often associated with a compensatory emergence of other immunosuppressive cell types and the presence of systemic toxicity, with limited therapeutic benefits. In this respect, reprogramming M2-like TAMs to acquire an immunostimulatory phenotype may represent a more effective strategy, in particular when this approach is combined with other treatments such as immune checkpoint inhibitors. Despite the fact that preclinical studies have demonstrated encouraging results, the use of animal models have some limitations, as they do not recapitulate the complex heterogeneity and full spectrum of TAM subtypes that are present in humans. At present, there are only a small number of clinical trials targeting TAMs. Nonetheless, their efficacy needs to be further assessed. Adoptive cell therapies using genetic engineered technologies such as CAR-M have also started to enter the field of cancer, also holding promise for the treatment of HCC. Accordingly, macrophages equipped with tumour antigen-specific CARs present increased anti-tumour activity. However, major challenges remain to be solved before their potential clinical application, including the selection of liver tumour cell-specific targets and their biosafety profiles. On the one hand, macrophages present a relatively weak proliferative capacity, which might affect the prolonged therapeutic benefit. Moreover, the high heterogeneity of HCC tumours might compromise the efficiency of CAR-Ms, as some HCC cells that do not express the target antigens may exist. In addition, although promising results might be obtained from experimental in vivo studies, further clinical trials in patients with a much more complex HCC heterogeneity are required to evaluate the efficiency of CAR-Ms. In the future, tailoring TAM-targeted therapies in combination with other therapeutic strategies may constitute a promising alternative treatment for patients with HCC.


TM) platform. Quality of prescribing was assessed based on local guidelines and clinical justification using published definitions . The objective of the study was to evaluate the impact of PAF+NAPS interventions on antibiotic use (AU) in acute leukemia patients at an academic cancer center, compared to PAF-only.Prospective audit-and-feedback (PAF) for hospitalized leukemia patients is resource-intensive, while data on stadardized assessment of the quality, i.e. appropriateness, of antimicrobial prescribing are scarce. We implemented a multifaceted program combining patient-level PAF with unit-level appropriateness reports generated by serial point-prevalence surveys using the web-based Canadian National Antimicrobial Prescribing Survey , the AMS team led 30-minute PAF rounds with prescribers 2x/week. After implementating PAF+NAPS (Jun 1 2019-Mar 31 2020), we met 2x/month for 30 minutes. At each meeting we presented an automated NAPS dashboard report  of aggregate appropriateness adjudication of antibiotic prescriptions, followed by targeted PAF on select patients with complex antimicrobial needs. We analyzed the impact of the two-step approach on the primary outcome of AU (defined daily dose [DDD]/100 patient-days [PD]) using interrupted time series models. Secondary outcomes were appropriateness of AU during PAF+NAPS, lenght of stay and in-hospital mortality. Target appropriatenesss was at least 90%. Categorical variables were compared using chi-square test and continuous variable using Mann Whitney U test.Patient characteristics and outcomes were presented in Table 1. AU data were presented in Table 2. AU of piperacillin-tazobactam and vancomycin decreased significantly after implementation of 2x/month PAF+NAPS, compared to 2x/week PAF-only interventions . Overall appropriateness was 91.4% (1757/1921 prescriptions).Despite reducing frequency of interventions from 2x/week to 2x/month, PAF+NAPS was an efficient and sustainable AMS model that promoted appropriate and judicious antimicrobial use in hematology-oncology patients , allowing the AMS team to expand to other specialized areas.Shahid Husain, MD, Astellas: Grant/Research Support|Avir: Grant/Research Support|F2G: Grant/Research Support|Gilead: Grant/Research Support|Merck: Grant/Research Support|Pfizer: Grant/Research Support|Pulmocide: Grant/Research Support|Synexis: Grant/Research Support|Takeda: Honoraria


