Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25 Accumulating evidence, in both humans and mice, indicates that specific immune responses to tumours require the activation, amplification, and cytotoxic function of antigen-specific T cells. Notably, a strong infiltration of CD8 T cells at the tumour site is needed to control tumour growth . However+CD25+Foxp3+Tregs (Tregs) is a well-documented mechanism of tolerance [DTR mice was shown to induce the differentiation of high numbers of pre-DCs and DCs, and their accumulation in LNs [in vivo mechanism involved in the Treg-mediated suppression of anti-tumour responses is still lacking and could be pivotal for the specific manipulation of Tregs.Suppression of immune responses by thymus-derived CD4olerance , 8. Foxpolerance . Mechaniolerance , 11, andolerance , where Tn in LNs , 14. Lasn in LNs  and the n in LNs , both re+Treg resulted in the strong activation/amplification of CD4 and CD8 effector T cells and the control of tumour growth [The role of Tregs in the suppression of the anti-tumour response was first demonstrated when the administration of a single dose of anti-CD25 antibodies (PC61) prior to tumour injection, induced tumour regression in the majority of treated mice . In anotr growth . Howeverr growth . However+ T cells in DLNs, characterized by their expression of CD11c and low levels of PD1. Tumour neo-angiogenesis was fully modified, allowing the direct access of these CD8+CD11c+ T cells from the DLNs to the tumour sites and elimination of the tumour in treated mice. When low numbers of these cells were injected i.v. in non-treated tumour bearing mice, they were able to control tumour growth. Additionally, tumour neo-angiogenesis appeared enhanced favoring direct access of CD8+CD11c+ effector T cells from the DLNs to tumour sites and elimination of the tumour in treated mice. Thus, our data demonstrates an unappreciated effect of Treg depletion in a coordinated expansion of DC subsets in tumour bearing mice, and suggest that the efficacy of therapeutic vaccines requires the partial depletion of Tregs allowing to increased number of DCs and the generation of large numbers of CD8+ CD11c+ PD1lo T cells, which have the capacity to infiltrate the tumour and control its growth.We used here the 4T1 mammary carcinoma and PC61-mediated Treg depletion as an experimental approach that allowed us to study the mechanism of tumour regression, with anti-tumour responses being evaluated simultaneously in the DLN and at the tumour site. We show that Treg depletion induced the sequential recruitment and expansion of the two main DC subsets, and gave rise to the expansion of cytotoxic CD85 cells were injected s.c. in the left flank of the mice in a final volume of 100μl PBS. Tumours size was measured by caliper every 3 days. After 4, 7, 10 and 14 days post-injection, mice were sacrificed and tumours resected. To isolate lymphocytes from tumour tissues, tumours were collected, cut into small pieces, and incubated in RPMI with 0.15 mg/mL Liberase TL (Roche) and 200ng DNase (Roche) for 45 min at 37°C. Tumour tissues were then homogenized until resolved into a cell suspension.All animal experiments were conducted according to European Directive (2010/63/UE) and were approved by the Ethical Committee of Paris Descartes University (registered number: 14–075). 6 to 8 week-old BALB/c mice were purchased from Charles River Laboratories. The 4T1 tumour cell line derived from a BALB/c spontaneous mammary carcinoma was supplied by Dr S. Ostrand-Rosenberg . 4T1 cells were cultured in RPMI supplemented 5% FCS and 1% antibiotics. 2x10For Treg depletion, mice were treated i.p. with 300μg anti-CD25 antibody PC61  4 days before tumour inoculation.For flow cytometry analysis, the inguinal lymph node from the tumour (left) was taken as the tumour-draining lymph node (DLN) and the contralateral inguinal lymph node as a non-draining lymph node (nDLN). LNs and tumours were excised at various time-points, cut into pieces, and digested into single cell suspensions using collagenase D1 (1mg/ml) and DNAse (100ng/ml)(Roche Applied Science) for 15–40 minutes at 37°C. T cells. DC subsets were then treated with EDTA 5mM for 5 min and stained with anti-CD11c (PE-Cy7), anti-MHC-II (FITC), anti-CD8α (Pacific Blue), anti-CD11b (PerCP-Cya5.5), anti-DEC-205 (PE) and anti-CD103 (APC) antibodies. To further delineate DC subsets, total cells were stained with anti-CD3 (APC), CD19 (APC), and B220 (APC). T cell subset was stained with anti-CD8β-PE or CD8β-FITC.Treg characterization and KI67 proliferation were assessed using a Foxp3 permeabilisation kit (eBioscience). Cell surface staining was performed with anti-CD4 (APC-AlexaFluor-780), anti-CD3 (PerCP-Cya5.5) and anti-CD25 (clone 7D4-FITC). Intracellular staining was performed with anti-Foxp3 (PE) or anti-KI67 (PE).hiMHCIIintCD11bhi sorted by using a cell sorter FACS Aria I (Becton Dickinson) from DLNs and tumours from untreated and treated mice. DCs (3x103) and CellTrace violet (CTV) labeled CD8 T cells (3x104) sorted from normal mice LNs were cultured for 3 days in 96 well round-bottomed plates (Costar) in RPMI 1640 medium supplemented with 5% FCS, penicillin (100U/ml), streptomycin (100μg/ml) and 50 μM 2-ME. Sorted CD4+CD25- T cells were added to the culture separately. Anti-CD3 mAb (145-2C11) at a final concentration of 0.1 μg/ml was added to the cultures for stimulation. CTV dilutions were used to determine the degree of CD8 T cell proliferation. After 3 days, cells were collected, treated with 5 mM EDTA and stained with anti-PD-1 mAb.CD11c+CD11c+ tumour-draining LNs were harvested on day 14 and incubated with sorted CD11chi dendritic cells, PE-conjugated CD107a mAb, and monensin for 6 hours at 37°C. Cell suspensions were then stained for CD3 and CD8β to identify CTLs, as well as CD107a and analysed by flow cytometry.CTL granule exocytosis was measured as mobilization of the lysosomal marker CD107a (Lamp-1) to the cell surface as described . To trig+CD8+CD11c+ single T cells were directly sorted into PCR tubes after staining with anti-CD3 FITC, anti-CD8β Pacific Blue and anti-CD11c Percp-Cy5-5 Abs. cDNA synthesis and PCR amplification with specific primers of CD3, CD11c, IFNγ, Granzyme B (Grzb) and Perforin (Prf) genes (CD3f) genes  were perInguinal draining LNs and tumours were harvested at day 12 after tumour inoculation, directly frozen in OCT and stored at -80°C. Multiple LN and 6 μm tumour sections were then prepared with a cryostat. Sections were fixed with 50% Acetone and 50% Ethanol (10 min at -20°C) and washed with PBS. Sections were blocked over night with Blocking Buffer Low Protein (eBioscience) and then stained with appropriate primary and secondary antibodies. Each incubation step was conducted for 2 hours in the dark at room temperature. The sections were first stained with monoclonal antibodies: purified anti-CD8β and biotinylated-CD11c (clone HL3), secondly stained with anti-rat Alexa fluor 555, Cy5-conjugated streptavidin, and Alexa fluor 488- CD31. Antibodies were purchased from Biolegend, BD Pharmingen, and eBioscience. Images were acquired using a Zeiss LSM 700 confocal microscope with ZEN 2009. Sections were analysed by ImageJ v1.47 software. Levels of angiogenesis were determined by quantitating the number of vessel on the tumour surface and the percentage of CD31-positive pixels per area of tumour.Means of two groups were compared with two-tailed Student t test for unpaired observation with Prism 4 software  and also by Fisher’s exact test using C-Stat 1.0 . Differences with p values < 0.05 were considered significant.5 to 2 105 cells per mouse. To gain quantitative information on tumour growth and the different cellular subsets involved in tumour regression, we first depleted CD25+ Tregs by one injection of PC61 antibodies in a group of 20 mice. 4 days later, PC61-treated and 10 untreated mice were injected subcutaneously in one flank with 2 x 105 4T1 mammary carcinoma cells. Tumour sizes were measured every 3 days until complete tumour regression. Comparison of tumour sizes between treated and untreated mice showed similar kinetics of tumour growth until days 9–11 after injection of tumour cells . These data indicate that all treated mice had the same level of Treg depletion before tumour inoculation.We previously reported that the prophylactic depletion of Tregs using anti-CD25-specific antibodies (PC61) resulted in a partial depletion of Tregs and the complete regression of the 4T1 mammary carcinoma in majority of tumour-bearing mice . The 4T1ur cells . Thereaf+Foxp3+ Tregs were reduced by 76% in all treated mice, and this depletion lasted a minimum of 14 days (+Foxp3+ Tregs cells (D14) in the DLN of treated mice indicates that Tregs suppressive effects are likely to occur mainly at the initial phase of the anti-tumour response.To investigate the rate of Tregs repopulation following PC61 treatment in tumour- bearing mice, we compared cell numbers in non-draining LNs (nDLN) and in the draining LNs (DLN) at different time points after tumour inoculation. In nDLNs, the percentages and absolute numbers of CD25 14 days . Convers 14 days . However+Foxp3+ Tregs led to increased numbers of LN-resident DCs [- cells were examined for CD11c and MHC II expression characteristic of the main subsets of CD11c+ DCs, i.e, CD11chiMHC IIint LN resident DCs including CD11bhi, CD8+DEC205+ and CD11chiMHC IIhi including CD103+ migratory DCs (mDCs).Dendritic cells (DCs) are promising targets for immunotherapy because they play an essential role in initiating and regulating T cell immunity. It has been shown that CD25dent DCs  but neitdent DCs , 23 and hi DCs at day 7 and day 10 after tumour injection while increased numbers of CD8+ DCs were only observed at day 10  of each staining. The level of MHC II expression on CD11bhi DCs from treated mice was lower than the level of MHC II of DCs from untreated mice at day 7 , while they were co-expressed by CD8+CD11c+ cells (+CD11c+ T cells were able to kill the sorted intra-tumour MHC-IIhiCD11chi DCs (which should present tumour antigens), as determined by mobilization of the lysosome marker CD107a to the cell surface upon encounter with these targets , and to assess the frequency of the cytotoxic T cells in DLNs and at the tumour sites. We observed that the proportion of single cells expressing the cytotoxic associated genes IFNγ, granzyme B (Grzb), and perforin (Prf) genes was similar in both groups at day 7 but however increased in Grzb at day 14 in treated mice. In contrast, co-expression of the three genes was markedly augmented at Day 14 from regressive tumours . Importantly CD8+CD11c+T cells (circle) co-localized with dendritic cells  from treated and untreated tumours were sorted and cultured with normal CD8 T cells sorted from LNs, labeled with CellTrace Violet (CTV), division being evaluated by CTV dilutions (Published data identified PD-1 as one of the most overexpressed negative receptors by exhausted CD8 T cells in chronic infections  as well ilutions . After aTo look for a possible connection between the effect of DC stimulation and the regulation of PD-1 expression, we next analysed PD1 expression on dividing cells elicited by the different DCs. Without addition of anti-CD3 mAbs in the culture, tumour DCs from untreated mice were unable to induce CD8 T cell divisions . In contThese results indicate that Treg depletion modifies the properties of DCs and showed that in the absence of Tregs, DLN and tumour DCs provided a stronger signal at the priming phase of activation improving the quality of CD8 T cell divisions and reducing the level of PD1.