Toxicologic evaluation of new drug candidates routinely utilizes healthy animals. In oncology, there remains a limited understanding of the effects of novel test candidates in a diseased host. For vascular modulating agents (VMAs), an increased understanding of preclinical tumour–host interaction, and its potential to exacerbate or alleviate ‘off‐target’ effects of anti‐angiogenic administration, could aid in the prediction of adverse clinical outcomes in a defined cancer patient. We have previously reported that the implantation and growth of a range of human‐ and mouse‐derived tumours leads to structural vascular and, potentially, functional signalling changes within host mouse endocrine tissues, indicating possible roles for tumour‐ and host‐derived cytokines/growth factors and the liberation of myeloid‐derived suppressor cells in this phenomenon. Here, we further demonstrate that the growth of the Calu‐6 xenograft is associated with a resistance to VMA‐induced mouse peripheral endocrine vascular rarefaction (toxicity), with potential functional impact, notably with respect to mixed tyrosine kinase inhibition. The pathogenesis of these findings indicates a potential role for both tumour‐ and host‐derived basic fibroblast growth factor (bFGF), with associated upregulation in the intra‐tumoural autotaxin‐lysophosphatic acid signalling axis. The growth of the Calu‐6 xenograft is associated with a resistance to vascular modulating agent‐induced mouse peripheral endocrine vascular rarefaction (toxicity), with potential functional impact, notably with respect to mixed tyrosine kinase inhibition. The pathogenesis of these findings indicates a potential role for basic fibroblast growth factor, with associated upregulation in the autotaxin‐lysophosphatic acid signalling axis. Both monotherapeutic and combinatory approaches have been utilized, to overcome various forms of treatment resistance and to increase drug efficacy. However, despite notable positive achievements, patient side effects, variably predicted from preclinical animal testing, have impacted the clinical utility of a number of these vascular‐targeted drugs  in this phenomenon  in the mouse.22.1To assess the repeatability of initial studies incorporating the Calu‐6 tumour, and to determine if this response was specific to M396647‐induced mixed TKI Figure , or if inu/nu genotype) mice were supplied by the Rodent Breeding Unit  and housed in negative pressure isolators with 12‐h light/dark cycles and provided with sterilized food  and water ad libitum. The mice weighed ∼25 g and were at least 8 weeks of age at study commencement. Animals were randomized into either vehicle control non‐tumour‐bearing, vehicle control tumour‐bearing, test article‐treated non‐tumour‐bearing or test article‐treated tumour‐bearing groups (n = 6 per group).All female athymic nude (Swiss nu/nu genotype) mice by subcutaneous injection of 1 × 106 cells in Matrigel TM . When tumours reached a volume of 0.1–0.7 cm3  xenografts were established on the flank of female athymic nude  was suspended in a 1% (v/v) solution of polyoxyethylene (20) sorbitan mono‐oleate in deionised water vehicle. AZ10167514 is a mixed vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI), and a structurally similar analogue to AZD2171 (Cediranib), with potent inhibition of the tyrosine kinase of VEGFR‐2, and additional inhibition of VEGFR‐3, c‐Kit and platelet‐derived growth factor receptor alpha and beta  , or 12 mg/kg/day (established maximum tolerated dose), for a period of 28 days. Mice within a corresponding oral gavage vehicle control group were administered vehicle in isolation.DC101  is a mouse‐specific monoclonal IgG, the administration of which leads to neutralization/blockage of VEGFR‐2  injection, once daily, at a dosage of 15 mg/kg/day, for a period of 28 days. Mice within a corresponding i.p. vehicle control group were administered phosphate‐buffered saline (PBS) vehicle in isolation (0.4 ml).The incorporation of the above dosing regimens intended to explore the effects of differential pharmacology, the influence of low versus high‐dose and the variability of an extended period of treatment, over and above that examined during preliminary investigations.2/O2 mixture, followed by exsanguination.Following study completion, all mice were culled by terminal narcosis with 5:1 COAll procedures were conducted in accordance with Home Office (UK) and local ethical review committee guidelines and complied with the Animals Scientific Procedures Act 1986. Animals were monitored throughout for body weight variations and clinical signs indicative of systemic toxicity/morbidity. Tumours were also measured to ensure that the mean diameter of growth did not exceed 10 mm and that necrosis‐associated skin breakdown or exudation did not arise.2.3in life phase, selected endocrine organs  were removed from all animals . Tissues were fixed, embedded and processed for CD31 immunohistochemical (IHC) staining, light microscopic analysis and 3D Fibrelength Density analysis, using the Definiens XD image analysis platform (version 2.0.4 Definiens AG), with Tissue Studio and Developer XD, as described previously  and thyroid stimulating hormone (TSH) were quantified using the Milliplex mouse pituitary/thyroid magnetic bead panel .In addition, to assess the induction of pro‐angiogenic signalling, a series of xenograft ‐1, FGF‐2, granulocyte colony stimulating factor (G‐CSF), interleukin‐8 (IL‐8), leptin, placental growth factor (PlGF) and VEGF‐A) or host ‐derived cytokines/growth factors were quantified using Milliplex MAP kits , on a Luminex 200™ System, using xPONENT software .Whole blood samples were taken from the tail vein prior to study termination. Samples were placed in a covered test tube, allowing the blood to clot at room temperature for 30 min. Clot separation was undertaken by centrifuging the samples at 10,000 rpm, for 10 min at 4°C, in a refrigerated centrifuge . The serum was then placed into clean polypropylene tubes using a Pasteur pipette, before immediate analysis, as per the manufacturer's guidelines .2.5In order to analyse distinct lineages of bone marrow‐derived cells, a combination of the differential expression of leukocyte common antigen  and transferrin receptor  was coupled with side scatter analysis and the DNA stain LDS‐751 , the latter separating immature versus mature red cells. For further analysis of endothelial precursor cells (EPCs), phycoerythrin (PE)‐conjugated CD202b  monoclonal antibodies (5 μl) were additionally incorporated into the protocol.Fresh bone marrow suspensions were prepared by obtaining femoral samples from all animals at termination, cleaning residual tissue with a scalpel and removing the diaphyseal portion. A 27‐gauge needle on a 3‐ml syringe was used to place 2 ml of PBS +50% fetal bovine serum (FBS) through the bone and into a 12 × 75‐mm tube. The expelled solution was drawn up through the bone and back into the syringe; the process repeated 5 times. The cell suspension was filtered through a 100‐μm nylon mesh filter before underlaying with 1‐ml FBS, centrifuging at 3000 rpm for 5 min at 4°C  and resuspending the pellet in 4 ml of PBS +0.5% bovine serum albumin (BSA).Fluorescein isothiocyanate (FITC)‐conjugated CD45 (5 μl) and PE‐conjugated CD71 (10 μl) monoclonal antibodies  were added to 100 μl of the bone marrow cell suspension and incubated on ice in the dark for 20 min. Cells were washed with PBS containing 0.5% BSA and re‐centrifuged at 3000 rpm for 5 min at 4°C. The resulting cell pellet was resuspended in 0.5‐ml PBS +0.5% BSA, plus 20 μl of LDS‐751 staining solution . Samples were then placed in the dark for 20 min, prior to flow cytometric analysis, incorporating negative control samples . Analysis was performed using a Becton Dickinson FACSCanto II, with FACSDiva software , as per the manufacturer's instructions.2.6To assess the relative levels of intra‐tumoural gene expression, ribonucleic acid (RNA) was isolated from tumour fragments using the RNeasy Mini Kit , according to the manufacturer's instructions. On‐column DNase digestion was performed using the RNase‐free DNase Kit . RNA concentration was measured using the NanoDrop ND1000 .For fluidigm profiling, mouse specific TaqMan Gene Expression Assays were designed and supplied by Life Technologies . Reverse transcription was performed with 50 ng of total RNA in a final volume of 20 μl, using the High Capacity Complementary Deoxyribonucleic Acid (cDNA) Reverse Transcription Kit . The following thermal profile was used in the reaction: 25°C for 10 min; 37°C for 120 min; 85°C for 5 s; and 4°C for 2 min. Pre‐amplification was performed with 1.25 μl of resulting cDNA in a final volume of 5 μl, using a pool of TaqMan assays at a final dilution of 1 in 100 and TaqMan PreAmp Master Mix . The following thermal profile was used in the reaction: 95°C for 10 min; 14 cycles of 95°C for 15 s; and 60°C for 4 min. Pre‐amplified samples were diluted 1:5 with Tris/Ethylenediaminetetraacetic acid buffer, prior to analysis on the Fluidigm BioMark System . Sample and assay preparations for 96.96 Fluidigm Dynamic Arrays (DA) were performed according to the manufacturer's instructions. Briefly, samples were mixed with DA Sample Loading Reagent  and TaqMan Gene Expression Master Mix . Assays were mixed with DA Assay Loading Reagent . The 96.96 Fluidigm Dynamic Arrays were primed and loaded on a Controller , and quantitative PCR (qPCR) was performed on a BioMark System , using the following thermal profile: 50°C for 2 min; 70°C for 30 min; 25°C for 10 min; 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s; and 60°C for 1 min. Data were analysed using the Fluidigm BioMark Real‐Time PCR Analysis software, version 3.1.3 , and gene expression values were calculated using the Delta C(T) method.2.7Fibrelength Density analysis, in terms of protein expression, the left adrenal gland of each test animal was snap frozen at necropsy and transferred to liquid nitrogen, before thawing in radioimmunoprecipitation assay buffer solution containing cOmplete™ Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail . Sonication was performed for 20 s and followed by centrifugation at 5000 rpm, for 10 min at, 4°C, in a refrigerated centrifuge . Supernatant was harvested, and the protein concentration determined using the Thermo Scientific Pierce Bicinchoninic Acid Protein Assay Kit ; 100‐μg cell extracts were resolved in 10% sodium dodecyl (lauryl) sulphate‐polyacrylamide gels. These were electrophoretically transferred onto a polyvinylidene difluoride membrane . Western blot analysis was performed for total CD31 and VEGFR‐2, using the following antibodies: CD31  and VEGFR‐2 . Anti‐glyceraldehyde 3‐phosphate dehydrogenase antibody (GAPDH); cat. no. sc‐32233, mouse monoclonal Ig  was used to verify