Background: Postoperative atrial fibrillation (POAF) is defined as new-onset AF in the immediate postoperative period. The relatively high incidence of POAF after cardiac surgery is well described, but pathophysiological mechanisms underlying the initiation, maintenance, and progression of POAF may be multifactorial and have not yet been comprehensively characterized. One of the mechanisms includes altered Ca2+ kinetics. Accumulating evidence has suggested that altered atrial cytosolic calcium handling contributes to the development of POAF, protamine reversibly modulates the calcium release channel/ryanodine receptor 2 (RyR2) and voltage-dependent cardiac RyR2. However, it is currently unknown whether such abnormalities contribute to the arrhythmogenic substrate predisposing patients to the development of POAF. Methods: We have retrospectively analyzed 147 patients who underwent cardiac surgery with cardiopulmonary bypass support. Of these, 40 patients were excluded from the analysis because of pre-existing AF. All patients received heparin followed by protamine at different dosing ratios of protamine-to-heparin, depending on the periods studied. Results: The dosing ratio of protamine-to-heparin = 1.0 was compared with higher dosing ratios of protamine-to-heparin >1.0 up to 1.7. POAF developed in 15 patients (15/107 = 14%), of these, 5 out of 57 patients (33.3%) in the dosing ratio of protamine-to-heparin = 1.0 and 10 out of 35 patients (66.7%) in the higher dosing ratios of protamine-to-heparin. Statistical significance was observed in patients with higher dosing ratios of protamine-to-heparin, compared with the dosing ratio of protamine-to-heparin = 1.0 . When types of diseases were analyzed in terms of higher dosing ratios of protamine-to-heparin, only valvular disorders were significantly associated with POAF (p = 0.04). Conclusions: Protamine is clinically utilized to reverse heparin overdose and has been shown to display immunological and inflammatory alterations. However, its association with POAF has not been reported. Our results provide evidence that higher dosing ratios of protamine-to-heparin may increase the incidence of POAF. Postoperative atrial fibrillation (POAF) complicates 20–40% of cardiac surgical procedures . TypicalThe mechanisms of POAF are complex and incompletely defined, but include intraoperative and postoperative phenomena, such as inflammation, sympathetic activation, hypoxia, acidosis, oxidative stress, electrolyte abnormalities, and intra-atrial conduction delay that combine to trigger atrial fibrillation, often in the presence of pre-existing factors, making the atria vulnerable to atrial fibrillation induction and maintenance , 2. To p2+ handling is a major determinant of cardiac contractile function, Fakuade et al. hypothesized that alterations in cellular Ca2+ dynamics contribute to impaired atrial contractility in patients who proceed to develop POAF [2+-ATPase (SERCA)-mediated Ca2+ reuptake into SR is a major contributor to impaired preoperative atrial contractile function [2+ leak via RyR2 lead to Ca2+ waves and possible re-entry loops that trigger atrial tachycardia and AF [Considering that cytosolic Calop POAF . They fofunction . Ryanodifunction . RyRs arfunction . Diaz-Syfunction . Shan eta and AF .In STS/SCA/AmSECT  clinical practice guidelines published in 2018 , recommeHere, we have retrospectively reviewed 147 patients who underwent cardiac surgery with cardiopulmonary bypass. All these patients received heparin followed by protamine at different dosing ratios of protamine-to-heparin, depending on the periods used. We have found that higher dosing ratios of protamine-to-heparin increased the incidence of POAF.Data Collection was conducted as a retrospective cohort study to clarify the prevalence and incidence of POAF in the patients who received cardiac surgery. The study protocol conformed to the Declaration of Helsinki and was approved by the institution’s ethics committee in Okanami General Hospital, Iga-City, Mie Prefecture, Japan. Okanami General Hospital is a secondary medical center in the Iga district. The study database was from the Okanami Hospital Medical Database which was composed of detailed medical and procedural information.®  from April 1, 2002, to March 12, 2015, and ACT Plus®  from March 13, 2015, and thereafter. ACT was determined before CPB, and then every 30 min. Additional heparin (1000–2000 IU) was administered if necessary. An ACT target of ≥ 480 s (≥ 480 s up to 600 s) was set in the hospital. All patients received unfractionated heparin  and protamine (10 mg/mL), both are purchased from Mochida Pharmaceutical Co. Ltd. . All patients received initial heparin doses of 300 IU/kg. The dosing ratios of protamine-to-heparin were determined by the volume-based method in the clinical setting, that is, the dosing ratio of protamine-to-heparin = 1.0 represents 1 mL protamine to 1 mL heparin of the first heparin bolus. This ratio is equal to 1 mg of protamine to 100 international units of heparin. The dosing ratios of protamine-to-heparin >1.0 (up to 1.7) were used from February 2004 to September 2009 and the dosing ratio of protamine-to-heparin = 1.0 from October 2009 and thereafter. The change of dosing ratios of protamine-to-heparin was made according to the instructions of the pharmaceutical company.Atrial fibrillation and its occurrence time were identified by the diagnosis of health records and electrocardiograms. Comorbidities including hypertension, diabetes mellitus, hyperlipidemia, heart failure, coronary artery disease, myocardial infarction, chronic obstructive