+FoxP3+CD25+ natural Tregs [in vivo Tregs depletion affects tumour growth and induces a vigorous effector response from the diverse repertoire of normal T cells remains poorly understood. In this study, we determined that Treg depletion by PC61 mAbs leads to tumour regression by triggering three major events: modifications of different dendritic cell subsets in the tumour draining lymph node, an enhancement of tumour neo-angiogenesis and the generation of polyfunctional cytotoxic T cells uniquely capable of infiltrating the tumour and controlling tumour growth.The failure of immune responses against tumours has been attributed to an active suppression by CD4al Tregs . We haveal Tregs . Neverth- and CD25lo FoxP3+ Tregs are not affected. Moreover, in the presence of the tumour, CD25+FoxP3+ Treg cells accumulate in the DLN of treated mice, reaching the same numbers as those found in untreated mice by day 14 after tumour inoculation. As this late accumulation does not prevent tumour rejection, our data indicate that CD25+FoxP3+ Treg cells only influence the priming phase of the immune response. It is well known that IL-2 is a central mediator of Treg suppression, essential for their survival and competition for limited amount of IL-2 during immune response [PC61-Treg depletion does not fully eliminate Tregs, as CD25response , 27. TheDTR mice promotes the differentiation of high numbers of pre-DCs and cDCs from bone marrow precursors and their accumulation in LNs [in vivo [+ DCs preferentially presented antigen to CD4 T cells rather than CD8+DCs [+ DCs have been described as capable of inducing a protective anti-tumour immune response [+ DCs were associated with antigen cross-presentation and CTL priming [+ DCs that could activate CD4 T cells to produce IL-2, necessary for the efficient generation of CD8 effector T cells. Then, CD8 T cell activation would be further sustained by CD8+ DCs, which are known to cross-present antigens to CD8 T cells. Thus, Treg depletion promotes DC modifications as a part of a program that optimizes the microenvironment for the generation of effector T cells.A previous study proposed that Tregs control the DCs homeostasis  as Treg n in LNs , 14. In [in vivo . It has  CD8+DCs . In addiresponse . Lastly, priming . Our datReleased from Treg control, the increase of DC numbers were expected to impact the number of CD8 T cells and the generation of effector T cells. In agreement with this hypothesis, DLN increased 4-5-fold in treated mice and the number of CD8 T cells increased proportionally. This increased DLN size correlated with the DCs accumulation, suggesting that high numbers of DCs promote the recruitment of higher numbers of CD8 T cells. This recruitment of CD8 T lymphocytes should increase the probability of DC and antigen-specific T cell contact, and therefore promote the generation of higher numbers of anti-tumour-specific effector cells. This observation is in agreement with previous reports, which show that mature DCs control lymphocyte entry to LN through HEVs, modifying the lymphocyte traffic and the magnitude of the effector response , 32. TheINFγ, Grzb, and Prf, proliferated vigorously and were able to kill DCs extracted from both tumours. Moreover, the adoptive transfer of only 6x105 of these cells was able to control tumour growth in untreated tumour-bearing mice. Overall, these results demonstrate that the CD8+CD11c+T cells present in the DLN are cytotoxic T cells involved in tumour regression. It is possible that their expression of CD11c, an integrin involved in cell adhesion, favour their migration into the tumour mass.Besides these modifications, we also found that Treg depletion lead to the generation of a subpopulation of anti-tumour cytotoxic effector CD8 T cells that could be identified by their expression of the integrin CD11c. These cells, that accumulated in the DLN and in the tumour of PC61-treated mice were found in close contact with DCs at tumour sites, co-expressed the cytotoxic associated genes as DTR mice to deplete Tregs, previous studies have observed an acute lymphopenia immediately after DTX injection and this was transient since T cell numbers recovered by day 6 [in vivo data establishes a direct link between Treg-mediated suppression and the expression of high levels of PD-1 on exhausted cells.Using Foxp3by day 6 . Althougby day 6 , 37. Theby day 6 . Tumour-by day 6 . Furtherby day 6 . Consistlo cells have the capacity to kill DCs presenting the tumour antigens. This effect would regulate the immune response by reducing antigen presentation to expanded CD8 T cells and prevent their chronic stimulation and exhaustion. This is supported by previous reports in which the killing of DCs was shown to be an important mechanism for the regulation of T cell responses [+CD11c+ T cell degranulation upon interaction with tumour DCs. Our results also highlight a correlation between Treg levels, CD4 help/IL-2 availability, DC properties, and the expression of PD1. It was previously reported that PD1 expression inhibited the IL-2 autonomous production by CD8 T cells [The finding that in PC61-treated mice, all CD8 effector cells accumulate inside the tumour with low expression of PD-1 raises the question of how these cells are not chronically stimulated. It is tempting to speculate that chronic stimulation does not occur because PD1esponses –40 and o T cells , yet a p+DC subset as well as the reduced Treg numbers would decrease considerably the Treg/DC ratio, providing up-regulation of co-stimulatory molecules, followed by increased CD8 T cell recruitment in DLN. These events should favour the efficient activation and expansion of CD8+CD11c+ cytotoxic T cells, activation of which in the presence of IL2 induces their rapid expansion and avoids PD-1 expression. A higher capacity of these cells to divide and invade the tumour, together with an extensive angiogenesis, would allow antigen-specific cell expansion and elimination of the tumour mass. Importantly, these CTLs could prevent chronic T cell stimulation, high level of PD1 expression and functional inactivation by reducing antigen loads through killing tumour cells and the DCs expressing tumour antigens.By combining the multiple reports cited above and our novel data, we propose a model of tumour regression providing a possible explanation for the complex events following the transient reduction of Tregs by PC61 treatment in tumour-bearing mice. The modifications of DC populations would be responsible for most of the modifications of the anti-tumour response leading to tumour rejection. Thus, the recruitment of CD11b+CD11c+PD1lo effector cells and their migration into tumours. With regard to cancer, new approaches for cancer therapies will be facilitated by the identification of cellular pathways involved in the role of Treg cells in the evasion of tumour immunity. Thus, the sequential and differential impact of DC subsets may guide the design of new protocols for partial Treg depletion and DC generation, in order to develop more efficient anti-tumour therapy.Our data highlight the effect of Treg depletion on tumour regression and the major role of three DC subsets that act at different time points of the anti-tumour response. Therefore, our results delineate an additional level at which DCs induce complete tumour regression by orchestrating all events leading to fully differentiated CD8


The present investigation focuses the flow and heat transfer characteristics of the steady three-dimensional Sisko fluid driven by a bidirectional stretching sheet. The modeled partial differential equations are reduced to coupled ordinary differential equations by a suitable transformation. The resulting equations are solved numerically by the shooting method using adaptive Runge Kutta algorithm in combination with Newton's method in the domain [0,∞). The numerical results for the velocity and temperature fields are graphically presented and effects of the relevant parameters are discussed in detail. Moreover, the skin-friction coefficient and local Nusselt number for different values of the power-law index and stretching ratio parameter are presented through tabulated data. The numerical results are also verified with the results obtained analytically by the homotopy analysis method (HAM). Additionally, the results are validated with previously published pertinent literature as a limiting case of the problem. Noting that inertial and viscous forces are of the same order of magnitude within the boundary layer, taking Defining the dimensionless variables and parameters asber, Eqs –7), in , in u*=uEqs –11) in  in 11) iz > 0. The fluid is set into motion by an elastic flat sheet in the plane z = 0, kept at a constant temperature, which is being continuously stretched with linear velocities cx and dy in the x- and y- directions, respectively. The constants c and d are positive real numbers relating to stretching of the sheet. The ambient temperature far away from the sheet is uniform and taken as T∞. The continuity, momentum and energy equations governing the steady three-dimensional flow of Sisko fluid, approximated by boundary-layer theory areConsidering the three-dimensional steady, laminar and incompressible flow of fluid obeying the Sisko model which occupies space u, v, w) are unknown velocity components x, y and z directions, T the temperature, ρ the fluid density, cp the specific heat of fluid at constant pressure and κ the thermal conductivity.Here (Eqs –19) are are19) aThe governing coupled partial differential Eqs –19) are are19) aη and α is the stretching ratio parameter. We are considering the range 0 ≤ α ≤ 1, since for α > 1, the x and y axes are interchanged [a, Reb are the local Reynolds number, A the material parameter of Sisko fluid and Pr the generalized Prandtl number, which are defined asThe momentum and heat transfer equations with the associated boundary conditions reduce toα → 0 the unidirectional case is obtained and motion of fluid is merely in xz plane, i.e. g and g′ in Eq . Thus the dimensionless skin friction coefficients along the x- and y- directions, respectively, are given byτxz and τyz are shear stresses along the x- and y- directions, respectively. These quantities in dimensionless form can be expressed asThe skin coefficient is an important boundary layer characteristic, which is the dimensionless shear stress at the wall -(27). Consequently, these equations are solved numerically by the shooting technique. The equations are written as a system of eight first order ordinary differential equations. The corresponding initial value problem is solved by adaptive Runge-Kutta method. The Newton’s iterative algorithm is used to assure quadratic convergence of iteration required to satisfy the boundary condition at infinity.Analytical results are sought using the homotopy analysis method (HAM) for certain values of parameters with a view to check the veracity of our numerical results. To apply the HAM, we selected the initial guesses agreeing the boundary data asf, ℏg and ℏθ assure the convergence of series solution. The optimal values of these parameters are chosen by minimizing the discrete squared residual error [The proper values of convergence control parameters ℏal error .The validation of results is essential to check the credibility of the numerical solution methodology. The presently computed results are compared with the analytical results obtained by HAM, and an excellent correspondence is seen to exist between the two sets of data as shown in n and results are presented graphically. The effects of various parameters like the power-law index n, material parameter A and stretching ratio parameter α are investigated for non-dimensional velocity components f′(η), g′(η) and temperature θ(η). Variation of the local skin friction coefficients and local Nusselt number are also observed for different values of n and α.This article predominantly focuses the flow and heat transfer characteristics of Sisko fluid past a uniformly heated and bidirectional stretching surface. To grasp both the phenomena, Eqs –25) wit wit25) wα on the velocity components f′(η) and g′(η) for Sisko fluid with shear thinning (n = 0.5) and shear thickening (n = 1.5) properties. It is evident from f′(η) decreases with increasing values of α, however, opposite behavior is observed