equal protein loading and transfer. Densitometric analysis was undertaken using the LI‐COR® Image Studio Lite version 3.1 . The relative intensities of CD31 and VEGFR‐2 expression were quantified relative to the protein loading control (GAPDH).To confirm the findings as observed by 3D 2.8t‐test analysis with Welch's correction . Comparisons of any groups found to have a non‐Gaussian distribution were completed using Mann–Whitney non‐parametric analysis. The significance level for all tests was 0.05.For statistical analysis, data were examined for normal distribution using the Shapiro–Wilk test. All comparisons containing normally distributed data groups were subject to unpaired 3Tumour growth was well‐tolerated in all animals over the duration of study, with no premature decedent animals, nor clinical signs indicative of systemic toxicity/morbidity in any group. No abnormal macroscopic findings were present within any harvested endocrine tissues, there were no tumour‐ or treatment‐related group variations in body weight gain/adrenal weight, and the light microscopic appearances of the selected endocrine organs displayed no differences in H&E staining characteristics.Fibrelength Density within the adrenal gland, pancreatic islets and thyroid interstitium, along with serological measurements of ACTH and TSH, indicated that the process of oral gavage versus i.p. vehicle dosing was unassociated with significant variation amongst these baseline values  branching/anastomoses, within the adrenal cortex, amongst a number of tumour‐bearing animals, when compared with non‐tumour‐bearing counterparts. This was similar to previous observations made when examining this tissue from xenograft‐implanted mice  and most pronounced amongst high‐dose AZ10167514‐treated mice (−67% group mean reduction).When tumour‐bearing comparative vehicle control and treated groups were contrasted, there was again a statistically significant reduction in adrenal cortical vascularity amongst animals from all treatment groups. However, when compared with non‐tumour‐bearing animals, the adrenal cortex retained higher vascular density measures for AZ10167514‐treated mice, notably at the low‐dose .Finally, as previously described and also observed here  serum concentrations, when compared with non‐tumour‐bearing comparative vehicle control mice. In tumour‐bearing animals administered DC101 or AZ10167514 (at both doses), there was a comparative increase in ACTH release, potentially indicative of attempted cortical stimulation. At the high‐dose of AZ10167514, ACTH levels were of greater magnitude amongst tumour‐bearing, versus non‐tumour‐bearing, animals.With no tumour burden, there additionally appeared to be relatively high serum levels of circulatory TSH amongst animals administered DC101 or AZ10167514 (at both doses). This was most marked in animals administered low‐dose AZ10167514. This trend was recapitulated in tumour‐bearing animals, albeit to a lesser degree Figure .3.3With DC101 administration, there was endogenous release of mouse‐specific VEGF‐A, for both non‐tumour‐bearing and tumour‐bearing animals. There were also higher serum concentrations of mouse‐specific PlGF‐2; also apparent, albeit to a lesser extent, with AZ10167514 administration. With AZ10167514 administration, for both tumour‐bearing and non‐tumour‐bearing animals, there were further increases in the serum concentrations of mouse‐specific basic fibroblast growth factor (bFGF), showing a greater elevation in tumour‐bearing animals, notably at the low dose. This was not evident with DC101 administration. Additionally, the serum concentrations of mouse specific bFGF in vehicle control tumour‐bearing animals appeared higher, when compared with their vehicle control non‐tumour‐bearing comparative counterparts. Finally, with both DC101 and AZ10167514 administration, there were treatment‐related increased serum concentrations of mouse‐specific G‐CSF , when compared with the response generated amongst Calu‐6 tumours exposed to DC101.Other genes with differential expression between DC101 and AZ10167514 treatment groups included apob (apolipoprotein B), apoc1 (apolipoprotein C1) and apoe (apolipoprotein E). These changes appeared more pronounced at 6 mg/kg AZ10167514 Table .3.6We previously demonstrated that the implantation and growth of a range of mouse‐ and human‐derived allografts/xenografts leads not only to structural vascular changes within the peripheral host endocrine tissues but also imparts potential functional consequence. We noted that histogenetically diverse subtypes of tumour appeared stratified in their ability to influence peripheral endocrine vascularity and indicated a potential role in these observations for tumour‐ and host‐derived cytokines/growth factors, and the liberation of MDSCs , in any group. Furthermore, FACS analysis of the bone marrow did not reveal any findings consistent with a stress leukogram, the presence of a tumour burden in isolation was associated with a decrease in measured ACTH, and there did not appear to be a dose–response regarding ACTH liberation with AZD10167514 administration. These features gave weight to the conclusion that stress was unlikely to have materially influenced the observations reported ‐derived VEGF‐A, as measured in the serum, was also markedly attenuated with AZ10167514 administration. These observations suggested that the VEGF‐signalling cascade may be of limited importance within the findings reported here.The administration of a mixed TKI (AZ10167514) demonstrated that paraneoplastic resistance to VMA‐induced systemic endocrine vascular rarefaction may be most pronounced at comparatively lower dose levels . Furthermore, despite a marked release of mouse‐specific VEGF‐A and PlGF‐2 with DC101 administration , amongst tumour‐bearing animals. Whilst these mediators might also theoretically contribute to the observations of increased systemic endocrine vascularity, amongst tumour‐bearing mice, there appeared a general suppression, amongst all treatment groups, irrespective of the test item. These relative decreases were considered likely a result of decreased functional tumour mass/volume, following VMA administration Table .There was also a general drug‐induced release of host (mouse)‐derived G‐CSF, with all treatment protocols. However, changes were not significant between non‐tumour‐bearing and tumour‐bearing vehicle control mice. Additionally, an assessment of host bone marrow, via FACS analysis, failed to demonstrate significant shifts in the proportion of tumour‐associated myeloid progenitor/precursor lineages (in addition to CD202b‐positive EPCs), at this time point. It was therefore considered that this finding was likely a non‐specific and mild toxicologic effect of VMA administration.The production of mouse‐specific bFGF was significantly increased with the administration of AZ10167514. Measured quantities were higher in treated tumour‐bearing animals, more notably at the lower dose. Additionally, there were increased concentrations in tumour‐bearing versus non‐tumour bearing vehicle control animals. Likewise, there was a marked release of human‐specific bFGF from the xenografts of AZ10167514‐administered animals, at both the low and high doses. These findings indicated that both host‐ and tumour‐derived bFGF may play a role in these observations of endocrine vascular rescue.Amongst other mediators, the FGF signalling cascade has been proposed as one of a number of significant contributors to anti‐angiogenic treatment resistance  expression amongst Calu‐6 tumours exposed to AZ10167514, over and above that observed following the dosing of DC101. The role of autotaxin, in both angiogenesis and in the systemic stabilization of blood vessels, has been documented, in addition to the ability of bFGF to upregulate this factor , with potential functional impact and notably with respect to mixed TKI. The pathogenesis of these findings indicates a potential role for both tumour‐ and host‐derived bFGF, with associated upregulation in the intra‐tumoural autotaxin‐lysophosphatic acid signalling axis.Further understanding of preclinical tumour–host interactions, and their potential to alleviate or exacerbate the off‐target effects of anti‐angiogenic (and other drug) treatments, could aid in the prediction of therapeutic efficacy versus potential adverse clinical outcome, in a defined cancer population.The authors have no conflicts of interest to declare.https://www.merckmillipore.com/GB/en/product/MILLIPLEX-MAP-Mouse-Pituitary-Magnetic-Bead-Panel-Endocrine-Multiplex-Assay,MM_NF-MPTMAG-49K; https://www.merckmillipore.com/GB/en/product/MILLIPLEX-MAP-Human-Angiogenesis-Growth-Factor-Magnetic-Bead-Panel-Cancer-Multiplex-Assay,MM_NF-HAGP1MAG-12K; https://www.merckmillipore.com/GB/en/product/MILLIPLEX-MAP-Mouse-Angiogenesis-Growth-Factor-Magnetic-Bead-Panel-Cancer-Multiplex-Assay,MM_NF-MAGPMAG-24K.Merck Millipore (n.d.). PROTOCOL_FOR_00003798MAN_MPTMAG‐49K; PROTOCOL_HAGP1MAG‐12K; PROTCOL_MAGPMAG‐24K. Retrieved from: Table S1: Mean tumor volume, standard deviation, and p value per vehicle control/treatment group at the start and end of study. i.p.; Intra‐peritoneal, LD; Low‐dose (6 mg/kg/day), HD; High‐dose (12 mg/kg/day), ns; not significant.Click here for additional data file.Figure S1: Plot showing that automated estimation of 3D Fibrelength Density detected an attenuated decrease in vascular bed density within the adrenal gland (A) and thyroid gland (B) of M396647‐treated Calu‐6 tumor‐bearing mice, from a previous study, when compared to treated non‐tumor‐bearing counterparts. There was no comparable trend noted for pancreatic islet vascularity (C). Median +/− 10‐90th percentile for 6 animals per group. *p < 0.05.Click here for additional data file.Figure S2: Plot showing that automated estimation of 3D Fibrelength Density within the adrenal gland (A), pancreatic islets (B), and thyroid gland (C), along with multiplex serological probing for ACTH (D) and TSH (E), in non‐tumor‐bearing and Calu‐6 tumor‐bearing vehicle control animals, did not detect a statistically‐significant variation within these baseline measures, between oral gavage vehicle and i.p. vehicle administration. The effects of DC101 administration upon these end‐points incorporated the i.p. vehicle control values for statistical comparison .Click here for additional data file.Figure S3: Plots demonstrating that automated estimation of 3D Fibrelength Density detected comparable trends in non‐tumor‐bearing versus tumor‐bearing mouse pancreatic islet (A) and thyroid interstitial (B) vascularity, as documented for the adrenal gland . LD; Low‐dose (6 mg/kg/day), HD; High‐dose (12 mg/kg/day). Median +/− 10‐90th percentile for 6 animals per group. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Click here for additional data file.