pulmonary disease, peripheral arterial occlusive disease, and chronic kidney disease were also coded from the records. Prescription information was categorized into antiarrhythmic agents, beta-blockers, angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARBs), mineralocorticoid-receptor antagonists, calcium blockers, digitalis preparation, sodium channel blocker, and anticoagulants. The open heart procedures included coronary artery bypass grafting, valvuloplasty, and valve replacement. The echocardiographic data and outcomes including transient ischemic attack, ischemic stroke, hospitalization, and mortality were also obtained from the electronic health records. Activated clotting time (ACT) was measured using HemochronPatient Selection. In this study, all patients above 18 years of age undergoing first-time open-heart surgery at Okanami General Hospital from February 1, 2004, to December 13, 2021, were included for analysis (n = 147). In this retrospective study, we did not include off-pump CABG cases (n = 91) who did not receive protamine. Thus, the annual surgical volume is 14–16 patients per year in the Department of Cardiovascular Surgery, Okanami General Hospital. Patients with previous documented AF (n = 40), who received anticoagulant therapy within 6 months before the open heart surgery or during the follow-up period, could not survive the open-heart procedures, and could not receive regular follow-up at the out-patient clinics were all excluded from the analysis. The definition of POAF was new-onset AF, which is sustained for over 30 s, and detected by either continuous telemetry in the intensive care unit, standard 12-lead electrocardiogram, or implanted devices. All medical records were followed until their last clinical visit, repeat cardiac surgery or death. The index date of outcomes was defined as the date of diagnosis.Statistical analysis. All statistical analyses were performed using EZR software  [P-value less than 0.05 was considered significant., Japan) , which ip = 0.848), gender , BMI , hypertension , and diabetes  in this cohort. The chances of taking anti-arrhythmic agents such as beta-blockers were similar in both groups . POAF developed in 15 patients (15/107 = 14%), of these, 5 out of 57 patients (33.3%) in the dosing ratio of protamine-to-heparin = 1.0 and 10 out of 35 patients (66.7%) in the higher dosing ratios of protamine-to-heparin. Compared to individuals using dosing ratios of protamine-to-heparin >1.0 (up to 1.7), dosing ratios of protamine-to-heparin = 1.0 was associated with reduced risk of POAF . As shown in p = 0.04).A total of 147 patients received cardiac surgery between February 1, 2004, to December 13, 2021, of which 40 patients were excluded because of pre-existing AF. After excluding patients with pre-existing AF, a total of 107 patients were enrolled in this cohort. 2+ kinetics in POAF. As for risk factors of POAF, Yamashita et al. recently performed a systematic review and meta-analysis and identified that older age and a history of heart failure were significant risk factors for POAF whether the included studies were prospective or retrospective data sets [In this study, we investigated retrospectively the development of POAF after cardiac surgery, based on the recent reports concerning altered Caata sets . Our cliThe advantages of unfractionated heparin are its rapid onset of action, clinical efficacy, rapid neutralization by protamine, safety, and low cost. The dose of heparin used to prevent blood clotting during cardiopulmonary bypass (CPB) is 300–400 U/kg plus additional doses to achieve and maintain an ACT of greater than 480 s . Histori2+ release from sarcoplasmic reticulum microsomes [2+ homeostasis [It has been reported that large doses of protamine (>20 mcg/mL) block single skeletal RyR1 channels and inhibit RyR1-mediated Cacrosomes . Diaz-Sycrosomes . They foeostasis . Our cliAdverse effects of protamine include anaphylactic reactions caused by an injection of protamine during cardiac surgery are well-known complications , 20. Gerp = 0.04). In 2+ uptake is a common underlying mechanism that contributes both to the impaired pre-existing atrial contractile function as an independent risk factor of POAF and to the arrhythmogenic substrate, which predisposes patients to the development of POAF [2+ leak, resulting in increased cytosolic Ca2+ and POAF. In the current retrospective study, POAF developed both in 5 of 57 patients (33.3%) in the dosing ratio of protamine-to-heparin = 1.0 and 10 out of 35 patients (66.7%) in the higher dosing ratios of protamine-to-heparin . POAF development in the dosing ratio of protamine-to-heparin = 1.0 may suggest that residual protamine has some adverse effect on inducing POAF. If this is the case, a lower protamine-to-heparin ratio (less than protamine-to-heparin = 1.0) may be used to prevent POAF, as discussed above [POAF, defined as new-onset AF in the immediate postoperative period, is a clinically