for the g′(η) component of the velocity  component more pronouncedly. velocity . This beA affects the velocity profiles f′(η) and g′(η) for Sisko fluid with shear thinning and shear thickening properties. A. The rest of the figures show the same qualitative behavior. Again, α on dimensionless temperature θ(η). From these figures, decrease in dimensionless temperature θ(η) is noticed for each increment in value of α. The reduction in temperature can be attributed to the increased entrainment process. As the value of α is incremented, the entrainment of ambient fluid increases toward the isothermal stretching surface, eventually the local fluid temperature decreases. The effect is more noticeable for shear thinning fluid (n = 1.5) fluid.ng fluid . Furtherα, the Prandtl number Pr and the material parameter A of the Sisko fluid. θ(η) for fixed values of the power-law index n and the stretching ratio parameter α. These figures reveal that the temperature profile decreases when Pr is increased. A larger Prandtl number fluid has lesser thermal diffusivity, hence it allows diminished thermal effects to penetrate deeper into the fluid. Further, α.The following figures of temperature profiles are plotted and discussed for selected values of stretching ratio parameter A of Sisko fluid on non-dimensional temperature profile, we have plotted A for the shear thinning and shear thickening fluids. Again, it is noticed that the thermal boundary layer thickness is markedly reduced for shear-thickening (n = 1.5) fluid. These figures also provide a comparison between the profiles of the power-law fluid (A = 0) with those of the Sisko fluid (A ≠ 0). From these figures, it is clear that the temperature profiles for the power law fluid is higher when compared with those of Sisko fluid.To exhibit the effects of the material parameter α and total entrainment rate, which is proportional to f(∞) + g(∞). This figure depicts that the quantity f(∞) + g(∞) tends to increase monotonically, when α is varied from 0 to 1, both for the shear thinning and shear thickening Sisko fluids. The ambient flow rate entrained perpendicular to the stretching sheet is about 43% for shear thinning Sisko fluids, with axis symmetric stretching (α = 1) than with unidirectional stretching (α = 0). This quantity, however, is 63% higher for shear thickening Sisko fluids.x and y directions are given in α. Further, it is apparent that the increase is more rapid for the power-law index n > 1. θ′(η) with the stretching ratio parameter α. It is observed that the value of −θ′(η) increases with each increment of α. This table depicts that the wall temperature gradient is higher for fluid with shear thickening properties and hence results in a higher heat transfer coefficient.The magnitude of the numerical values of the skin friction coefficients in the f′(η) and g′(η) for increasing value of the stretching ratio parameter.A qualitatively opposite trend was observed in the velocity components A substantial reduction in the momentum and thermal boundary layer thickness was noticed for shear thickening fluid.Thinning of the thermal boundary layer was seen for increasing Prandtl number and stretching ratio parameter and hence resulted in better heat transfer at the wall.The skin friction drag and wall temperature gradient were increased with the stretching ratio parameter.The entrainment of ambient fluid, towards the heated plate was much larger for shear thinning Sisko fluids as compared to that of shear thickening Sisko fluids.The steady three-dimensional flow and heat transfer characteristics within boundary layer of Sisko fluid past an isothermal bidirectional stretching surface have been studied numerically. The effects of the stretching ratio parameter, the material parameter and the Prandtl number on the velocity and temperature profiles were studied. Our computations have indicated that:


There is a lack of data related to activity uptake and particle track distribution in targeted alpha therapy. These data are required to estimate the absorbed dose on a cellular level as alpha particles have a limited range and traverse only a few cells. Tracking of individual alpha particles is possible using the Timepix semiconductor radiation detector. We investigated the feasibility of imaging alpha particle emissions in tumour sections from mice treated with Thorium-227 (using APOMAB), with and without prior chemotherapy and Timepix detector. Additionally, the sensitivity of the Timepix detector to monitor variations in tumour uptake based on the necrotic tissue volume was also studied. Compartmental analysis model was used, based on the obtained imaging data, to assess the Th-227 uptake. Results show that alpha particle, photon, electron, and muon tracks were detected and resolved by Timepix detector. The current study demonstrated that individual alpha particle emissions, resulting from targeted alpha therapy, can be visualised and quantified using Timepix detector. Furthermore, the variations in the uptake based on the tumour necrotic volume have been observed with four times higher uptake for tumours pretreated with chemotherapy than for those without chemotherapy. The relationship between the characteristics of a particular radiation type and its impact on irradiated biological tissues and cells plays an important role in estimating the efficacy and applicability of targeted radiotherapies . In a spα-camera”) combines autoradiography with a scintillation detector and optical registration using a charge-coupled device. In the study of Bäck and Jacobsson, the distribution of At-211 labelled antibody and antibody fragments in human tumour, mouse kidney, and whole-body sections were examined [α-camera had a resolution of 35 ± 11 µm, and the quantitative analysis proved that the pixel intensity has a linear relationship with the activity of the imaged tissue. The results demonstrated the ability of the α-camera to be employed in small-scale dosimetry for TAT as it was able to provide quantitative data on a microscopic scale [α-camera was also used to examine the accumulation of an At-211 radioimmunoconjugate administered to mice with ovarian cancer micrometastases. The activity level and the number of tumour cell clusters were determined by imaging one section with the α-camera and by staining a consecutive section with hematoxylin and eosin [α-camera.To date, there is a lack of methods that can quantify alpha particle emissions in biological systems. One of the few devices presently available  technology for constructing integrated circuits. Timepix consists of a silicon semiconductor layer, divided into an array of pixels, which is bumped-bonded to an electronics integrated layer . Each piβ-particles froma C-14 sample [μm thick Mylar, and being read by the detector [β particle with the detector. The result showed that Timepix was highly sensitive with a minimum detectable activity of 0.0077 Bq and with a spatial resolution of 76.9 μm at full width at half maximum (FWHM) [Timepix is a sophisticated microdosimeter that can be used for a wide range of experiments with photons, electrons, and other charged particles and has applications in fields such as space physics, nuclear physics, radiotherapy physics, imaging, and radiation protection. One of its main advantages is that it can measure energy deposition directly and in real time . For exa4 sample , 13. C-1detector . The ima ex vivo in mouse tumour sections. As mentioned above, the α-emitting radioimmunoconjugate binds to its cancer-specific antigen. The emitted α particles deposit their kinetic energy inside a target cell, as well as in surrounding cells, potentially resulting in cell death. In the current work, the DAB4 murine monoclonal antibody (trademarked as APOMAB), which binds to necrotic tumour cells [In this paper, we present the first results for using Timepix to visualise TATur cells , 15, wasur cells . In ordeμm [β-particles and a low percentage of X- and gamma rays prior to reaching a stable isotope (Lead-207). The Th-227 decay chain and the mean energies of major particles produced in this decay chain are presented in The radionuclide Thorium-227 is an alpha emitter produced from actinium-227 with a half-life of 18.7 days, making it, attractive for use in therapeutic applications , 18. Alpμm . Along wThe La-specific murine monoclonal antibody DAB4 (APOMAB)  in conju227Th was purified as previously described [escribed  immediat6 LL2 cells (this cell line is derived from transplantable murine Lewis lung carcinoma). Tumour size was measured using electronic callipers, and tumour volume was determined using the following calculation: tumour  volume = (a2 × b)/2, where a is the shortest diameter and b is the longest diameter of the tumour. Treatment commenced when tumours reached 45–60 mm3. To generate more necrotic tumour cells for Th-227-DAB4 binding, some mice also received chemotherapy prior to injection with Th-227-DAB4. These were treated intravenously with 50 mg/kg gemcitabine  on days 1 and 2 and 2.5 mg/kg cisplatin (Hospira) on day 1. 18 kBq of 227Th-DAB4 was administered on day 3. Mice were euthanised 2 days after administration of Th-227 labelled antibody via cervical dislocation, and tumours were collected and fixed in 10% neutral-buffered formalin. Tumours were paraffin embedded, and 5 µm sections were cut for imaging.All animal experiments were approved by the SA Pathology Animal Ethics Committee, Adelaide, and conducted following institutional ethical guidelines. Six- to eight-week-old female C57Bl/6 mice were injected subcutaneously in the right flank with 102 in size containing 256 × 256 pixels, with each pixel having an area of 55 × 55 µm2 and 300 µm depth.The Timepix radiation detector, used in the current work, was purchased from Amsterdam Scientific Instruments (ASI), the Netherlands. The device consists of a silicon chip, 1.408 × 1.408 cmThe chip can collect positive or negative charges , and theThe ASI Timepix detector is combined with the beam data acquisition software, SoPhy (Software for Physics), developed by the provider. The SoPhy software allows one to control Timepix acquisition modes such as selection of the operation mode, adjustment of suitable energy thresholds, cluster sizes, and other acquisition settings using multiwindows. The recorded data and frames/images can be exported to and processed using other programs such as Matlab or ImageJ.In the current work, before any measurements and following manufacturer recommended procedure, pixel equalisation was performed to ensure a uniform response of all pixels. Subsequently, tumour sections mounted on glass microscope slides were placed 2 cm away from the detector with the front face of the detector uncovered to allow the emitted alpha particles to penetrate the Timepix silicon chip . A simplB), tumour volume (T), and a free volume (E), where the radioactive source will escape from the blood. It is assumed that, after injection, the Th-227-DAB4 will distribute throughout all volumes. The blood and the escape volumes are open volumes where the radiolabelled immunoconjugate can escape from. The tumour volume, however, is a closed volume where the taken-up Th-227-DAB4 concentration will remain steady as shown in The compartmental model  was usedCBo, is equal to the administered activity, Ao; that is, all of the injected activity has been taken up by blood. Subsequent changes of the Th-227-DAB4 concentration in the blood volume, dCB/dt, are due to isotope uptake by the tumour and due to excretion/depletion through escape volume. This can be expressed as [kT and kE are the so-called transfer (uptake) coefficients for tumour and for the escape volumes.For simplicity, it is assumed in the current work that, at time zero, the initial activity concentration of the isotope in blood, dCT/dt, are due to Th-227 uptake from the blood volume and due to its depletion through radioactive decay of Th-227. This can be then written asλ is the decay probability