The primary goal of this article is to analyze the oscillating behavior of Maxwell Nano-fluid with regard to heat and mass transfer. Due to high thermal conductivity of engine oil is taken as a base fluid and graphene Nano-particles are introduced in it. Coupled partial differential equations are used to model the governing equations. To evaluate the given differential equations, certain dimensionless factors and Laplace transformations are used. The analytical solution is obtained for temperature, concentration and velocity. The temperature and concentration gradient are also finds to analyze the rate of heat and mass transfer. As a special case, the solution for Newtonian fluid is discussed. Finally, the behaviors of various physical factors are studied graphically for both sine and cosine oscillation and give physical meanings to the parameters. Some of the fluids show both the viscous and elastic properties such fluid are called viscoelastic fluids like toothpaste, polymers solutions, paints, clay etc.2. The three major types of non-Newtonian fluids are Integral form, rate and differential type fluids. The Maxwell fluid is the principal viscoelastic rate type liquid, which is likewise broadly used. The differential structure and rate type models have stood out enough to be noticed among them. Due to the straightforwardness of rate type liquid, numerous examiners are giving specific consideration to Maxwell fluid6. Riaz et al.8 studied the Maxwell fluid of heat and mass transport in term of local and non-local differential operators. The semi analytical solution was obtained via Laplace transform. A fractional Maxwell fluid was analyzed by10 using numerical techniques. Natural convection flow of Maxwell fluid between vertical plates was investigated by Na,W et al.11. A closed- form solution was acquired through Laplace transform. Khan et al.13 studied mixed convection Maxwell fluid ordinary and fractionally over oscillating vertical plate. Exact solution and some special cases for Newtonian fluid was obtained through Laplace transform. Abro, K. A. et al.14 also obtained analytical solution of Maxwell fluid over vertical plane. Numerical solution of comparative Maxwell and Casson fluid was illustrated by Kumar et al.15 using Runge- Kutta based shooting method. Sodium alginate (SA-NaAlg) based (MoS2) nanofluid was researched by Ahmed et al.16 utilizing Maxwell Garnetts and Brinkman models. Physically, mixed convection is induced because of upgrade force and abrupt plate motion. Farooq et al.17 analyzed the generalized Maxwell model flow of magnetic hydrodynamic (MHD) fluid through porous duct. The solution was obtained via double Fourier sine and Laplace transform. Exact solutions for unsteady MHD flow of Maxwell fluid over oscillating plate have been illustrated by19. Sandeep et al.20 discussed the comparative study of Jeffery, Maxwell and Oldroyd-B fluid through extended surface utilizing similarity transformation and solution was acquired numerically via Runge–Kutta dependent shooting method. A fractional Maxwell model in porous medium was illustrated by Aman, S et al.21. The numerical solution was obtained using Stehfest’s algorithm. Fetecau et al.22 studied the second problem of stokes for Maxwell fluid via Laplace transform.A large number of scientists and engineers are keen in knowing the computational and anatomical features of industrial fluid in recent times, because of their increasing usage in engineering and industrial sciences. Such fluids are categorized as non-Newtonian fluid, and their sub-division contains cement, drilling mud, synthetic oils, asphalts and many more23 considered the Maxwell fluid on an extended sheet over Darcian medium. The general solution for natural convection flow past on a vertical plate with heat and mass transfer was discussed by25. Free convection flow with heat and mass transfer over fluctuating and accelerated vertical plate through porous medium was studied by28. Closed-form solution was obtained via Laplace transform method. Rajput et al.29 researched the impact of radiation on an impulsively vertical plate with heat and mass transmission of MHD flow. Pattnaik, J. R et al.30 addressed the MHD flow over exponentially inclined plate via porous medium. For solving the given equations Laplace transformation was utilized. Seth et al.31 illustrated the MHD convected flow of Soret and Hall effects in a rotating system with heat and mass transmission. Kumam, P et al.32 explained the comparative study of fractional Maxwell fluid. Semi analytical solution was obtained via Laplace transformation. Fetecau et al.33 examine the impact of radiation and porosity on MHD fluid on an oscillating vertical layer. Tang et al.34 have given the comparison of two different fractional definitions  of Maxwell fluid.Coupled heat and mass transport is a process that happens commonly in nature. It exists not only as a result of temperature variations, but also because of concentration variations or the combine effect of these two. The impact of a compound response is dictated by, whether it is homogeneous or heterogeneous. The incorporation of unadulterated water and air, are inconceivable in nature. It's conceivable that any external matter is normally there, or that it's blended in with air or water. At the point when an external mass is available in air or fluids, it prompts a synthetic response. Numerous substance advances, like the assembling of pottery, the creation of polymers and food handling, benefits from the investigation of related synthetic responses. Shateyi35 studied the natural convection flow of Maxwell fluid with graphene nanoparticles. Murtaza et al.36 examined the concrete nanoparticles in fractional Maxwell fluid. Exact solution was acquired via LT. The Maxwell hybrid nano-fluid of convective flow in a channel was discussed by38. Asjad et al.39 investigated the clay-nanoparticles of generalized Maxwell fluid in heat transmission via infinite flat surface. Wang et al.40 argued the Oldroyd-B nanofluid of MHD natural convection flow via permeable medium. Arif et al.41 studied the Maxwell hybrid nanofluid (engine oil) in vibratory vertical cylinder. Kumar et al.45 studied the impact of magnetic dipole on thermophoretic particle deposition in the flow of Maxwell fluid and nanofluid over a stretching sheet. Prasannakumara43 focused on the nuumerical simulation of heat transport in Maxwell nanofluid flow over a stretching sheet considering magnetic dipole effect. Through the stretchable disks slip flow of Casson–Maxwell nanofluid was studied by Gowda et al.44. Also, many other authors focused to studied Maxwell nanofluid, see e.g.48 for better understanding. Cheng et al.50 proposed the heat transfer analysis of elastoviscoplastic non-Newtonian generalized fluid with hybrid nanofluid and dust particles. Numerical solution of the model is acquired via shooting method. Kaneez et al.52 investigated the numerical solution of micropolar fluid including dusty, mono and hybrid nono-structures. Khan et al.53 demonstrated the upper convected Maxwell MHD micropolar fluid with the impact of Joule heating and thermal radiation utilizing a hyperbolic heat flux.Nanofluids are being used to improve the thermal conductivity of base fluids such as water, engine oil, propylene glycol, and ethylene glycol, among others. They have a wide range of uses in engineering and biomedicine including cooling, cancer treatment and industrial plants. The use of solid particles suspension to improve the thermal conductivity of traditional heat transfer fluid is a relatively new advancement in engineering technology. This technology has been recently paired with advance nanofluids and liquid nanoparticles suspensions technologies, to establish a new group of nanofluids based on solar collectors. Aman et al.To the best of author’s knowledge no one has consider the oscillating Maxwell nanofluid with the heat and mass transfer. So, motivated by this we study this problem analytically. The aim of this work is to explore oscillating Maxwell nanofluid with heat and mass transfer. The suspension of graphene nanoparticles and engine oil (base fluid) is taken in consideration. The governing equation is solved through LT. The solution for temperature, concentration and velocity are calculated analytically. Temperature slope and concentration gradient in the form of Nusslet number and Sherwood number are also acquired. Finally, the influence of various embedded factors on temperature, concentration and velocity shows graphically as well as theoretically.x-axis. Both the fluid and the plate are initially at rest with ambient temperature Let us assume Maxwell nanofluid passed on an infinite oscillating vertical plate with heat and mass transfer. mentclass2pt{minimHere, 49:The corresponding initial conditions (ICs) and boundary conditions (BCs) are of the following form33 and gaining the small temperature variation between the temperature Using Rosseland approximationsandwhere Substituting (5) into (2) we obtain the following form35;The thermo-physical characteristics of nanoparticles were given byThe dimensionless parameters are given below;After substituting the above dimensionless parameters in Eqs. , 3) and and3) anwhereHere, The dimensionless ICs and BCs are as follow and skip The thermo-physical property of graphene (nanoparticles) and engine oil (base fluid) are tabulated in table. 1Taking LT on Eq.  and alsoThe inverse LT of Eq.  has the The Nusslet number, measure the rate of heat transfer at the plate can be acquired by differentiating Eq.  with resThis shows the heat is transfer due to purely conduction.Applying LT on Eq.  and alsoThe Laplace inverse transform of Eq.  as;17\doThe Sherwood number measure the mass transfer at the plate. The Sherwood number defined and represented by,Now to obtain Sherwood number we differentiate Eq.  with resThe Laplace transform of Eq.  and alsoHere,The inverse LT of Eqs. –27), we, we27), The Laplace inverse transform of Eq.  and util33.In the absence of nanoparticles, we obtained the solution of Fetecau et al.When we put 1 in Eq. , we get In the absence of Maxwell fluid coefficient (Newtonian fluid whereIn order to see the physical meaning of the problem, we use the LT method to obtain the solution for temperature, concentration, velocity, rate of heat transfer and rate of mass transfer. These solutions have been studied graphically by giving numerical values to various embedded parameters like radiation factor, chemical reaction factor, thermal Grashof number, mass Grashof number, Maxwell fluid coefficient, Schmidt number, Prandtl number. The value of volume fraction parameter is taken 0.01.Figure Figure Figures , are presented. It can be observed if the volume fraction parameter 13 for both sine and cosine oscillations and the temperature solution of Fetecau et al.33.In order to authenticate our present solutions, Figs. 13Sc.The concentration profile is decreasing function for Schmidt number Decrease occurs in concentration with increasing the estimation of chemical reaction factor The velocity of the nanofluid grows, when thermal Grashof number The nanofluid’s velocity enhances with the enhancement of mass Grashof number The velocity field is accelerated as we accelerate the estimation of Maxwell fluid parameter Reduction occurs in velocity with higher value of Schmidt number The nanofluid’s velocity is increasing function as we increase the value of radiation parameter Volume function also reduces the nanofluid’s velocity.The temperature of fluid is growing function when radiation parameter Nusslet number and Sherwood number are constant.The aspiration of this work was to evaluate the oscillating Maxwell nanofluid with heat and mass transfer. The analytical solution for temperature, concentration, and velocity were obtained through LT method. The rate of heat and mass transfer was also measured in the form of Nusslet number and Sherwood number. Finally, the effect of different physical factors were shown in discussion section graphically and theoretically for both sine and cosine oscillation. The solution of Newtonian fluid was also analyzed as a special case. Following are the key concepts of this work (Supplementary Information Supplementary Information.