relevant problem, occurring in 20–40% of patients after cardiac surgery, with the peak incidence between postoperative days 2 and 4 . Intra-  of POAF . Based o-heparin . Statisted above . FurtherThe study has some limitations. First, this small study is a retrospective analysis of an integrated database from a secondary medical center in the Iga district, Mie, Japan. Second, AF was identified by diagnostic code, electrocardiogram, and medical history, not by a long-term recorder, some patients with AF might be underdiagnosed. Furthermore, although potential arrhythmogenic mechanisms for POAF development may be associated with higher dosing ratios of protamine-to-heparin as shown in this study, there may be additional factors predisposing to the development of POAF, including genetic, type of surgery, autonomic nervous system, inflammation, and pre-existing fibrosis.In conclusion, our results provide evidence that higher dosing ratios of protamine-to-heparin may increase the incidence of POAF. In this regard, STS/SCA/AmSECT and EACTS/EACTA clinical guidelines on anticoagulation during cardiopulmonary bypass are useful in clinical practice.


To summarise the current knowledge regarding diagnostics, prognostication and follow-up in upper tract urothelial carcinoma (UTUC).A scoping review combined with expert opinion was applied to provide an overview of the current research field. Based on the published literature and the experts’ own experience and opinions, consensus was reached through presentations and discussions at the meeting Consultation on UTUC II in Stockholm 2022.The strongest prognostic factors in UTUC are tumour grade and stage. They are correlated, and grade is used for indirect staging. The diagnostic examinations should include multiphase computed tomography urography (CTU) with corticomedullary phase, and urethrocystoscopy with cytology. If there is no clear diagnosis for clinical decision-making, ureterorenoscopy (URS) with focal cytology and biopsies should be performed. Both WHO classification systems (1973/1999 and 2004/2016) should be used. Novel biomarker tests are not yet widespread nor recommended for the detection of UTUC. Long-term, regular follow-up, including URS in patients who have had organ-sparing treatment, is important to check for tumour recurrences, intravesical recurrences, metastases and progression of the tumour.Proper diagnostics with correct grading of UTUC are necessary for appropriate treatment decisions. The diagnostics should include CTU with corticomedullary phase, urine or bladder cytology, URS with focal barbotage cytology, and biopsies when needed for proper diagnosis and risk stratification. Regular, long-term follow-ups are fundamental, due to the high rate of recurrence and risk of progression. Urothelial carcinoma (UC) is the sixth most common cancer in developed countries. Upper tract urothelial carcinoma (UTUC) in the renal pelvis or the ureter is comparatively rare and represents 5–10% of UC with an estimated incidence of 2/100,000 in Western countries , 4. RiskFurthermore, there must be a clear aim of organ-sparing treatments: elective, relative, imperative, or palliative. For palliative organ-sparing treatment, indications may be to achieve local disease control, or to give guidance regarding what systemic therapy may be indicated whilst the patient still has preserved renal function.The 5-year disease-specific survival (DSS) for patients with UTUC is relatively low compared to patients with UC in the bladder as approximately 60% of the patients have invasive cancer at the time of diagnosis and approximately 9% have metastases . Since UThe objective of Consultation on UTUC II Stockholm 2022 was to assemble expert clinicians and scientists within the field to discuss present guidelines, share current knowledge on diagnostics, prognostication, and follow-up regimens. Based on the published literature and the experts’ own experience and opinions, consensus was reached through presentations and discussions at the meeting.Accurate diagnostic workup is important to optimise risk stratification and minimise suboptimal or over-treatment of patients with UTUC. In the diagnostic workup, multiphase computed tomography urography (CTU) including the corticomedullary phase is the recommended imaging modality for diagnostics and staging . UrethroConsensus reached at the Consultation on UTUC II Stockholm 2022: diagnostic URS should be performed if imaging with CTU and cytology are not sufficient for clear diagnosis or risk classification.It can be challenging to assess invasiveness of UTUC with CTU, unless the tumour is advanced. However, for staging, CTU is informative regarding lymph node involvement and distant metastasis. In the upper urinary tract, CTU has a sensitivity of 89% for detecting UTUC; however, with smaller lesions, the sensitivity is lower, decreasing to 40% for lesions sized less than 3 mm . When peConsensus reached at the Consultation