constant for Th-227.Variations in Th-227-DAB4 concentration in tumour volume, kT, is much higher than escape volume transfer coefficient, kE, the previous equation becomest can be expressed ast can be expressed asλ is much smaller compared to the transfer coefficient kT, the above ratio can be reduced totmax), the amount of radioisotope in the tumour volume, CT, will only vary as a result of radioactive decay:CT,chemo and CT,nochemo, during measurements. As a result, from the number of recorded alpha hits measured at different time intervals, the transfer coefficients for the two scenarios could be determined.Assuming, for simplicity, that the tumour volume transfer coefficient, 227Th-DAB4 alone ((a), (b)) and two tumours from mice treated with chemotherapy followed by 227Th-DAB4 ((c), (d)). Even though the collimator was positioned around the tumour section, the collimator was not touching the detector, leaving a small (~2 mm) air gap. As a result, particles emitted at smaller angles, compared to a trajectory perpendicular to the detector, can still reach the detector. These are observed as hits outside the red circle in High LET alpha particles release their kinetic energy in more than one pixel as they pass in the semiconductor detector, forming a charge cluster. The small dots recorded in the image represent X- and gamma rays. An electron passage will result in a wavy and short track, and a muon track will be detected as a straight, long line .An automated method using ImageJ was used to determine the number of alpha particle hits. These were also counted manually for comparison. The differences between the two methods varied between 4 and 20 hits. Using the automated method, the number of hits varied between 93 and 251, for the sections that did not receive chemotherapy, and between 445 and 582, for the sections that were treated with chemotherapy before administering Th-227-DAB4.2·hour for tumour sections of mice not previously given chemotherapy and between 94.0 and 206.8 hits/cm2·hour for sections that were treated with chemotherapy followed by 227Th-DAB4.As shown in  P value of 0.026 , confirming that the observed differences between the two sample groups are significant.Corrected alpha particle acquisition rates were statistically analysed using one-way ANOVA for their significance, using GraphPad Prism software, yieldingEnergy spectra of alpha particles detected from decay of Th-227 and its daughters were also acquired and an example is presented in Using the compartmental analysis, corresponding transfer coefficients for the two samples can be determined when repeating the measurements for at least one sample type at different times. The data can be plotted using  or 5) a a5) and β particles, electrons, and X-ray tracks. The Timepix measurements also showed an increased uptake of Th-227–DAB4 following chemotherapy due to an increase in necrotic tissue volume. The experiment showed that the Timepix detector can be used effectively for autoradiography in TAT to provide high-resolution images. Development of a fine collimator can improve the definition of the tumour boundaries and identify the geometrical origin of detected alpha particles within the sample. It was also shown that, in principle, the data acquired by Timepix can be used for compartmental analysis to quantify the uptake of radioimmunoconjugate in targeted alpha therapy.Tumour sections were imaged in the current work to characterise the pattern of uptake and distribution of Th-227-DAB4. High resolution autoradiographs of radiolabelled tumour sections were acquired, showing alpha particles,


Photodynamic therapy (PDT) combines visible light and photosensitizing dyes. Different animal models have been used to test PDT for cancer, infectious disease and cardiovascular disease. Mouse models of tumours include subcutaneous, orthotopic, syngeneic, xenograft, autochthonous and genetically modified. Photodynamic therapy (PDT) employs non-toxic dyes called photosensitizers (PSs), which absorb visible light to give the excited singlet state, followed by the long-lived triplet state that can undergo photochemistry. In the presence of ambient oxygen, reactive oxygen species (ROS), such as singlet oxygen and hydroxyl radicals are formed that are able to kill cancer cells, inactivate microbial pathogens and destroy unwanted tissue. Although there are already several clinically approved PSs for various disease indications, many studies around the world are using animal models to investigate the further utility of PDT. The present review will cover the main groups of animal models that have been described in the literature. Cancer comprises the single biggest group of models including syngeneic mouse/rat tumours that can either be subcutaneous or orthotopic and allow the study of anti-tumour immune response; human tumours that need to be implanted in immunosuppressed hosts; carcinogen-induced tumours; and mice that have been genetically engineered to develop cancer (often by pathways similar to those in patients). Infections are the second biggest class of animal models and the anatomical sites include wounds, burns, oral cavity, ears, eyes, nose etc. Responsible pathogens can include Gram-positive and Gram-negative bacteria, fungi, viruses and parasites. A smaller and diverse group of miscellaneous animal models have been reported that allow PDT to be tested in ophthalmology, atherosclerosis, atrial fibrillation, dermatology and wound healing. Successful studies using animal models of PDT are blazing the trail for tomorrow's clinical approvals. Photodynamic therapy (PDT) employs non-toxic dyes called photosensitizers (PSs), which absorb visible light of the correct wavelength to first give the excited singlet state, followed by a transition to the long-lived excited triplet state that can undergo photochemistry . In the in vivo mechanisms, namely direct PDT cytotoxicity to tumour cells, destruction of the tumour microvasculature and induction of an acute local inflammatory response leading to activation of the host immune system , followed by a waiting period known as the ‘drug light interval’ to allow the tumour to accumulate at the site of the tumour. Light can be delivered to the tumour site either by shining a focused spot of light on to the tissue  or by inserting fibre optics into the tumour tissue in a technique called interstitial light delivery. The anti-tumour effects of PDT result from the combination of three different e system .Many different types of photoactivable molecules have been synthesized and tested as possible PDT agents. These PS are often based on the tetrapyrrole backbone such as porphyrins, chlorins, bacteriochlorins and phthalocyanines . The latin vitro and, more recently, in 3D tissue culture models [Since the first pioneering studies of PDT to cure tumours in the 1970s by Diamond et al.  and by De models ,6, more in vitro cell culture and laboratory animals is the chorioallantoic membrane (CAM) of fertilized chicken eggs that have had a window of eggshell removed. This model allows the growth of tumour cells that are applied as a suspension on to the surface of the membrane and turn into ‘tumours’ that go on to develop their own blood supply by the process of angiogenesis . PS can be injected into these blood vessels, allowed to accumulate in the tumours and then light can easily be delivered and changes in blood flow in the tumour and normal vessels can be observed in real time. PS can also be topically applied to the xenografted tumours on the CAM. The advantages of this model include the ease of in vivo microscopy to study PDT-induced vascular damage and the lack of regulatory controls on experiments involving eggs [One very simple intermediate model that lies in between ing eggs –11.Mus musculus) have particular combinations of major histocompatibility complexes  and the tumour cells should have the same combination of MHC molecules as the host mouse to allow them to grow without instant rejection. This syngeneic mouse tumour approach is often used because the mice have intact immune systems and, therefore, immunology and anti-tumour immunity effects after PDT can be studied. There are many inbred mouse strains available (over 400), but the most commonly used examples in anti-cancer PDT studies are BALB/c , C57BL/6 (black), C3H (brown) and DBA2 (grey). Tumour types include adenocarcinoma lines , squamous cell carcinoma, fibrosarcoma, lymphoma, melanoma, malignant glioma and many others. Rat tumour cell lines are less common than murine tumour lines, but there are several known belonging to inbred laboratory rat strains (Rattus norwegicus) such as Wistar, Lewis, Sprague–Dawley, Fischer 344 etc. Subcutaneous tumours differ in their rate of growth, degree of vascularization, amount and kind of tumour stroma and potential to form spontaneous metastasis. In addition to injecting the tumour cells beneath the skin, they can also be injected between the layers of the epidermis and dermis to form intradermal tumours. It has been shown that intradermal tumours are more easily recognized by the immune system compared with subcutaneous tumours [Using subcutaneous tumours in laboratory mice is very common among investigators aiming to test various PDT regimens . There a tumours . In some tumours . Many inPDT of subcutaneous mouse tumours is usually carried out by IV injection of the PS (often in the tail vein) followed after a certain period of time by light delivery to the tumour including a certain amount of surrounding normal tissue. This time period, called the ‘drug light interval’, is an important parameter that governs the amount of vascular damage that occurs during the treatment and therefore whether the PDT regimen is ‘vascular’ or ‘cellular’. Less common is the injection of the PS into the abdominal cavity  mice that are T- and B-cell-deficient, allows the engraftment of human tumour cells known as ‘xenografts’ (or sometimes xenographs). Due to the increased susceptibility of these mice to infections, the cell lines must be free of mouse pathogens and the mice must be kept in specific pathogen-free conditions. Even so, innate immunity, particularly natural killer (NK) cells and tumouricidal macrophages, can limit tumour growth and prevent metastasis in nude mice . A largeThere has been criticism levelled at the widespread use of subcutaneous tumours in PDT research, to the effect that they fail to replicate the normal biology of human tumours by not being situated in their organ of origin (orthotopic) and are therefore not subject to the appropriate environmental cues and signalling from the host tissue. The blood supply that develops to supply the tumour is very different in orthotopic models and the propensity to spontaneously metastasize is also higher. For instance brain tumours behave very differently when implanted in the brain, than when grown subcutaneously. Investigators have therefore injected the tumour cells into the tissue of the organ of origin in the mouse or rat. This procedure may need a surgical approach that depends on the actual organ to be implanted with the tumour. This has been done with colon carcinomas , renal cell cancers (into the kidney), mammary carcinomas (into the mammary fat pad), bladder carcinomas , prostate carcinoma (into the prostate), pancreatic carcinoma (into the pancreas) and lung cancer (into the bronchi). Moreover, the actual PDT treatment approach necessitates being able to get the light to the tumour, which is more difficult for orthotopic tumours.Orthotopic tumours used to test PDT can either be syngeneic in mice or rats or can be xenografts . There iTumours that arise naturally in the host are called ‘autochthonous’. In the laboratory studies, these tumours are usually induced by application of chemical carcinogens, but viruses and physical carcinogenic stimuli (for instance UV radiation) have also been used. These models effectively recapitulate the time-dependent and