While many tumour OoC systems have been developed to date, there have been limited validation studies to ascertain whether drug responses obtained from tumour OoCs are comparable to those predicted from patient-derived xenograft (PDX) models. In this study, we established a multiplexed tumour OoC device, that consists of an 8 × 4 array (32-plex) of culture chamber coupled to a concentration gradient generator. The device enabled perfusion culture of primary PDX-derived tumour spheroids to obtain dose-dependent response of 5 distinct standard-of-care (SOC) chemotherapeutic drugs for 3 colorectal cancer (CRC) patients. The in vitro efficacies of the chemotherapeutic drugs were rank-ordered for individual patients and compared to the in vivo efficacy obtained from matched PDX models. We show that quantitative correlation analysis between the drug efficacies predicted via the microfluidic perfusion culture is predictive of response in animal PDX models. This is a first study showing a comparative framework to quantitatively correlate the drug response predictions made by a microfluidic tumour organ-on-chip (OoC) model with that of PDX animal models.Inter-patient and intra-tumour heterogeneity (ITH) have prompted the need for a more personalised approach to cancer therapy. Although patient-derived xenograft (PDX) models can generate drug response specific to patients, they are not sustainable in terms of cost and time and have limited scalability. Tumour Organ-on-Chip (OoC) models are  One of the goals in precision oncology is to identify and administer effective treatment regimen suitable for individual cancer patients. Due to the nature of tumour heterogeneity arising from genetic  and non-in vitro models are predictive of in vivo drug response, which takes a longer time (>4 weeks). This has in turn limited their routine application in cancer drug testing applications.Microfluidic Organ-on-a-Chip (OoC) systems are widely regarded as a potential animal-alternative platform for drug screening applications. OoC systems leverage on microfabrication techniques to mimic different aspects of tissue microenvironment and physiology (shear stress and microenvironment)  in a micin vitro-in vivo comparative study to develop a comparative framework for drug response predictions made from a tumour OoC models against that of matched PDX models in a patient-specific manner. PDX-derived 3D tumour spheroids were introduced into an Integrated Microfluidic Tumour Culture Array (IMITA) device, comprised of an 8 × 4 array of 3D tumour spheroid culture-chambers fed by a microfluidic concentration gradient generator. We demonstrate that the IMITA device can support perfusion culture of primary tumour spheroids isolated from PDX models generated from 3 colorectal cancer (CRC) patients, and simultaneously test the response of 5 standard-of-care (SOC) chemotherapies at 8 concentrations with 4 repeats. Importantly, the efficacy rank order of the 5 SOC drug combinations for individual patients predicted from the 3D spheroid cultures grown in the IMITA device correlated well with that obtained from the treatment of matched PDX models. Overall, this study demonstrates that microfluidic OoCs platforms, such as the IMITA device, offer a means to efficiently upscale drug testing with various drug combinations using minimal patient-derived or PDX tumour samples, and are predictive of in vivo response.Here, we report the first instance of a direct All chemicals and reagents were purchased from Sigma-Aldrich Pte Ltd., Singapore unless otherwise stated.The Integrated Microfluidic Tumour Array (IMITA) array was designed using AutoCAD . The IMITA device comprised a cell culture array with 32 circular chambers arranged as 8 × 4 (row × column). Each circular perfusion chamber was 2 mm in diameter and housed a cell culture chamber, which was 500 µm in diameter. The perfusion chambers were fed by an orthogonal network of microchannels, namely the cell seeding channels and the outputs of a concentration gradient generator. The volume of the cell culture chamber was defined by an array of micro-pillars (100 µm in length and 30 µm in width with an inter-pillar spacing of 20 µm), which was arranged into a circular cup. The circular micro-pillar array had a 250 µm wide opening, which faced the flow direction from the cell seeding inlet. Cells that were dynamically seeded into the device would be trapped by the micro-pillar array and packed at high density in the cell culture chamber. This physical trapping mechanism is a well-established method to form 3D spheroids in a non-cell type specific manner . The per2, for 50 s)  to a 75 cm × 50 cm glass slide.The microfluidic devices were produced by moulding polydimethylsiloxane (PDMS)  on DRIE-etched silicon templates. The microfluidic device was capped with either a glass or PDMS substrate using plasma bonding  to 3 ml fluid dispensing reservoirs  and 1-way stopcocks with Luer connections . Quick connect Luer adaptors  connected the tubing to the stopcocks. Prior to assembly, all parts were sterilized by autoclaving at 121°C for 20 min except for the stopcocks, which were sterilized with 70% ethanol for 1 h before rinsing with sterile 1X PBS. The assembled device was then mounted on a 3D printed support frame, printed with an Objet260 Connex3 Printer , and using acrylonitrile butadiene styrene (ABS) plastic, whereby the inlet and outlet reservoirs were supported on a 3D printed insert with 2 support pillars with a height difference of 6.3 cm.The perfusion inlet and outlet of the device were connected −1.CFD simulation was performed using Autodesk CFD . The device geometry was adapted from previously established design . Differe3 fragments and suspended in a mixture of 5% Matrigel  in DMEM/F12 . The tumour fragment mixtures were then implanted subcutaneously into the left flanks of 5–7 weeks old female NSG   mice, using 18-gauge needles. Tumours were excised and passaged when they reached 1.5 cm3 . For passaging, tissues were cut into small fragments of 1 mm3 prior to resuspension in a 20% Matrigel/DMEM/F12 mix, before subcutaneous inoculation of tumour fragments into 5–7 weeks old NSG mice. Animals were housed in individually ventilated cages in the Biological Resource Centre, A*STAR, Department 2. Room lighting was set to a 12-h light-dark cycle as recommended by the National Advisory Committee for Laboratory Animal Research (NACLAR). Animals were provided with an irradiated Altromin 1324 diet and autoclaved water, ad libitum. Protocols for all the animal experiments described were approved by the A*STAR Biological Resource Centre (BRC) Institutional Animal Care and Use Committee (IACUC) under protocol #171207.Tumour samples were obtained from patients’ post-surgery with informed patient consent in accordance with SingHealth Centralised Institutional Review Board (CIRB: 2015/2165). Tumours were minced into ∼1 mm−1 in 50% Polyethylene Glycol 400 (Sigma) and administered at a dosage of 50 mg·kg−1 i. p. Once a week (IRI) and twice a week (5-FU). Oxaliplatin  was prepared to a concentration of 1 mg·ml−1 with the same vehicle and administered at a dosage of 5 mg·kg−1 i. p. Once a week. Fluorouracil + Oxaliplatin (5-FU + OXA) and Fluorouracil + irinotecan (5-FU + CPT-11) were prepared with same concentration and dosed with the schedule of their single agent counterpart with 5-FU being administered first. Control groups for all compounds were treated in their corresponding vehicle (50% Polyethylene Glycol 400), following standard procedures  and Irinotecan  were prepared to a concentration of 5 mg·mlocedures , in the 2). Mice were euthanised when tumours in the control group reached 2.0 cm3. The tumour growth inhibition percentage (TGI%), the ratio of the change in treated tumour volume (ΔT) to the average change in control tumour volume (ΔT/Average ΔC) at each time point, was used to determine drug treatment efficacies, and calculated as follows:The length and width of tumours were measured by calliper once every 2 days. Tumour volumes were determined using the following modified ellipsoidal formula: Tumour volume = ½  in DMEM/F12, at 37 °C for 2 h. Cells were washed using the cyclical treatment of pelleting and resuspension in phosphate-buffered saline  for 3 cycles. The final cell suspensions were strained through 70 µm cell strainers , prior to pelleting and resuspension in RPMI , supplemented with 10% foetal bovine serum , and 1% penicillin-streptomycin . Cells were kept in a humidified atmosphere of 5% CO2 at 37°C. Cells were routinely screened for mycoplasma contamination using the Venor®GEM OneStep mycoplasma detection kit .Tumour tissues excised from PDX were minced prior to enzymatic dissociation using 4 mg ml−1) from the cell reservoir through the common outlet with a withdrawal syringe pump at a flow rate of 0.03–0.08 ml h−1 , and the perfusion inlets and outlets closed. Once the microfluidic device was almost full, the cell-free media was added into the cell seeding inlet to wash off cells not trapped within the central chamber before closing the seeding inlet and outlet. The perfusion outlet reservoir was connected to the stopcock at the common outlet and medium perfusion was initiated by turning on the perfusion inlet and the common outlet of the device. The seeded device was then mounted on the 3D printed support and incubated at 37 °C with 5% CO2 for 24 h with the culture medium before any drug dose-response study.The culture medium was formulated using RPMI 1640 Medium  supplemented with 10% foetal bovine serum , and 1% Pen-Strep . Cell seeding was initiated by withdrawing a cell suspension , Oxaliplatin (OXA), irinotecan (IRI), Fluorouracil + Oxaliplatin (5-FU + OXA), and Fluorouracil + irinotecan (5-FU + IRI). All compounds were dissolved in DMSO . For each drug dose-response, 500 μM of the drugs were diluted using DMSO into the cell culture media and loaded into the drug inlet medium reservoir. For combinatorial drugs, 500 μM of the individual drugs were mixed into the cell culture media. The cell culture medium infused with 1% (v/v) DMSO was loaded into the diluent medium reservoir. The seeded devices were then incubated for 3 days at 37 C with 5% COIn situ labelling of viable cells was performed using Calcein AM . Culture medium containing 2 µM Calcein AM was perfused at 0.08 ml·h−1 from both perfusion inlets using a syringe pump for 1 h at 37°C and 5% CO2. The devices were then washed with the culture medium at 0.08 ml·h−1 for 30 min before viewing with the fluorescence microscope  anda CoolLED pE-2 LED-excitation light source  at wavelength of 470 nm for Calcein AM. For each chamber, the transmission image and fluorescence image were taken for each chamber. Exposure time was kept at 300 ms for all fluorescent microscopy imaging steps.All microscope images were processed as 16-bit images for