on UTUC II Stockholm 2022: the diagnostic examination should include multiphase CTU including corticomedullary phase if no contraindications are present.Diagnostic URS provides information regarding tumour characteristics such as localisation, size, architecture and focality and enables collection of samples for cytology and histopathology , 24. In The development of flexible ureteroscopes with digital technology has ameliorated both diagnostic and therapeutic URS. As the ureter is narrow and long, instruments must be sized in a way to fully access the whole urinary tract system, but still be able to capture digital images and harbour enhancement technologies . TechniqConsensus reached at the Consultation on UTUC II Stockholm 2022: diagnostic URS should be performed when needed for proper diagnosis and risk stratification.The objective of taking biopsies is to unveil tumour grade, which may be used for indirect tumour staging as well, and thus is very important for treatment decision . Local sBefore performing organ-sparing treatment, it is fundamental to rule out high-grade lesions. Retrieving URS biopsies may be challenging; the narrow working channel in the most frequently used flexible ureteroscopes is 3.6 Fr, and the forceps must be thinner, as shown in Fig. Consensus reached at the Consultation on UTUC II Stockholm 2022: endoscopically taken biopsies are of high importance to unveil tumour grade and are highly valuable for proper risk classification.Cytology for the detection of malignant cells in UTUC can be performed using voided urine, selective ureteral catheterisation, or intraoperative collection of saline barbotage fluid. Due to the heterogeneity of the tumour, grading can be improved with cytology, which potentially represents a larger part of the tumour. For voided urine, the sensitivity is rather low—43% to detect grade 2 malignant cells and 80% for grade 3 (WHO 1973/1999) . CytologConsensus reached at the Consultation on UTUC II Stockholm 2022: due to the heterogeneity of the tumour, grading can be improved with cytology. The Paris system should be used to report cytological findings.There are several biomarker tests on the market for the detection of UC using analysis of proteins, DNA and mRNA in urine or blood. Series have been reported in UTUC for use of several molecular tests including FISH, BTA, NMP22, uCyt, Bladder EpiCheck and Xpert Bladder Cancer, although all these series are small with variable sensitivity and specificity . They miConsensus reached at the Consultation on UTUC II Stockholm 2022: biomarker tests might provide extra information as a complement to histopathology at diagnosis and follow-up, but still need further evaluation.Prognostic factors to aid treatment decision are of utmost importance. Tumour stage and grade are prognostic factors with strong evidence in UTUC, whereas other criteria in the EAU Guidelines are supported by less strong evidence. Thus, proper diagnostics with correct grading and staging are important for appropriate treatment decisions .As mentioned before, there is a strong correlation between grade and stage. UCs are currently classified according to two classification systems: WHO 1973/1999, by which tumour grade is categorised as grades 1–3, and WHO 2004/2016, by which UC is described as either low grade or high grade and arrived when WHO adopted the International Society of Urological Pathology (ISUP) criteria, with new stratification of the different categories , 52. TheIn the EAU Guidelines, several known prognostic factors of survival are stated, such as lower cancer-specific survival with old age, tobacco consumption, comorbidity and performance status , 56. To Consensus reached at the Consultation on UTUC II Stockholm 2022: proper diagnostics, especially with correct grading, is necessary for appropriate treatment decisions.The aim of follow-up is to check for upper tract tumour recurrences, intravesical recurrences (IVR), metastases and progression of tumour stage and grade. If organ-sparing treatment has been performed, follow-up including long-term surveillance with repeated CTU, urethrocystoscopy and URS is essential . The EAUConsensus reached at the Consultation on UTUC II Stockholm 2022: regular and long-term follow-up, including URS if organ-sparing treatment has been carried out, is crucial for handling recurrences, IVRs, metastases and tumour progression.Proper diagnostics with correct grading are necessary for accurate risk stratification, thereby enabling treatment decisions. The diagnostic workup should include multiphase CTU with corticomedullary phase and urine cytology or cytology taken at cystoscopy. URS with focal cytology, preferably taken as a barbotage, and biopsies may add valuable diagnostic information, which should not be underestimated. Both WHO classification systems (1973/1999 and 2004/2016) should be used due to their added prognostic value. Biomarker tests on the market are not yet recommended in