multi-stage progression of cancer formation in response to relevant environmental carcinogens and tumour-promoting agents . These mGenetically-engineered mouse models (GEMMs) of cancer have been constructed to more closely mimic the development of human disease in a predictable manner . Since tMany GEMM models of cancer have been developed including lung, colon, stomach, oesophagus, pancreas, liver, breast, ovary, prostate, bladder, kidney, brain and skin . In someTable 5.in vivo imaging modalities are based on adaptation of those clinical modalities that are familiar to oncologists to be used in small animals. This classification includes such modalities as micro-positron emitting tomography (microPET) [in vivo imaging modalities relies upon genetically-encoded reporter molecules and falls into the category of molecular imaging. The two most often used modalities in this class are (1) in vivo fluorescence imaging relying on engineered fluorescent protein reporters such as GFP; and (2) in vivo bioluminescent imaging relying on engineered luciferase reporters such as the firefly or marine luciferase enzymes.In recent years, there has been an explosion of non-invasive imaging techniques applied to laboratory studies of cancer and anti-cancer therapy. Many of these icroPET) , micro-sicroPET) , micro-cicroPET) , high reicroPET) , photoacicroPET) . Howeverin vivo bioluminescence imaging to non-invasively monitor the progression of the infection. Sensitive cameras can record non-invasively, in real time and with longitudinal monitoring, the anatomical location and growth of infectious microorganisms in living hosts [Escherichia coli infection mediated by pL-ce6 conjugate and red light, in excisional wounds on the back of BALB/c mice [After cancer, infections represent the next most frequent application of PDT. The use of PDT against infections is motivated by the widespread and growing problem of antibiotic resistance, often called ‘the single biggest problem facing global health’ . A very ng hosts . Figure B/c mice .Galleria mellonella, the greater wax moth. The pathogens are injected into the larva, followed by injection of the PS and then by light delivery. This has been reported for Enterococcus faecium [Candida albicans [An interesting alternative animal model to study the pathogenesis and therapy of infections is the use of invertebrate hosts . The inv faecium  and for albicans  both of There has been a diverse range of diseases other than cancer and infections that have been treated with PDT in animal models. Some of these are listed in It is almost a universally accepted axiom in cancer research that enormous numbers of mice and rats have been cured of cancer by a wide range of experimental therapeutics, but the translation into clinical practice has been disappointing more often than not. Nevertheless, the importance of designing and selecting the most appropriate animal models to investigate the ever-increasing range of new experimental therapeutic approaches has never been greater. This consideration applies even more to the rather complex techniques of PDT and aPDI , than it does to the more traditional testing of new pharmaceutical or biologic therapies. The multi-target nature of anti-cancer PDT  means that thought must be given to understanding the specific tumour biology of the model chosen. Moreover, since the light delivery is by definition a localized process, the anatomical location of orthotopic tumours becomes important.When the testing of PDT in animal models of infectious disease is undertaken, some other points need to be considered. Firstly it is important to realize that human infections generally develop gradually from a relatively small initial infectious inoculum, rather than from a sudden large application of millions or tens of millions of CFU. Secondly, a high level of selectivity for microbial cells over host mammalian cells is needed, since the actual fraction by weight of microbial cells in even a serious infection is still very low. Thirdly, when the light is switched off, the generation of microbicidal ROS is halted and the microbial cells may be free to resume their growth unhindered.The popularity of testing PDT for non-traditional indications involves designing animal models of non-cancer, non-infectious disease. A diverse assortment of conditions in cardiology, atherosclerosis, ophthalmology, neuroscience could in principle be treated with PDT.Finally, for those laboratories that are not easily able to carry out studies on traditional mammalian animal models, there are new models described using invertebrate hosts and fertilized eggs. Large animal models have not been much studied in the PDT field, but as more new indications are being studied, studies on large animals can be expected to become more important.


The article is missing grant details in the Funding section. Please see the correct Funding Statement here:This work was funded by the Swiss National Science Foundation (grant 310030–130699) to AT, AC and NB, by a Grand Challenges Exploration award from the Bill and Melinda Gates Foundation, and by the European Union's Seventh Framework Programme FP7/2007–2013/ under grant agreement n°305762. HFG is supported by SNF grant 127317. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Hyponatremia is the most common electrolyte abnormality. Its diagnostic and therapeutic approaches are in a state of flux. It is evident that hyponatremic patients are symptomatic with a potential for serious consequences at sodium levels that were once considered trivial. The recommendation to treat virtually all hyponatremics exposes the need to resolve the diagnostic and therapeutic dilemma of deciding whether to water restrict a patient with the syndrome of inappropriate antidiuretic hormone secretion (SIADH) or administer salt and water to a renal salt waster. In this review, we briefly discuss the pathophysiology of SIADH and renal salt wasting (RSW), and the difficulty in differentiating SIADH from RSW, and review the origin of the perceived rarity of RSW, as well as the value of determining fractional excretion of urate (FEurate) in differentiating both syndromes, the high prevalence of RSW which highlights the inadequacy of the volume approach to hyponatremia, the importance of changing cerebral salt wasting to RSW, and the proposal to eliminate reset osmostat as a subtype of SIADH, and finally propose a new algorithm to replace the outmoded volume approach by highlighting FEurate. This algorithm eliminates the need to assess the volume status with less reliance on determining urine sodium concentration, plasma renin, aldosterone and atrial/brain natriuretic peptide or the BUN to creatinine ratio. We intend to briefly discuss the pathophysiology of RSW and SIADH, current methods of differentiating SIADH from RSW, the failure of the volume approach to address hyponatremia which has resulted in misconceptions and mismanagement of many hyponatremic patients, present data to support our proposal to change CSW to RSW, and removing reset osmostat (RO) as a subtype of SIADH, and present an algorithm which eliminates the need to assess volume, determine urine sodium concentration (UNa), plasma renin, aldosterone or atrial/brain natriuretic peptide (A/BNP).Hyponatremia, defined as serum sodium <135 mEq/L, is the most common electrolyte abnormality encountered worldwide and is an independent risk factor for higher morbidity and mortality rates . SymptomThe initiation of RSW starts with the stimulation of a natriuretic factor that reduces sodium transport to induce RSW and extracellular volume (ECV) depletion, which in turn stimulates secretion of antidiuretic hormone (ADH), renin and aldosterone and decreases atrial/brain natriuretic peptide (A/BNP). The volume stimulus for ADH secretion is common to any state where there is ineffective circulatory volume be it heart failure or true volume depletion. The volume stimulus is more potent than the osmolar stimulus so a volume depleted patient continues to secrete ADH despite becoming progressively hyponatremic as long as the patient continues to take in free water . Removalet al., who defined the consequences of administering vasopressin to normal human subjects [SIADH evolved as a clinical entity by the demonstration of a clinical correlate to the seminal work by Leaf subjects . The prosubjects . This hysubjects . Moreovesubjects . An incrsubjects ,20,21. IDifferentiating SIADH from RSW has been extremely difficult to accomplish, in part because of significant overlapping clinical findings between both syndromes. As noted in There is universal agreement that we cannot assess ECV with any degree of accuracy by usual clinical criteria, yet the approach to hyponatremia starts with an assessment of volume. The ineffectiveness of this volume approach is becoming even more evident by an objective review of the literature and recent publications of RSW occurring in patients without clinical cerebral disease ,12. SincCalculation of fractional excretion (FE) of urate determines the percent excretion of the filtered load of urate at the glomerulus. It determines the net transport of urate without distinguishing what is secreted or reabsorbed and can be readily determined by collecting blood and spot urine at the same time. FEurate in percentage excretion of the filtered load of urate can be determined by dividing the ratio of urine to plasma urate by the ratio of urine to plasma creatinine and multiplying by 100. It can also be derived by dividing the product of UNa (×) serum urate by the product of serum sodium (×) urine creatinine and multiplying by 100. FEurate, normal 4%–11%, has been consistently increased to >11% in SIADH and RSW and has a unique relationship to serum sodium in SIADH and RSW. In SIADH, correction of hyponatremia will normalize FEurate to 4%–11% as compared to being persistently increased to >11% in RSW, Two unequivocal cases of RSW without clinical evidence of cerebral disease not only verified the persistent increase in FEurate with correction of hyponatremia, but served as the basis for us to make a clinically important proposal to change CSW to RSW ,12,34. OThe relationship between serum sodium and FEurate as noted in The value of determining FEurate in hyponatremic conditions has been further amplified by a normal FEurate being observed in every hyponatremic patient with RO . The norBased on a large database, we would like to introduce a new, updated algorithm which centers on the determination of FEurate outlined in This algorithm omits a reliance on plasma renin, aldosterone, A/BNP, UNa or even the BUN to creatinine ratio. Plasma renin, aldosterone and A/BNP have not been consistently reliable indices because of the effect of saline infusions that are often utilized in hyponatremic patients, the presence of chronic kidney disease and frequent use of ACE inhibitors, angiotensin receptor blockers, diuretics and beta blockers. The determination of UNa has been utilized to evaluate patients with hyponatremia, but UNa <20 and even <40 mEq/L are being observed with greater frequency so this parameter has not proven to be effective in the evaluation of hyponatremic conditions. The low UNa in our experience has been observed much more commonly in patients with RSW as compared to patients with SIADH and RO. Since UNa reflects salt intake in conditions such as SIADH, RO and RSW, the low UNa suggests that these subjects have reduced intake of salt, probably reflecting the severity of their co-morbid condition. In some cases, the reduced salt intake could be iatrogenic. This has been reported with increased morbidity when the patient with RSW was fluid restricted for an erroneous diagnosis of SIADH ,24,25,30There is mounting evidence to prove the ineffectiveness of the volume approach to hyponatremia. Surprisingly, about a third of hyponatremic patients outside of the neurosurgical intensive care unit had RSW; the majority demonstrating no clinical evidence of cerebral disease; thus providing additional support for our proposal to change CSW to RSW ,49. As dIt is extremely difficult to differentiate SIADH from RSW largely because of significant overlapping of clinical and laboratory findings and the perception that RSW is a rare clinical entity. This differentiation is extremely important because of divergent therapeutic goals of appropriately water restricting those with SIADH and increasing salt and water with RSW to avoid iatrogenic increases in morbidity and mortality. The recent recommendations to treat most or all patients with hyponatremia introduce an urgency to resolve this diagnostic and therapeutic dilemma. Changing CSW to RSW is an important modification in nomenclature that will expand our consideration of a large number of RSW patients without evidence of clinical cerebral disease and to avoid mismanagement and possibly reduce morbidity and mortality. The volume approach to hyponatremia and perception that RSW is a rare clinical entity should be abandoned in favor of a more open-minded approach that will lead to better diagnosis and treatment of hyponatremic conditions. To this end, we propose a new algorithm that utilizes FEurate as a pivotal determination, . Determi