consistency. Transmission images of the CRC organoids were used to identify and measure the area of the organoid within the chambers. Cells located outside the cell culture chambers were not considered. The identified organoid area was used to create a mask in ImageJ  and overlaid against the fluorescence images. The mean fluorescent intensities of the mask-overlaid fluorescent images were quantified, and then normalised to the measured organoid area. Across the different concentration of drugs down the row, the area-normalised fluorescence intensities for each chamber where normalised again to the mean normalised intensity measured from the chambers in the first row which served as a control 50 values. A two-tailed Pearson correlation was computed using the GraphPad PRISM 8.0. All graphs were plotted on GraphPad PRISM 8.0.All results were obtained from at least 3 independent experiments with values expressed as mean ± standard deviation (SD). Curve fitting was undertaken with a GraphPad PRISM 8.0  using a 4-parameter variable slope, inhibition dose-response fitting to identify the ICThe IMITA device serves as a proof-of-concept multiplexed OoC platform, that can provide a suitable 3D tumour microenvironment to support the growth of primary PDX-derived tumour cells while being amenable to conducting dose response studies of chemotherapy drugs. The device was designed to handle parallel tumour spheroid cultures in an 8 × 4 array of culture chambers so that 32 tumour samples could be maintained simultaneously . The culThe 8 × 4 cell culture array  served an = (1-(n-1)/7) x C0 ; across each row of output (R2 = 0.9863) and concurred with our simulation results. By changing the inlet drug concentration, C0, we were able to modify the range of drug concentrations. Our subsequent drug dose response study examined the ML drug dose response with 8 drug concentrations with C0 = 500 μM  for all drugs.The perfusion circuit consisted of 2 inlets (1 drug and 1 diluent inlet) connected to a concentration gradient generator . Branchi2O was sufficient to drive a perfusion rate of 0.02 ml·h−1 (−2 at the cell culture chamber (Pe) of model chemotherapeutic drugs in the cell chambers were in the convection dominated transport regime (Pe >> 1) and at least one order of magnitude higher than that in the seeding channels linking adjacent cell chambers in a row  into the array followed by 4 days of perfusion culture. We observed that the PDX tumour cells underwent remodelling, changing from a loosely packed configuration  to a denp = 0.0891), suggesting that cells seeded into the device can be uniformly distributed across all channels in the cell seeding flow circuit. The relative uniformity in the spheroid size is ideal for drug dose response studies as it ensures consistent diffusion lengths as the drugs diffused into the tumour spheroid. This uniformity will also aid in future automated measurement and analysis such as viability imaging and quantification, which facilitates multiplexed experiments. We further characterised the viability of the PDX-derived tumour spheroids within the IMITA device to investigate the duration over which the device can maintain the  and tumour growth inhibition percentage (TGI%), were used to indicate the in vitro and in vivo anti-tumour efficacies, respectively, following our previous work  patients , maintained in the IMITA device. A parallel o models . Specifi 4 weeks . 5 standous work .max) values of the 5 SOC drugs in human, which range from 4.96 µM for (OXA) to 426 µM (5-FU)  . The druM (5-FU) . The assM (5-FU) . MoreoveM (5-FU)   . QuantifM (5-FU) . Using tWe observed differential responses for SOC chemotherapies across the IMITA devices housing PDX tumours derived from different CRC patients . For allThe inter-patient variability of inherent tumour characteristics across the 3 CRC patients was evident from the PDX models. Of the control animals, CRC1030 exhibited the fastest tumour growth rate  whereas in vitro drug response observed in the IMITA device over a 4-day period and that of the PDX models over a 40-day period provides insightful window of opportunity to identify the degree of similarity between in vitro tumour OoC models and in vivo PDX models. Since the conventional measurement metrics to indicate treatment efficacies are different for in vitro  and in vivo models , we first ranked the SOC therapies in terms of anti-tumour efficacies before performing correlation analysis. Using IC50 values obtained from the IMITA dose-response studies  to indicate anti-tumour efficacies.Despite limitations associated with cost, time and engraftment rates , PDX modterparts . While tin vitro models to be a suitable alternative in drug screening platform, it is necessary for the established model to be able to recapitulate physiological microenvironment conditions, such as 3D architectures (in vitro and in vivo predictions (For tectures , fluid itectures , extracetectures , as welltectures . The higdictions  are stroin vivo PDX drug responses for CRC935. We postulate this may be due to the lack of ECM support within the IMITA device. Between the 3 patient samples, CRC935 tumour tissues showed a higher amount of ECM (in vitro and in vivo models (in vitro-in vivo correlative framework could potentially be deployed to assess the suitability of various OoCs in immunotherapy testings by co-culturing of tumour with effector cells such as NK (Nonetheless, the IMITA device did not adequately predict the t of ECM  comparedt of ECM . This apt of ECM . Since tt of ECM . Througho models . The demch as NK  and CAR-ch as NK .in vitro models predict the drug-response obtained from PDX animal models. In this study, we have successfully established a multiplexed tumour OoC device that can recapitulate a dynamic 3D culture environment to maintain PDX derived primary tumour tissues from 3 CRC patients. This enabled us to systematically compare the drug responses to 5 different SOC chemotherapeutic regimes between the in vitro microfluidic tumour OoC model and PDX in vivo model. In addition, we established a framework to statistically examine the correlation between ranked drug efficacies based on in vitro IC50 values and in vivo TGI values. It was found that in vitro-in vivo correlation coefficient was >0.70 for 2 out of the 3 CRC patients. This indicated the potential application of in vitro microfluidic platform in drug screening applications towards personalised cancer treatment.One of the key appeals of tumour OoC devices is their potential to reduce the reliance of animal models for cancer drug testing. To achieve this goal, there is a need to systematically validate how well these


Dear Editor,Eccrine poroma (EP), as first described in 1956 by Goldman et al.,Reflectance confocal microscopy (RCM), which has shown to be an important in vivo, non‐invasive diagnostic technique for skin lesions. Effectiveness of RCM for inflammatory and neoplastic skin diseases has been widely reported in literature.Herein, we report a 25‐year‐old Asian male presented with an asymptomatic papule on his right chest for 1 year, without history of trauma, chronic irritation or radiation at the site of the tumour. Upon cutaneous examination, the lesion was sessile, firm, reddish‐pink, well‐circumscribed and elevated at the periphery, and a small amount of bleeding can be seen on the surface Figure . After cConfocal microscopic analysis revealed a well‐demarcated tumour consists of bright tumour islands with surrounding homogeneous dark stroma. Tumour cells were highly refractile, uniform in size and shape, round and dark nuclei and smaller than epidermal keratinocytes. Tumour cells arranged in an honeycombed structure. Hyperreflective inflammatory cells can be seen in the dermal papillary layer Figure . Owing tDermoscopy of the lesion showed linear irregular, hairpin, glomerular and poorly visualized vessels. White structureless area and perivascular light pink halos were also be observed Figure . MarchetOur case serves as one of the few reported cases of using RCM and dermoscopy observed EP lesion and this young patient's lesion located at an unfrequent site. Given the uncommonness of the neoplasms, the diagnosis mostly relies on the histopathological examinan. RCM and dermoscopy may assist in the correct diagnosis of this neoplasm and help rule out some differential diagnoses. Due to about 18% of poromas will progress to porocarcinomas,The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


Background: Previous studies have shown that a large number of valuable and functional cell-free RNAs (cfRNAs) were found in follicular fluid. However, the species and characteristics of follicular fluid cfRNAs have not been reported. Furthermore, their implications are still barely understood in the evaluation of follicular fluid from follicles of different sizes, which warrants further studies.Objective: This study investigated the landscape and characteristics of follicular fluid cfRNAs, the source of organization, and the potential for distinguishing between follicles of different sizes.Methods: Twenty-four follicular fluid samples were collected from 20 patients who received in vitro fertilization (n = 9) or ICSI (n = 11), including 16 large follicular fluid and 8 small follicular fluid samples. Also, the cfRNA profile of follicular fluid samples was analyzed by RNA sequencing.Results: This result indicated that the concentration of follicular fluid cfRNAs ranged from 0.78 to 8.76 ng/ml, and fragment length was 20–200 nucleotides. The concentration and fragment length of large follicular fluid and small follicular fluid samples were not significantly different (p > 0.05). The technical replica correlation of follicular fluid samples ranged from 0.3 to 0.9, and the correlation of small follicular fluid samples was remarkably (p < 0.001) lower than that of large follicular fluid samples. Moreover, this study found that cfRNAs of the follicular fluid could be divided into 37 Ensembl RNA biotypes, and a large number of mRNAs, circRNAs, and lncRNAs were observed in the follicular fluid. The number of cfRNAs in large follicular fluid was remarkably (p < 0.05) higher than that of small follicular fluid. Furthermore, the follicular fluid contained a large amount of intact mRNA and splice junctions and a large number of tissue-derived RNAs, which are at a balanced state of supply and elimination in the follicular fluid. KEGG pathway analysis showed that differentially expressed cfRNAs were enriched in several pathways, including thyroid hormone synthesis, the cGMP-PKG signaling pathway, and inflammatory mediator regulation of TRP channels. In addition, we further showed that four cfRNAs  serve as a potential indicator to distinguish the follicles of different sizes. The ROC curve shows great potential to predict follicular fluid from follicles of