the EAU Guidelines, since they are developed for UC of the bladder, and more validation for UTUC is needed. Regular and long-term follow-up, including URS if organ-sparing treatment has been carried out, is crucial for handling recurrences, IVRs, metastases and tumour progression. During the Consultation on UTUC II in Stockholm, discussions were held regarding the recommendations in the EAU Guidelines for the follow-up of patients after organ-sparing treatments. They were considered to represent the bare minimum, and it was suggested they should be updated with more stringent recommendations regarding length and frequency of the follow-up, bearing in mind the high rate of recurrence and risk of progression. The AUA Guidelines state from expert opinion that follow-up should be performed longer than 5 years in the absence of recurrence, based on shared decision-making between the patient and clinician. In case of a high-risk tumour, a follow-up exceeding 5 years is encouraged .


We report a non-resonant piezoelectric microelectromechanical cantilever system for the measurement of liquid viscosity. The system consists of two PiezoMEMS cantilevers in-line, with their free ends facing each other. The system is immersed in the fluid under test for viscosity measurement. One of the cantilevers is actuated using the embedded piezoelectric thin film to oscillate at a pre-selected non-resonant frequency. The second cantilever, the passive one, starts to oscillate due to the fluid-mediated energy transfer. The relative response of the passive cantilever is used as the metric for the fluid’s kinematic viscosity. The fabricated cantilevers are tested as viscosity sensors by carrying out experiments in fluids with different viscosities. The viscometer can measure viscosity at a single frequency of choice, and hence some important considerations for frequency selection are discussed. A discussion on the energy coupling between the active and the passive cantilevers is presented. The novel PiezoMEMS viscometer architecture proposed in this work will overcome several challenges faced by state-of-the-art resonance MEMS viscometers, by enabling faster and direct measurement, straightforward calibration, and the possibility of shear rate-dependent viscosity measurement. Elevated viscosity of whole blood is also associated with diminishing renal functions4. The estimation of viscosity is also essential for monitoring the quality of lubricants during operation5. In chemical processing industries, the viscosity of the byproducts and the end product is used as a quality control measure. Currently, most of these applications utilise a rotating cylinder-based bulky instrument for viscosity sensing which is expensive, time-consuming, and incompatible with the paradigm of internet-of-things (IOT) based distributed and online sensing. The use of bulky equipment for fluid-property measurement are expensive, require large operating power, and are slower. There is a need for cheaper, low cost and in-line measurement techniques for accurate viscosity measurement without any post-processing. Moreover, there is much emphasis on the fourth industrial revolution (Industry 4.0) led by a large number of interconnected smart devices for self-monitoring6.At a fixed temperature and pressure, the mass density and the viscosity have unique values for a given fluid. Accurate characterisation of fluids using these properties is of immense importance in healthcare, processing industries, and liquid (lubricant) health monitoring. For example, in healthcare applications, the viscosity of blood plasma can be a marker for several conditions such as cardiovascular diseases and autoimmune disorders like rheumatoid arthritis12. Moreover, PiezoMEMS devices offer low-voltage operation and high electromechanical coupling and hence are suitable for such applications.Micro-scale solutions have emerged in the last decade to overcome the above challenges because they offer low cost, small footprint, and on-site installation for monitoring many properties and parameters. Piezoelectric MEMS resonators have become popular devices for the measurement of fluid properties because of their excellent ability to operate at a reasonable quality factor in a fluidic environment as evident from several publications over the last few yearsfr, and quality factor, Q, to estimate the density and viscosity of the fluid12. The obtained values depend on the fluid density and viscosity through secondary parameters13,Most MEMS-based fluid-property sensors use a single resonator (such as a cantilever) and track its resonance frequency, g1 and g2 are the secondary parameters which are a series expansion in terms of density, viscosity, and their products. For example, 13. This method is time-consuming and demands extensive post-processing of the experimental data. These single-cantilever systems suffer from large errors, particularly for viscosity measurement13, because changes