In hematopoietic cells, serglycin proteoglycans mainly contribute to proper storage and secretion of inflammatory mediators via their negatively charged glycosaminoglycans. Serglycin proteoglycans are also expressed in cancer cells where increased expression has been linked to poor prognosis. However, the serglycin-dependent mediators promoting cancer progression remain to be determined. In the present study we report that genetic ablation of serglycin proteoglycan completely blocks lung metastasis in the MMTV-PyMT-driven mouse breast cancer model, while serglycin-deficiency did not affect primary tumour growth or number of mammary tumours. Although E-cadherin expression was higher in the serglycin-deficient primary tumour tissue, indicating reduced invasiveness, serglycin-deficient tumour cells were still detected in the circulation. These data suggest that serglycin proteoglycans play a role in extravasation as well as colonization and growth of metastatic cells. A microarray expression analysis and functional annotation of differentially expressed genes identified several biological pathways where serglycin may be important. Our results suggest that serglycin and serglycin-dependent mediators are potential drug targets to prevent metastatic disease/dissemination of cancer. We hcompetent, 11, 12.een found–17. Sergeen found. On the een found–21. The een found. To date-/- C57BL/6 mice (N15)[+/- F1 males were selected and crossed with 2 females each of SG-/- C57BL/6 (N15) to produce the littermates for the experimental breast cancer groups, that is PyMT+ SG+/- and SG-/- F2 female mice (now 25% FVB/n). The Neomycin resistance gene, i.e. the knockout cassette replacing exon 1, is still integrated in the genome of the serglycin-deficient mice, why we decided to breed for and analyse heterozygote SG+/- mice, and not wild type SG+/+ mice, in comparison with the SG-/- mice. All mice were scored blindly, i.e. the genotype was not known to the assessor until determined at endpoint, and the animals were euthanized when primary tumour burden reached 2 cm3 or when general health status was affected, i.e. body condition scoring[2 Macrolon IIL cages) with aspen shavings for bedding, paper for nesting and a small house of paper. A 12 hour light cycle was used and water and rodent chow were ad libitum.Congenic male FVB/n MMTV-PyMT+ transgenic mice were cron scoring or when Primary antibodies: PyMT-antibody , anti-CD31 , anti-Ki67 , anti-cleaved caspase-3 , anti-F4/80 Alexa Fluor 488 conjugate , anti-human fibrinogen , E-cadherin , CCL2/MCP-1 , anti-HGF , anti-beta actin . Secondary antibodies: goat anti-mouse Alexa Fluor 488 , goat anti-rabbit Alexa Fluor 594 , goat anti-rat Alexa Fluor 680 , goat anti-rat Alexa Fluor 568 , donkey anti-rabbit Alexa Fluor 555 , anti rabbit IR-Dye 800CW, anti goat IR-Dye 680CW, anti-mouse IRDye 800CW, anti-mouse IRDye 680CW® Plus microscope slides.Mice were anesthetized with avertin and then sacrificed with whole body perfusion using 10 ml PBS followed by fixation with 10 ml 2% paraformaldehyde. Some anesthetized mice also received FITC-conjugated tomato lectin (0.1 ml of 1 mg/ml in PBS)  that was administered by retro orbital injection and left to circulate for 2 minutes before perfusion. Blood was collected during the initial 10 ml PBS perfusion and mixed with Trizol for RNA preparation. After perfusion tumours, lungs and liver were excised, collected in 30% sucrose solution and then stored frozen at -80°C until further use. For histological evaluation, tissues were dehydrated at 4°C in 30% sucrose in PBS, embedded in OCT compound and cryo-sectioned at 7–10 μm for collection onto SuperfrostPrimary tumours were dissected and after removal of normal breast tissue, number of tumours and tumour weight were recorded to assess the total tumour burden. Morphological assessment and necrosis determination was done by an expert pathologist on 7 μm tumour sections stained with haematoxylin and eosin (H&E) in a blinded fashion. The tumours were in general distinctly circumscribed, but also extended outside colonizing normal mammary ducts. The tumour sections displayed variable amounts and different types of necrosis. Occasional large areas of dead tumour cells could be seen in few areas while in other parts the necrotic lesions were more of the comedo type that were localized in the central parts surrounded by vital cells. Between tumours a lucent stroma containing scanty inflammatory cells could be noticed. The grade of differentiation was usually moderate with a low-grade atypia among individual cells.H&E staining was done to quantify metastasis in the lungs and necrosis in the primary tumours. Chloroacetate esterase staining was used to quantify mast cells in the primary tumour. Immunofluorescence staining was performed to visualize and quantify PyMT positive cells  in lungs and primary tumours, as well as to assess proliferation , apoptosis , macrophage infiltration , endothelial cells , and plasma leakage  in the primary tumours. For immunostaining, sections were fixed with ice-cold methanol, washed and then blocked for 1 h at room temperature with 3% BSA in PBS followed by overnight incubation at 4°C with primary antibody diluted in blocking buffer. Hoechst (diluted 1:5000 in PBS) was used to visualize nuclei. Alexa Fluor secondary antibodies at 1:1000 dilutions were used for detection (45 min) of the primary antibodies. Prolonged washing between each step was done with PBS. Immunostained sections were mounted with Fluoromount G. Negative controls were done without primary antibody. High-resolution images for each section were taken using a Nikon 90i microscope and quantification of fluorescent regions in every section were done using ImageJ software. Approximately 1–3 tumours (depending on size) per mouse were selected for staining. For quantification of stained areas (% of 20X field using ImageJ64 software), 3–8 pictures of each tumour section depending on the tumour size were randomly taken with a 20X objective. Alternatively, cells staining positive for the respective marker were counted per field (20X) and the average of 7 to 10 fields calculated. For necrosis quantification from H&E stained sections, tumour areas and the percentage of necrotic and viable areas per section were assessed. Because the tumours were variable in size, percentage of necrosis was calculated in each tumour section.® blocking buffer  and all antibody dilutions were done in the blocking buffer. Washing of the membrane in between every step was done with PBS/0.01% Tween 20. IR-Dye secondary antibodies from LI-COR Biosciences were used for detection. Membranes were scanned using the Odyssey® CLx imager (LI-COR Biosciences) scanner for visualization. Quantification of relative intensity was done using ImageJ software.Frozen tissue sections (100 mg) from the lungs and primary tumours were crushed in liquid nitrogen and suspended in RIPA buffer containing 2% Triton X-100 and proteinase inhibitors. The tissue lysate was left on ice for 15 min, centrifuged at 13,000 rpm for 20 min at 4°C, and supernatants were used for western blot analysis. Membranes were blocked with OdysseyFrozen tissue sections (50 mg) from the primary tumours were crushed in liquid nitrogen and suspended in PBS supplemented with protease inhibitors and stored in -20°C until used. Samples from 3 mice per genotype were pooled to perform the analysis of the cytokine (ARY006 from R&D systems) and angiogenesis (ARY015 from R&D systems) arrays. Analysis of the arrays was carried out according to the manufacturers instructions with a slight modification. In short, a secondary biotin labelled antibody was used to detect the primary antibody and streptavidin-Alexa was used for detection and visualization of the cytokine spots on the membranes. The filter layout and the area of the positive control spots was defined and used for all the other spots. All signal spots falling outside the filter layout were considered as artefacts. Quantification of the duplicate spots on the filters was done using ImageJ software as instructed by the manufacturers.RNA extraction from lung tissue, primary tumours, blood and liver followed the Trizol method. During the perfusion step blood was collected and mixed with Trizol. Frozen tissue sections (100 mg) were crushed in liquid nitrogen and suspended in Trizol. Ten ng of the purified total RNA was subjected to reverse transcriptase (Life Techonologies) with PyMT or GAPDH reverse primers and then PCR (KAPA biosystems) was performed using PyMT primers: forward 5’-CTGAGCCCGATGACAGCATA-3’, and reverse 5’-TCTTGGTCGCTTTCTGGATAC-3’. As baseline control of the RT-PCR mouse GAPDH primers were used: forward 5’-TGCCCCCATGTTTGTGATG-3’, and reverse 5’-TGTGGTCATGAGCCCTTCC-3’.HS- and CS-chains were isolated from tumours and surrounding tissues and characterized essentially as described by Ledin et al., 2004. In shorhttp://www.affymetrix.com) using the robust multi-array average (RMA) method first suggested by Li and Wong in 2001[http://www.r-project.org) using packages available from the Bioconductor project (www.bioconductor.org). In order to search for the differentially expressed genes between SG+/- and SG-/- groups an empirical Bayes moderated t-test was then applied, using the ‘limma’ package. To address the problem with multiple testing, the p-values were adjusted using the method of Benjamini and Hochberg[Database for Annotation, Visualization and Integrated Discovery)[RNA from tumour tissue was extracted using Trizol extraction protocol. RNA concentration was measured with ND-1000 spectrophotometer  and RNA quality evaluated with the Agilent 2100 Bioanalyzer system . 