different sizes [area under the curve (AUC) > 0.88].Conclusion: Overall, our study revealed that a large number of cfRNAs could be detected in follicular fluid and could serve as a potential non-invasive biomarker in distinguishing between follicles of different sizes. These results may inform the study of the utility and implementation of cfRNAs in clinical practice. The World Health Organization (WHO) predicts that infertility will become the third most serious disease worldwide in the 21st century, with an average incidence of 9% among couples of reproductive age . During in vitro fertilization (IVF) attempts (Previous studies have shown that the components of follicular fluid (FF), secreted by granulosa cells and theca cells and diffused by capillaries, were investigated as potential biomarkers for predicting the quality of oocytes and embryos . Actuallattempts . Therefoattempts .Recent studies have showed that a large number of cell-free RNAs (cfRNAs) are observed in follicular fluid. Among them, microRNAs (miRNAs) have been studied extensively, which could be used to distinguish high-quality embryos from low-quality embryos and affect ovarian function and follicular development, and as non-invasive tools for the prediction of blastulation . HoweverHence, the present study was conducted to observe the following: 1) the characterization of follicular fluid cfRNAs was first carried out, and its existence pattern and regularity were analyzed. The potential to distinguish between oocytes of different sizes was further studied; 2) the complexity of follicular fluid cfRNA transcripts was analyzed, which includes 37 Ensembl RNA biotypes. Also, a large number of mRNAs, circRNAs, and lncRNAs were observed in the follicular fluid; 3) the integrity of follicular fluid mRNA was analyzed. This result showed that follicular fluid contains a large number of intact mRNAs and spliced junctions; 4) the analysis of tissue-specific gene sources in follicular fluid. This result indicated the presence of RNAs derived from tissue-specific genes in follicular fluid; 5) follicular fluid cfRNAs can be used as a potential indicator to distinguish the quality of follicles. Overall, this study revealed the types of follicular fluid cfRNAs and fragment distribution, source of organization, and the potential to predict different follicle sizes. These results may inform the study of the utility and implementation of cfRNAs in clinical practice.All patients participating in this study provided written informed consent. Ovarian follicular fluid was collected at the Clinical Center of Reproductive Medicine in the First Affiliated Hospital, Nanjing, China. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University  and conducted in accordance with the Declaration of Helsinki.2 (22.1 ± 2.29 kg/m2). Except for two patients with ovarian insufficiency, the ovarian reserve was normal in 80.9% of patients. Anti-Mullerian hormone (AMH) was between 1.03 and 10.33 (4.72 ± 3.36). The baseline hormonal levels, including FSH, LH, and 17 β estradiol (E2), were assessed on day 3 of the menstrual cycle.In this study, samples were collected from 20 patients who received IVF (n = 9) or ICSI (n = 11) cycles at the Clinical Center of Reproductive Medicine in the First Affiliated Hospital, Nanjing, China. The clinical features of patients are shown in 2) concentration. When at least three follicles were 18 mm or more in diameter, injection of human chorionic gonadotrophin (hCG) promotes ovulation. Then, 36 h after hCG injection, the follicles larger than 10 mm in diameter were aspirated by ultrasound guidance. Among them, follicles greater than 18 mm in diameter were considered large follicles, and follicles of 10–18 mm in diameter were considered small follicles. To minimize maternal contamination, the cumulus cells of oocytes surrounding them should be removed as much as possible before ICSI or IVF cycles.A total of 30 follicular fluid samples were obtained from 20 patients (a mean of 1.5 ± 1.1 follicular fluid samples can be collected per patient). The follicular growth was evaluated by measuring transvaginal ultrasound and serum 17 β-estradiol  was used, according to the manufacturer’s instructions, to extract total RNA from follicular fluid samples within 4 h and was treated with RQ1 RNase-free DNase  to eliminate genomic DNA contamination. Then, the quality and quantity of total RNA were measured using an Agilent 2100 Bioanalyzer . The OD260/OD280 ratio was used as the RNA purity index. If the ratio of OD260/OD280 is 1.8–2.1, the RNA purity is acceptable. Its integrity was further determined by 1.5% formaldehyde denaturing agarose gel electrophoresis. Then, total RNA was stored at −80°C until further use.Only clear follicular fluid samples were included; blood-stained and cloudy follicular fluid samples were excluded to avoid contamination. Thereafter, the samples of follicular fluid were centrifuged at 12000 × g for 10 min to remove cell fragments and then immediately stored at −80°C for standby application. TRIzol@ RNA Clean Beads . 2) The purified RNAs are fragmented at 85°C for 2 min. 3) Then, 10 cycles were used for the first round of PCR to add Illumina Adapters and Indexes (PCR products could be stored at −20°C for up to 2 weeks). 4) Depletion of ribosomal cDNA with ZapR and R-Probes. 5) The final RNA-seq library was amplified by using 16 cycles. 6) The final RNA-seq library was purified by using VAHTS@ DNA Clean Beads (Vazyme Biotech Co. Ltd.). 7) The cDNA library concentration was measured using a Qubit® 2.0 Fluorometer (>1 ng/μl). The constructed library was sequenced by using the Illumina HiSeq 2500  sequencer (2 × 150-bp paired-end pattern (PE150)).We used SMARTer-seq® Stranded Kit User Manual  and modified some procedures to prepare the RNA-seq library. Briefly, 1) the total RNA was treated with DNase I (NEB) for 15 min at 37 °C and purified by VAHTSPrior to sequencing data mapping, raw reads (raw data) with adapters, > 5% unknown nucleotides, and low-quality reads containing >20% of bases with qualities of <20 were removed. The results of quality control are shown in The raw sequencing reads were filtered by FastQC (v0.11.4) and aligned using the spliced read aligner HISAT2 (version 2.1.0) , which whttps://david. ncifcrf.gov/).For the first time, clean paired-end RNA-seq reads were mapped to the human reference genome (UCSC hg19) with BWA . The cirp-value of each transcript was adjusted by the Benjamini–Hochberg method to control the false discovery rate (FDR). The genes of FDR < 0.01 and fold change > 2 were retained. Only RNAs upregulated in these follicular fluids  were considered valuable for oocyte classification.The RNA-seq raw read counts were normalized by FPKM. Variates with a frequency of <25%  were omitted, and the remaining transcripts were used for subsequent analyses. The identified genes were analyzed for differential expression by the Mann–Whitney U test in the large follicular fluid and small follicular fluid samples, and the p-value <0.05 was considered statistically significant. Figures were created by GraphPad Prism 7.0  and R software package.All statistical analyses were student’s t-tests, and the As shown in p > 0.05) difference in the distribution range of cfRNA fragments in follicular fluids of different-sized follicles, but small-sized follicular fluid fragments were more diffused . The small follicular fluid sample profile showed a poor correlation between two technical replicas  lower than that of large follicular fluid samples, with the median of R2 = 0.6515 and 0.418 in large follicular fluid and small follicular fluid samples, respectively (p > 0.05) (As shown in ectively . In addiectively . Neverth > 0.05) .We analyzed the cfRNAs of follicular fluid, and a large majority of the mapped reads corresponded to protein_coding RNAs and lncRNAs, respectively . Most ofp < 0.05) changes were observed in the sequenced read counts of protein_coding, lncRNA, and miRNA (p > 0.05)  . Further > 0.05) . This re,. In large follicular fluid samples, the expression levels of protein_coding genes and lncRNAs were higher than those of small RNAs and other ncRNA expression levels. In contrast, the expression level of lncRNAs in small follicular fluid samples was slightly higher than that of the other three RNAs. For large follicular fluid samples, we detected 40903 mRNAs, 360 circRNAs, and 11560 lncRNAs in the median , 5′-untranslated (5′UTR), and 3′-untranslated (3′UTR) regions. The result showed that 5′UTR regions in large follicular fluid samples were remarkably higher (p < 0.05) than CDS and 3′UTR regions (p > 0.05) in small follicular fluid samples (p < 0.05) in large follicular fluid than CDS and 3′UTR regions (p < 0.05) (p < 0.05) , respectively . The len regions . The rel samples . For oth regions . Moreove regions . Further < 0.05) . For oth < 0.05) ., respectively. Also, the known RNA was remarkably higher (p < 0.05) in small follicular fluid than in large follicular fluid, whereas the opposite was found for novel junctions  in follicular fluid were considered . The random forest algorithm was used to analyze the selected cell-free RNA markers to reduce the number of variables. Finally, four cfRNA markers  were selected. To evaluate the feasibility of using the selected four RNAs in follicular fluid as a difference between different follicular fluid sizes, we analyzed the sensitivity and specificity of these RNAs. As is presented in To our knowledge, this study is the first to assess the transcriptome range of cfRNA in follicular fluid. Previous work mainly focused on circulating miRNAs , which iIn this study, the biological variation of cfRNA content in different follicular fluid samples may be due to the fact that different follicles have different abilities to release RNAs into the follicular fluid. In addition, this study showed that the fragment length of large follicular fluid cfRNAs was higher than that of small follicular fluid, but there was no significant difference. This was supported by the fact that in mammals, including humans, the concentration of magnesium ions in small follicular fluid was higher than that of large follicular fluid, which might lead to RNA degradation . FurtherFurthermore, this result indicated that between-sample correlations were always lower than within-sample correlations. The low correlation may be due to the fact that the complexity of the follicular fluid composition results in great variation between samples. Additionally, the degree of development of different follicles can also lead to great differences between samples. Moreover, this study found that the sizes of follicular fluid cfRNA sequenced fragments were about 200 bp, and the maximum read fragment length was 173361750. This suggests that the follicular fluid may contain intact fragments of no degraded RNAs. In addition, we