in quality factor can be due to a complex combination of several factors including density, viscosity, acoustic damping, substrate damping, and temperature14. Moreover, the resonators used in the current viscometers are operated in higher-order modes to achieve a better Q-factor15, since higher modes have less energy dissipation16. However, at very high frequencies, viscoelasticity can affect the measurement results.In Eq. , g1 and 4. To overcome these challenges associated with SAW-based sensors, flexural modes of cantilever devices are actively being explored17.Another popular category of sensors for liquid property measurement is acoustic wave sensors. However, the response of the SAW-based sensors depends on the square root of the product of density and viscosity (Q-factor and associated complexity in the estimation of viscosity.We report a unique tip-coupled two-cantilever (TCTC) sensing system where the kinematic viscosity of any fluid can be obtained directly from the output parameters (velocity/displacement) of the device. Such a measurement technique offers a quick and direct measurement of viscosity. The use of two cantilevers in the proposed tip-coupled configuration allows us to eliminate the need for measurement of the The design of the viscometer mimics a conventional rheometer/viscometer where the test fluid is subjected to shear between a fixed and a rotating object (plates or cylinders). In the conventional rheometer, the relationship between the applied torque and the velocity of the moving object is used as a measure of the liquid viscosity. The sensor design used in this study consists of two micro-cantilevers integrated with piezoelectric, lead zirconate titanate (PZT) thin film as the active element. Figure 3 and 2.59 cSt, respectively. The frequency response of each cantilever shows two peaks corresponding to the cantilevers’ resonance frequencies. We observe two peaks for each of the cantilevers because, at the respective resonance frequencies of the individual cantilevers, there is enhanced fluid movement, resulting in more fluidic coupling between the two cantilevers.Although split electrodes were designed and fabricated on the cantilevers for simultaneous actuation and sensing of the response, the sensor output was corrupted due to the capacitive feedthrough between actuation and sensing electrodes. Due to this difficulty, the cantilevers’ vibrational responses were measured using a laser Doppler vibrometer (LDV), Polytec MSA-400. The active cantilever was actuated by applying a unipolar electric potential of 0.75 V across the top  and bottom electrodes of the PZT thin film while the sample was placed in a liquid (test fluid) filled petri-dish. The responses of the cantilevers  solutions. Figure 21. A fit model, aηb with the exponent b = 0.12, fits the experimental results. Such power law dependence results allow for a straightforward calibration of the sensor. This is considerably easier than the current resonant sensors where a Taylor series expansion of the hydrodynamic function is utilised for calibration requiring three or more experimental points13.Figure Q-measurement, thereby enabling faster measurements. Moreover, a frequency sweep away from the resonance may provide information on the shear rate dependence of the viscosity of the fluid. Figure This method enables the viscosity measurement without the frequency sweep and the requirement of any peak fitting for Since the viscosity measurement can be carried out at a single actuation frequency, it is important to discuss the critical factors for the selection of the actuation frequency. The two most important considerations for frequency selection that are discussed in this section are, (i) sensitivity, and (ii) the penetration depth of the shear waves generated by the oscillating active cantilever.m1 and m2 represent the effective masses of the active and passive cantilevers, respectively. The springs, k1 and k2 represent effective spring constants for the active and passive cantilevers, respectively. The dampers, c1 and c2 represent the damping coefficients associated with the vibration energy dissipation of each of the cantilevers. The fluid coupling can be incorporated in the model by introducing a coupling spring, kf and a coupling damper, cf. For an incompressible flow of fluid, the presence of the coupling spring, kf can be ignored. The damping coefficient cf for the coupling damper depends on the fluid properties. Please refer to the The cantilever system can be modelled as a two-degree-of-freedom spring mass-damper system, as shown in Fig. R) were calculated and plotted for different parameters. Predominantly, fluid density contributes to the added mass and viscosity contributes to the damping. The mass sensitivity, R with respect to m2, this sensitivity is valid for added mass also . Specifically, c2). The sensitivity is calculated for a cantilever exhibiting a resonance frequency of 34 kHz. It is well known that the response of the vibrating system is dominated by the damper only near the resonance frequency and hence we see that the amplitude ratio also follows the same trend.To understand the effects of fluid density and viscosity on the amplitude ratio, the sensitivities of the amplitude ratio  for fluid-property measurement. However, the lowest frequency that can be used will be dictated by the noise-required sensitivity for that particular measurement. Fortunately, the sensitivity of the device can be varied (if the application demands) by just changing the operating frequency of the device and designing the resonance frequency of the cantilever.