250 nanograms of total RNA from each sample were used to generate amplified and biotinylated sense-strand cDNA from the entire expressed genome according to the Ambion WT Expression Kit (P/N 4425209 Rev C 09/2009) and Affymetrix GeneChip® WT Terminal Labeling and Hybridization User Manual . GeneChip® ST Arrays (GeneChip® Mouse Gene 2.0 ST Array containing 33794 gene transcripts) were hybridized for 16 hours in a 45°C incubator, rotated at 60 rpm. According to the GeneChip® Expression Wash, Stain and Scan Manual  the arrays were then washed and stained using the Fluidics Station 450 and finally scanned using the GeneChip® Scanner 3000 7G. The raw data was normalized in the free software Expression Console provided by Affymetrix (g in 2001. Subsequiscovery). The hea+/- and SG-/- mice were assessed using the non-parametric two-tailed Mann Whitney test and differences indicated with their p values in the figure panels. Data are presented as mean ±SEM, except for Statistical analyses were performed using GraphPad Prism. Differences between SG-/- mice. Lung is the major site for metastasis in the MMTV-PyMT model[+/- and SG-/- mice, respectively. Strikingly, not a single macroscopic or microscopic metastatic nodule was detected in cryo-sectioned lungs of SG-/- mice when metastasis was quantified with H&E staining  was reached between 14 and 18 weeks in both PyMT+ SG+/- and SG-/- mice. The variations in survival curve and age at the humane end point were similar in SG+/- and SG-/- mice  lectin that binds to the lumen of perfused blood vessels. Furthermore, vascular leakage was assessed by immunostaining for extravasated fibrinogen in the tissue. This was combined with immunofluorescent staining for CD31, a pan vascular marker. No significant difference in the extent of vascularization (CD31) , perfusir tissue . Taken t+/- and SG-/- primary tumours  is a hallmark of invasive and metastatic tumour cells, 40 and r tissue , indicat control . Interes-/- mice , suggest-/- lung  or the P/- liver , suggest+/- and three SG-/- PyMT+ mice. A principal component analysis (PCA) of the data showed SG+/- and SG-/- RNA expression to be differentially clustered. The gene expression analysis identified 664 genes with a significantly altered expression  but when setting the expression level at a two-fold difference only 61 genes were differentially expressed in the SG-/- tumour tissue compared to the SG+/- tumour tissue. Of these 61 genes 51 were annotated genes (including serglycin). Importantly, the expression level of serglycin in the SG-/- tumour tissue was 4.89 ±0.1 (log2), i.e. at background levels, whereas serglycin expression in the SG+/- tumour tissue was 10.52 ±0.4 (log2). Of the 51 differentially expressed genes 13 were down-regulated and 38 were up-regulated in the SG-/- mammary tumour tissue . In contrast, although at a lower level other cadherins and protocadherins like Cdh3  and Pcdh18  were significantly down-regulated while Cdh5 , Pcdh6 , Pcdh12 , Pcdh13 , and Pcdh15  were significantly up-regulated. We also checked the transcriptional levels of cadherin-associated catenins but found no significant differences in expression between the SG+/- and SG-/- tumour tissues. However, the shift in the expression levels of all of these mediators requires further investigation at the proteome level to define the relationship to serglycin and their involvement in the metastatic process.To identify potential serglycin-regulated mediators of tumour growth and metastasis, we performed a global gene expression analysis (GSE67806) of breast tumour tissues from three SGr tissue . Interesr tissue  we next e.g. “Signal” and “Glycoprotein” (ES = 9.75); “Cell communication” and “Secreted” (ES = 5); “Chemotaxis” and “Inflammatory response” (ES = 3.74)  of the 51 genes identified several cancer related processes with a significant enrichment score (ES):  = 3.74) . These e-/-) strain[-/- mice seemed to be completely protected from pulmonary metastasis.A high level of expression of serglycin in tumour cells has been correlated to poor prognosis and an increased metastatic potential, 16. The-/- mice suggesting that metastasis-induced inflammation were not present in the SG-/- lungs. Since we could not detect any tumour cells in the SG-/- lung by immunohistochemistry or by RT-PCR our results suggests that extravasation indeed may be blocked in the serglycin-deficient mice. However, at this time it could not be excluded that simply the metastatic latency was increased in the serglycin-deficient F2 mice (75% C57BL/6 and 25% FVB/n) as tumour onset is delayed at PyMT expression in congenic C57BL/6 mice compared to congenic FVB/n mice[-/- model. Nevertheless, to counteract eventual influence of a mixed genetic background or the gene-targeting cassette, which remains inserted in the serglycin-deficient mice, we strictly used heterozygote SG+/- and homozygote SG-/- PyMT+ female littermates to study how serglycin affects the metastatic potential in the model.Although serglycin-deficient tumour cells were found in the circulation they failed to establish metastatic growth in lungs and liver at the experimental endpoint Figs  and 4. IVB/n mice. This laVB/n mice–45. ImpoVB/n mice, 46. Forin vitro, tumour cell growth in vivo and vascularization when injected subcutaneously into immunocompromised CB17 SCID mice, suggesting an essential role for serglycin in tumour growth and vascularization[+/- and SG-/- PyMT+ mice had similar numbers and weight of primary mammary tumours, expressed comparable levels of CD31 and displayed equal fibrinogen leakage, suggesting that genetic ablation of serglycin not affected the overall primary tumour growth or vessel formation. However, increased apoptosis and a trend towards increased necrosis were noted in the SG-/- mammary tumours, which may explain why the overall tumour size was not significantly increased in the SG-/- PyMT+ mice. In agreement with the present results, no correlation was found between serglycin expression and tumour numbers and size, when analysed in human hepatocellular carcinoma[Targeting of serglycin with siRNA in human myeloma cells decreased cell adhesion arization. In the carcinoma or in nacarcinoma.e.g. matrix metalloproteinases (MMPs), tumour necrosis factor (TNF)-alpha, platelet derived growth factor (PDGF), tissue type plasminogen activator, as well as several cytokines and chemokines[+/- and SG-/- mice, SG-/- mice had decreased mast cell numbers and increased E-cadherin levels in the primary tumour tissue. However the increased E-cadherin levels was not matched with increased transcription of Cdh1 in the SG-/- tumour cells suggesting that serglycin may have a role in E-cadherin degradation during tumour progression. In addition, we found small but significantly changed mRNA expression levels of other cadherins indicating that serglycin may be involved in the regulation of protocadherin and cadherin levels in the growing tumour tissue.Previous studies in the serglycin proteoglycan deficient mouse strain has shown that the deletion impacts on the secretion of many tumour growth and metastasis promoting agents hemokines, 49, 50.-/- tumour cells and the complete absence of metastases in the PyMT+ SG-/- lung tissue could reflect a lower degree of tumour cell intravasation into the circulation. This is however less likely because circulating tumour cells were detected in blood also in SG-/- mice suggesting that intravasation was not dependent on serglycin. The failure of SG-/- tumour cells to metastasize could instead be due to a reduced adhesion to matrix proteins like collagen and fibronectin, or a reduced protease activity affecting vascular permeability. Furthermore, serglycin is expressed by platelets[-/- and SG+/- primary tumour cells in SG -competent and -deficient mice, respectively, will address if serglycin expression is required in the tumour cells or in the normal cell compartment to support metastasis. Our results point either to that the serglycin-deficient lung/liver microenvironment is unsuitable to support colonization and growth of the circulating SG-/- tumour cells or that SG-/- tumour cells cannot extravasate, and that other proteoglycans are unable to compensate for the lack of serglycin in these processes.Furthermore, the higher E-cadherin expression in SGplatelets and it hplatelets. Subcuta-/- macrophages[+/- and SG-/- mice with multiplex antibody arrays. The PDGF levels, which may modulate tumour angiogenesis[-/- tumour tissue, while MMP-3, -8, -9 and TNF-alpha protein levels did not differ between SG+/- and SG-/- tumours. However, despite changed protein levels of some angiogenic and inflammatory mediators, the tumour progression, i.e. overall vascularization and tumour growth, was intact in the SG-/- mice suggesting that expression of serglycin is not essential for primary tumour growth. The different effects of serglycin-deficiency found in the PyMT model and the RipTag2 model may thus reflect differences in the tumour growth conditions between the two models. Potentially, other proteoglycans may compensate for the serglycin-deficiency in growth and vascularization of the primary tumours[Deletion of serglycin affects strongly mast cell granulogenesis and granular retention/storage of the mast cell proteases, 52, andcrophages. To ideniogenesis, were sly tumours, 55.+/- and SG-/- tumours, indicating that heterozygotes are appropriate as controls. Interestingly, a metastatic gene signature involving around 17 genes has been described to arise already in primary spontaneous tumour cells[-/- and SG+/- primary tumours (GSE67806), the global transcription analysis identified 664 differentially expressed genes  of which 61 were expressed at a two-fold difference. The top 51 genes, including serglycin, were used for the downstream gene sets enrichment analysis (GSEA) on the DAVID platform  and the C-5 epimerases Dse and Dsel, or most MMPs were found when we selectively compared their expression level in SG+/- and SG-/- mammary tumours. However, although at lower transcriptional levels, MMP3 and 19 were significantly upregulated whereas MMP13 were significantly downregulated in the SG-/- primary tumour tissue. The increased expression of MMP3 was found to cause a slight increase at the protein level in the SG-/- tumour tissue  showed that the expression levels of genes upstream  or downstream  of serglycin did not differ significantly between SGour cells–58. Whilplatform . The GSE7806 showr tissue . The Aff tumours , but no  tumours  Figs sugr tissue . Thus, tIn summary this is the first report on the role of serglycin in tumour progression using an immune competent spontaneous and orthotopic mammary tumour mouse model that develops lung metastases. Our results show that serglycin is essential for the metastatic capacity of mammary tumour cells. Although we found tumour cells in the circulation, no tumour cells were detected in lungs of the serglycin-deficient PyMT+ mice, suggesting that extravasation is blocked. Hence, the role of serglycin in colonisation and metastatic growth cannot be assessed in the current model. Our finding warrants further investigations on the role of serglycin in metastatic cancer growth, with the aim to characterize the serglycin-dependent metastatic pathways and to identify novel drug targets.S1 Fig+/- mice (upper photos) and the absence of metastases in PyMT+ SG-/- mice (lower photos). Nucleus was stained with DAPI, blue. Note that the staining produces some background, i.e. in bronchiole.Representative photographs showing metastatic growth in the lung tissue of PyMT+ SG(TIF)Click here for additional data file.S2 Figa) To assess tumour vasculature numbers, tumour sections were immunofluorescently stained against CD31  and fibrinogen  and merged with DAPI stain . (b) The CD31 positive staining was quantified as percent of total area  . (c) Quantification of plasma leakage presented as ratio of fibrinogen (red) positive staining/CD31 (green) positive staining . (d) Quantification of FITC-lectin perfused vessels in the primary tumours . p values for the statistical differences between SG+/- and SG-/- are indicated in the graphs.((TIF)Click here for additional data file.S3 Fig+/- and SG-/-) were pooled for the array. Quantification of the relative intensities was done using ImageJ. (white columns show SG+/- and black columns SG-/-)Samples from 3 mice per genotype (SG(TIF)Click here for additional data file.S4 Fig+/- and SG-/-) were pooled for the array. Quantification of the relative intensities was done using ImageJ. (white columns show SG+/- and black columns SG-/-)Samples from 3 mice per genotype (SG(TIF)Click here for additional data file.S5 Figa) and HS in (b). In (c) and (d) the total GAG and disaccharide content was compared in SG+/- (n = 3) and SG-/- (n = 3) primary tumours with comparison of CS in (c) and HS in (d).The total GAG and disaccharide content in primary tumour tissue (n = 3) compared against normal breast tissue (n = 2), showing total CS in ((TIF)Click here for additional data file.