compared the correlations between follicular fluids of different sizes. This result indicated that the correlation between large follicular fluid samples was significantly higher than that between small follicular fluid samples. This may be due to the fact that the composition of the follicular fluid is highly variable and closely related to the stage of follicular development. For mature large follicular fluid, its components are relatively stable . HoweverMost previous analyses focused on miRNAs . HoweverIn this study, we found that most follicular fluid mRNAs were complete fragments (full-length coverage of protein_coding genes >83%). This result was different from previous studies, in which the RNAs of body fluids are fragments . TherefoThis study found that most tissue-specific RNAs, including the ovary, follicle, granulosa cells, and blood-specific RNAs, were detected in the human follicular fluid. The proportion of tissue-specific genes in large follicular fluid was remarkably higher than that of small follicular fluid. This result might be attributed to the release of various metabolites and molecules into the follicular fluid by tissues during follicular development, thus promoting follicular development. Furthermore, this study demonstrated that the proportion of tissue-specific genes from granulosa cells in the follicular fluid was significantly higher than that of tissues from other sources. This result can interpret that the proportion of granulosa cells in follicular fluid was significantly higher than that in other types of cells. This also proves that the contamination of granulosa cell source is the bottleneck problem to improve the detection of cfRNAs in the follicular fluid. Moreover, the RNA abundances (FPKM) of tissue-specific genes indicated unimodal distributions and positive patterns. These distributions show that tissue-derived cfRNAs are at a balance supply and clearance in follicular fluid.PCA results indicated that the genes of different-sized follicles’ follicular fluid could be completely separated, which suggested that the follicular fluid of different-sized follicles could be distinguished by using some genes. Moreover, we performed unsupervised clustering of differentially expressed genes and found that the follicular fluid of follicles of different sizes could be clearly separated. This again supported that cfRNAs could be used to predict different-sized follicles’ follicular fluid. Previous studies have shown that cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase G (PKG) were expressed in neonatal ovaries of pre-granulosa cells, promoting primordial follicle activation, oocyte growth, and granulosa cell proliferation . This stIn conclusion, our study revealed that a large number of cfRNAs could be detected in follicular fluid and could serve as a potential non-invasive biomarker in distinguishing between follicles of different sizes. These results may inform the study of the utility and implementation of cfRNAs in clinical practice.


Height identification of water-permeable fractured zone (WPFZ) is one of the decisive influence factors for mining safety, especially in some specific conditions, such as mining under aquifer. In order to demonstrate the formation process of the WPFZ, the scaling model experiment is carried out. Through the analysis of movement and breaking in overlying strata, the WPFZ height is significantly affected by mining range, movement characteristics of key strata and its follow-up strata. Based on the research findings, a new theoretical method, " overlying strata synchronous movement method " (OSSM) is established to predict the WPFZ height. Taking 3301 mining face of Zhujiamao Coal Mine in China as the engineering background, the WPFZ height is estimated by OSSM. Additionally, the field detection is carried out by the downhole segmented water injection method combined with borehole camera method. By comparing the results of different methods, the accuracy of OSSM is verified and the WPFZ height is determined finally. What´s more, various methods for determining WPFZ height are evaluated. As presented in Fig. 5. Caving zone and fractured zone are collectively referred to as WPFZ, in which a large number of mining-induced fractures are developed, which are the main channels for fluid migration and infiltration, so the WPFZ is extremely permeable. If the WPFZ height is greater than or equal to the distance between the coal seam and the aquifer, the aquifer will be destroyed and the water in the aquifer will pour into the mining face or roadway through the WPFZ, resulting in groundwater loss and mine disaster7. Therefore, the accurate prediction of WPFZ height is the basic work for determining the mining upper limit, coal mining technology and its parameters. Likewise, the determination of WPFZ height is of great significance for water-preserved-mining9.In subsurface coal mining, the overburden will be moved, deformed and destroyed after the coal seam is mined11. Therefore, the WPFZ height is affected not only by the thickness of mining coal seam, mining methods, but also by the lithologic characteristics, engineering geological conditions and other factors13. In view of these factors, many scholars have made a lot of exploration to predict the WPFZ height. Currently, there are many methods to determine the WPFZ height, such as simulation experiment method, field detection method and theoretical analysis method15. Generally, simulation experiment method, that is, simulating the failure height of overburden through numerical analysis or scaling model experiment can provide a basis for determining WPFZ height17. Field detection methods mainly include ground borehole flushing fluid method, geophysical exploration method, downhole segmented water injection method and borehole camera method19. The ground borehole flushing fluid method determines the WPFZ height by detecting the leakage of borehole flushing fluid during drilling, which has complex structure and low detection accuracy. Geophysical exploration method can determine the WPFZ height by analyzing the variation of resistivity in collapsed and cracked rock mass, but its exploration cost is high and it is easily affected by high temperature, high humidity and other factors. Downhole segmented water injection method is to determine the WPFZ height by detecting the water seepage of upward-inclined boreholes above the mining goaf, and the borehole camera technique is to put the detector into the boreholes to observe the separation of rock mass. The theoretical analysis method is characterized by simplification. Therefore, many theoretical analysis methods have been established20.The distribution of new fractures in overlying strata is very complex after the coal seam is mined out23. The distance between key strata and coal seam and the thickness of mined coal seam are considered in the existing methods based on key strata. However, we find that the WPFZ height is also significantly affected by mining range, movement characteristics of key strata and its follow-up strata, especially the zoning of follow-up strata after the key strata fracted. On the basis of this finding, the new method of "overlying strata synchronous movement method" is established to predict the WPFZ height. In the new method, the influence of the mined coal seam thickness, the mining range, the residual fragmentation coefficient of overlying strata, the key strata and its follow-up strata movement characteristics on the WPFZ height are considered comprehensively. Therefore, the WPFZ height predicted by this new method is more accurate, which can provide more convenient guidance for coal mines as well.For various theoretical analysis methods, although the traditional empirical formula method in China is widely applied because of its simplicity, the prediction methods based on key strata is more objectiveThe field engineering example applied in this paper is 3301 mining face of Zhujiamao Coal Mine in China. The identification of WPFZ height follows a framework that applies several progressive and mutually validated methods. Firstly, the "overlying strata synchronous movement method" is applied to predict the WPFZ height. Secondly, the field detection is carried out by the downhole segmented water injection method combined with borehole camera method to verify the reliability of the new method. These methods verify each other and determine the height of WPFZ comprehensively.24, the empirical formulas only include two factors of coal thickness and lithology. The overlying strata are divided into hard, medium-hard, soft and significantly soft categories according to the comprehensive compressive strength. Furthermore, the WFZ height could be calculated from the mining thickness by the corresponding empirical formulas for different roof lithology25. Although this method is simple, many key factors that affect the height of WPFZ are ignored, resulting in inaccurate height. Therefore, more accurate theoretical methods need to be explored.H is the WPFZ height and ΣM is the accumulated mining thickness.The empirical formulas included in the “Coal mining regulations under the buildings, water and rails” are widely utilized to predict WPFZ height in China. As presented in Eq.  to Eq.  24, the 28, the geometric similarity ratio is 100 and the bulk density similarity ratio is 0.6. The model material takes sand as aggregate, gypsum and lime as binder, and mica is scattered at the junction of rock stratum to separate strata. The material ratio is shown in Table In order to intuitively reflect the synchronous movement of overlying strata during the mining, that is, the influence of the key strata and its follow-up strata on the WPFZ height, the scaling model experiment is adopted. This experiment is carried out on bench with sizes 120 cm × 8 cm × 100 cm (length × width × height). The objective of the experiment is to study the influence of the key strata and its follow-up strata on the WPFZ. Therefore, the model is simplified by laying the key strata and the follow-up strata alternately, as presented in Fig. The experimental results are presented in Fig. When the face is advanced to 63 m, the key stratum 2 still remains stable due to the caving angle of overburden, although the fracture length reaches 36 m. Synchronously, the follow-up strata above key stratum 2 remains stable as well. The WPFZ is still within the range of 20 m above the coal seam. This experimental process is presented in Fig. As presented in Fig. Due to the size limitation of the experimental bench, the experimental process and results need to be further inferred. Multiple key strata are assumed to be located above the key stratum 2. After the fracture, the key stratum 2 sinks greatly, and the upper follow-up strata moves synchronously, due to the broken rock which is gradually compacted below. Therefore, it will lead to cavities below the higher key strata. With the increase of the mining range, the corresponding key strata will break when they reach the limit hanging lengths. Otherwise, they will remain stable.As we can see from the experimental results, the stability of the key strata could be affected by the mining space, and the WPFZ height is determined by the synchronous movement of the key strata and its follow-up strata. The height of the caving zone or fractured zone is fixed before the key strata approach to the limit hanging length. However, at the limit hanging length, the key strata break and the follow-up strata above it move synchronously. Whether the key strata can form a hinged structure determines the zone to which the key strata and its follow-up strata belong after fracture. That is, if the key stratum forms a hinged structure, the key stratum and its follow-up strata belong to the fractured zone; on the contrary, the key stratum and its follow-up strata belong to the caving zone.Based on the above experiment and theory, the OSSM is proposed to predict the WPFZ height. As is presented in Fig. 29. At the same time, we also need to pay additional attention to the strength conditions of key strata. As presented in Fig. L1, and the fracture develops upward along a certain span angle. If there are still key stratum in the upper strata (key stratum 2), it shall meet the requirements of Eq.  