δ22,ω is the frequency of oscillations and η is the kinematic viscosity of the fluid. The penetration depth of the shear waves follows an increasing trend with kinematic viscosity and a decreasing trend with frequency. Since the gap between the cantilevers is always fixed for a previously fabricated system, there can be an upper limit on the frequency and a lower limit on the viscosity.The transfer of energy from the active cantilever to the passive cantilever is through the shear waves generated by the active cantilever. The flow of shear waves created by an oscillating plate is governed by the characteristic length The concept of penetration depth also governs the gap between the cantilevers. Since there is an inverse relationship between the actuation frequency and the depth of penetration, as we increase the actuation frequency, the gap has to be reduced, to ensure effective coupling. The gap should be decided based on the minimum desired viscosity and maximum operational frequency.axb (with b < 1) dependence on kinematic viscosity. Such nonlinear dependence results in variable sensitivity in different measurement ranges. Since the exponent term in viscosity dependence is always smaller than unity, the sensitivity will follow a strongly decaying The amplitude ratio, which is the metric for viscosity measurement for TCTC viscometer, follows an with b <  dependenaxb, the sensor can be calibrated by performing two calibration experiments to obtain the two coefficients “a” and “b”. The precision of the sensor will be governed by the ability of these coefficients to capture the true viscosity values for other experiments. The expected curve for the dependence of the amplitude ratio on the kinematic viscosity of the fluid is shown in Fig. 13.Since the experimental curve follows a power law There are three modes of energy transfer from the active cantilever to the passive cantilever, namely structural coupling, coupling due to fluid flow, and acoustic coupling. The structural coupling has no effect on the viscosity measurement because it just adds a constant bias in the measured response. Both the acoustic coupling and the viscous coupling, affect the response of the passive cantilever. Moreover, both of these coupling mechanisms are affected by the kinematic viscosity of the fluid medium.22. The acoustic pressure wave, on the other hand, follows an exponentially decaying curve. The origin of these expressions is explained in the From a basic Stokes-flow assumption, the force on the passive cantilever due to the oscillations of the active cantilever should follow a The corresponding fit along with the experimental data points are plotted in Fig. aηb dependence on the kinematic viscosity of the fluid. Such a dependence allows for calibrations of the sensor using only two data points as opposed to four or more in the current MEMS viscometer. Another significant advantage of the TCTC viscometer is its ability to measure the viscosity of the liquid at different operating frequencies thereby allowing shear rate-dependent measurement. Although the test fluid used in this paper is a Newtonian fluid, the viscometer’s ability to measure viscosity at different frequencies is explained. We believe these initial results are encouraging for the MEMS community to explore the avenues of viscometry using TCTC design.The TCTC viscometer design presented in this paper utilises a novel two-cantilever architecture, where the fluid coupling between the tips of the cantilevers is exploited for viscosity sensing. This unique approach is a significant improvement over the current MEMS viscosity sensors, since it enables a direct, faster, and potentially, more sensitive measurement. The relative response of the passive cantilever follows a power law, In this paper, we have also presented an understanding of the energy coupling mechanism between the two cantilevers. It is proposed that in addition to the shear flow of fluid, the energy transfer between the cantilevers can also be due to the structural coupling (originating because the two cantilevers share a common frame) and due to the flow of acoustic waves. This hypothesis has been validated by fitting the experimental data to the appropriate expression which includes all three coupling mechanisms.Supplementary Material