The experimental results showed that the effects of the shape and vibrating mode of micro-cantilever on the measurement accuracies of fluid density and viscosity were analyzed in detail.Methods to calculate fluid density and viscosity using a micro-cantilever and based on the resonance principle were put forward. Their measuring mechanisms were analyzed and the theoretical equations to calculate the density and viscosity were deduced. The fluid-solid coupling simulations were completed for the micro-cantilevers with different shapes. The sensing chips with micro-cantilevers were designed based on the simulation results and fabricated using the micro electromechanical systems (MEMS) technology. Finally, the MEMS resonant sensor was packaged with the sensing chip to measure the densities and viscosities of eight different fluids under the flexural and torsional vibrating modes separately. The relative errors of the measured densities from 600 kg/m The density and viscosity of fluid are the most important parameters in the oil ,2, chemi−3) and the measurement accuracy ranged from 0.4% to 0.6%. To improve the sensitivity of microcantilever sensors, Ansari  is the real part of the hydrodynamic function evaluated at the frequency in the liquid. However, the dimensions of the resonator in [ffluid)] approaches 1 at Re >> 1:For Re >> 1, the frequency response of a rectangular cantilever in a viscous fluid is given by :(9)fflunator in  are 2.8 k1 and k2 are constants and calculated by experimental calibrations. However, both k1 and k2 are different under flexural and torsional resonant modes. Therefore, this paper used Equation (11) as working equation to measure the fluid density under different conditions such as the flexural and torsional resonant modes.Therefore, Equations (6)–(8) and (10) can be all simplified as the following Equation (11) since the parameters of cantilever itself can be all confirmed:Q is an important parameter for fluid viscosity measurement. When a resonator with random shape oscillates in a liquid, the inertial and viscous forces apply to the motion [Q is given by:ηf is the viscosity of the liquid.The resonance quality factor e motion , and Q ik3 is a constant and obtained by the experimental calibration, gfluid and gvac are half peak widths of the microcantilever in the fluid and vacuum, respectively. For a specific micro-cantilever, fvac and gvac are also constants and can be determined via calibration, so the ratio of gvac/fvac can be replaced by k4. Then Equation (13) becomes:Based on the above viewpoint, the relationship of the resonant frequency and fluid viscosity can be described as :(13)ηf=ρffw/4ηf), where f is the resonant frequency of the microcantilever) [Microcantilevers with rectangular and trapezoidal shapes were proposed. The design dimensions of the two different microcantilevers were a length of 1500 μm, width of 2500 μm, thickness of 20 μm, and the free end width of the trapezoidal micro-cantilever was 1000 μm. It is proven that a microcantilever with a larger width could help to improve the sensitivity  and qualtilever) . If The 3, and the Poisson’s ratio was 0.064. The fluid was described by the FLUID30 acoustic fluid element (the green part), and the fluid elements were coupled with solid elements. The fluid domain covered the microcantilever except for the fixed end, and the dimension of the fluid domain was increased by 5000 μm over the dimensions of the microcantilever. The mapping method was used to mesh the structure with the number of solid elements, which was 300, and the number of fluid elements which was 2000. The resonant frequency f was obtained by modal analyses in the fluid-solid coupling simulation. However, the quality factor Q has not been obtained from the current simulation because the peak width at half height cannot be calculated by the simulation results.Fluid-solid coupling simulations were carried out by the finite element method (FEM) as shown in n-pentane, as shown in The resonant modes of different micro-cantilevers were obtained by fluid-solid coupling simulation in The rectangular micro-cantilever was taken as an example to analyze the relationship of its resonant frequency and the measured density, as shown in ffluid = a (a > 0) of Equation (11) can be written as:Rn(f) is the high order infinitesimal of (f − a)n, and Rn(f) approaching zero, thus Equation (15) can be simplified as follows:In a, and the coefficients of higher order term are all close to zero. Then, Equation (16) is changed as follows:The resonant frequency of sensor in this paper are much larger than 4000 Hz, so the quadratic coefficient is very small due to the negative fourth power of −3)−1 in [In addition, it’s shown that the slopes of the fitting straight lines under the second order mode were larger than those under the first order mode. This means the variation of resonant frequency under higher order mode was larger with the same fluid density fluctuation. Thus, the sensitivity of fluid density measurement was higher under the second order mode than that under the first order mode. The density sensitivity under the second order mode in this paper was about 2 times larger than that with the value of −2.6 Hz·(kg·m−3)−1 in .Two different micro-cantilever chips with rectangular and trapezoidal structures were designed according to the simulation results as shown in The sensing chip with the rectangular microcantilever was taken as an example to demonstrate the layout in The schematic diagram and object diagram of the experimental system are shown in versus the sweeping frequency are shown in The resonant frequency and half peak width should be obtained to calculate the fluid density and viscosity using Equations (11) and (14). The Stanford S830 detected the output signals of the Wheatstone bridge when the sinusoidal AC voltage excited the metal coil from low frequency to high frequency. The signal amplitude of the Wheatstone bridge could be intensified when the input frequency of AC voltage was close to the resonant frequency of the micro-cantilever chip in the fluid. The obtained data of the amplitude n-pentane, n-hexane, n-heptane, isooctane, n-octane, 0.65 cs silicone oil, cyclohexane and methylbenzene. A specific fluids has different densities and viscosities under different temperatures. For the alkane fluids, their physical properties are the most stable from 20 °C to 25 °C. Their reference values of density, viscosity and sonic speed under standard atmospheric pressure and different temperature (293.15 K and 298.15 K) were calculated using the Reference Fluid Properties (REFPROP) software. In the field of thermophysical properties, the REFPROP software [In order to test the sensor’s performance, different fluids with various densities and viscosities should be measured by the proposed sensor. The measured fluids included software ,32 is ofFor the rectangular microcantilever chip, a frequency swept curve was obtained in 0.65 cs silicone oil under different resonant modes, as shown in Q were calculated by Q = ffluid/2gfluid using the MATLAB fitting method. The reference values of ρref and ηref for the fluid density and viscosity were obtained from the REFPROP software.It was easy to make sure that this curve had two resonant peaks in the frequency range from 1 to 100 kHz. In theory, another resonant peaks should also appear in the frequency range from 1 to 100 kHz, but the strains of the piezoresistors in the microcantilever chip were so small under higher order resonant modes, so the Wheatstone bridge’s outputs were also small and difficult to detect. The vibration modes could be estimated by the coupled fluid-solid simulation results. For example, Here only experimental results and data acquisition method of the rectangular microcantilever chip were discussed. The deviations between the experimental values and reference values of the measured densities and viscosities under different resonant modes are shown in In order to describe and evaluate the measurement results of fluid density and viscosity, the average relative deviation (ARD) and maximum relative deviation (MRD) were introduced to analyze the measurement accuracy. The ARD is the average relative errors of all corresponding measurement results of the eight fluids at two temperature points. The MRD is the maximum relative deviation of all corresponding measurement results of eight fluids as well. 3 to 866.87 kg/m3. In As shown in In In fluid parameter measurement, the main factor affecting the resonance frequency of a resonant sensor was the fluid density, and the main factor affecting the resonance amplitude was the fluid viscosity. In this paper, a fluid density measuring equation had been deduced from the resonant frequency of a microcantilever, and the fluid viscosity measuring equation had been deduced from the quality factor. In this research a simulation method was built to guide the design of a microcantilever resonant chip. An experimental system had been set up for the fabricated MEMS resonant sensor to measure the densities and viscosities of different fluids. The experimental results showed that the fluid density measurement accuracy could not be improved by using microcantilever chips of different shapes or high order resonant mode, but the fluid viscosity measurement accuracy under the torsional vibration mode was obviously better than that under the flexural vibration mode due to the higher quality factors under the former mode. Therefore, it’s necessary to investigate methods to improve the quality factor such as optimizing coil pattern and piezoresistors location or selecting a high order resonant mode.