Condition for non-sliding instability of fractured blocks:σp is compressive force at the interlocking areas of fractured rock blocks, σc is the compressive strength of rock blocks, k is the proportional coefficient determined by experience and β is the allowable subsidence angle of rock blocks after fracture.(2) Condition for non-deformation instability of fractured blocks:If the key stratum 1 after fracture satisfies Eq.  and Eq. One is that the next key stratum does not break within the mining range. In this circumstance, there are only caving zone and bending zone above the mining face, and there is no fractured zone.Another possibility is that the next key stratum breaks within the mining range. Furthermore, if hinged structures can be formed after the fracture, then the key stratum and its follow-up strata belong to the fractured zone. On the contrary, if the hinged structures can´t be formed after the fracture, then the key stratum and the follow-up strata belong to the caving zone. Therefore, steps III to IV to need to be repeated to identify the other key strata above.Step V: When the key stratum 1 is determined as belonging to the fractured zone after the fracture, steps III to IV need to be repeated to calculate the next stratum . Additionally, when the key stratum 1 is determined as belonging to the caving zone, and there is only one key stratum in the overlying strata, the caving zone extends from the mining face to the surface. Generally, many shallow coal seams present this mine pressure behavior. However, when there are multiple key strata in the overburden, steps III to IV need to be repeated to calculate the next stratum as well. Moreover, there are several situations that need to be considered:The OSSM is easy to calculate, but it has some application requirements. Firstly, the coal seam in the face should be fully mined at one time and the fallen method should be applied to control roof. Secondly, the application of the new method requires a complete grasp of many factors, such as the physical and mechanical properties of the overlying strata and the mining parameters of the mining face.As presented in Fig. Zhujiamao Coal Mine mainly mines No. 3 coal seam, with average buried depth of 160.37 m, dip angle of approximately horizontal and average thickness of 2.87 m, belonging to medium thick coal seam. The mining face is arranged with strike longwall, the comprehensive mechanized mining face with full-seam mining is adopted, and the roof is controlled with fallen method. As presented in Fig. In order to study the developing height of WPFZ in overburden of No. 3 coal seam, the exploration is carried out in No.3 panel. 3301 mining face is the first mining face in No. 3 panel. Thus, selecting 3301 mining face as the detection location can determine the WPFZ height after the first mining. Moreover, the risk of roof sandstone aquifer and Quaternary aquifer of No. 3 coal seam can be predicted and evaluated as well. Ultimately, all these findings can provide support for mine water prevention.The rock strata of 3301 mining face in Zhujiamao mine is presented in Fig. In order to more accurately determine the WPFZ height in the study area and verify the accuracy of the application of the OSSM, downhole segmented water injection method and borehole camera method are implemented in the field.(1) Detection principle33. The main equipment adopted is the water injection tube. Multiple holes are fabricated along the tube to allow high-pressure water injection into the borehole. Correspondingly, a water injection console is utilized to control the high-pressure water injection. In addition, two connected capsules are set at both ends of the tube simultaneously. The capsules can be pushed to any depth of the borehole because they are contracted in the non-working state. During the detection, these two capsules are filled with gas to seal the detecting segments of the boreholes. Similarly, a gas injection console is utilized as well to control the gas. The tube and the two consoles constitute the probe which is connected by pipelines and drill pipes, and the drilling rig enables the probe to move25.Figure (2) Detection schemeThe goaf of 3301 mining face is selected as the location to detect the WPFZ height. Factors, such as stability of surrounding rock, ventilation, pedestrian, water supply and court size should be taken into account in the selection of detection chamber. Combined with the engineering geological conditions, the detection chamber is arranged in the middle of rail crossheading of 3302 mining face. Three detection boreholes with different coordinate azimuths and dip angles are drilled on the roof of the same detection chamber. As a pre-mining detection borehole, borehole B1 is applied to detect the development of original fractures in overlying strata. For comparisons, boreholes B2 and B3 are applied to detect the development of fractures after mining. By comparing the water seepage in boreholes before and after mining, the WPFZ height can be accurately determined.The design of borehole parameters is the key to successful detection. Therefore, these following parameters must be satisfied: the dip angle must ensure that the probe can get into the WPFZ; the length must ensure that the probe can pierce through the WPFZ . The WPFZ height determined from the "overlying strata synchronous movement method" is 59.6 m. Considering the possible errors in the calculation, the WPFZ height is predicted to be 70 m, so as to fully guarantee the reliability of field detection. Hence, the lowest vertical heights of the detection boreholes are 70 m. According to the design requirements, the parameters are obtained by mathematical trigonometric calculation, as presented in Table (3) Detection resultsThe detection results indicate that the difference between the water seepage in detection boreholes before and after mining is apparent. Furthermore, the water seepage in detection boreholes presents obvious segmented characteristics after mining.The detection result of borehole B1 is presented in Fig. The detection result of borehole B2 is presented in Fig. Segment I: This borehole segment is located above the coal pillar. Correspondingly, the overlying strata are unaffected by the mining activity, and the water injection in borehole mainly seeps into the pre-existing fractures as well. The water seepage is basically the same as that of borehole B1, and the values are all below 5 L/min. This phenomenon is presented in the oblique length range of 36.2–38 m.Segment II: Compared with segment I, the water seepage increased significantly. Among them, the increase of water seepage is the highest in the oblique length range of 29–42 m, which is generally about 19.8–22.7 L/min, indicating that the borehole has penetrated into the fractured zone at this time. However, the water seepage decreases along the borehole, its value is still generally higher than that of borehole B1 or borehole B2 in the segment I, which indicates that the whole segment II is located in the WPFZ. This phenomenon is presented in the oblique length range of 29–74.4 m.Segment III: The water seepage generally decreases to less than 5 L/min, indicating that the water permeability of this segment is unsmooth and the borehole has penetrated the WPFZ. This phenomenon is presented in the oblique length range of more than 74.4 m.According to the detection result of borehole B2, the water seepage variation is the most obvious at the borehole oblique length of 74.4 m , which is the demarcation point. Therefore, it can be concluded that the WPFZ height detected by borehole B2 is 52.6 m.The detection result of borehole B3 is presented in Fig. To sum up, when the overlying strata are not affected by mining, the water seepage is low. However, a large number of fractures as water channels are generated in the overburden affected by mining. Ultimately, the comprehensive detected results indicate that the WPFZ height is approximately 52.6–55.8 m.(1) Detection scheme37. Borehole B3 is selected for this borehole camera detection, the detection scheme and equipment are present in Fig. In order to accurately determine the WPFZ height, the field detection follows the principle of one borehole for multiple purposes. On the basis of quantitative detection, the overlying strata fractures are observed directly by Panoramic Borehole Camera. Through borehole imaging, the development of rock fractures in the borehole is directly displayed in the form of images(2) Detection resultsThe images in Fig. Correspondingly, the range of WPFZ is presented beneath the images. It can be seen that the oblique length of the borehole is 88.7 m, that is, the vertical length is 54.6 m, which is the boundary of the WPFZ, from which the overburden enters into the outer part of the WPFZ and the inner part of the bending zone. Through the same borehole B3, the WPFZ height determined by borehole camera method is 54.6 m, while that determined by downhole segmented water injection method is 55.8 m, with an error of only 2%. Thus, it can be found easily that the research results are highly consistent, which verifies the reliability of the in situ detections.Based on the movement synchronous movement in overlying strata, a new method of OSSM is proposed to predict the WPFZ height. The method of OSSM is theoretical method. Compared with other methods, when it is applied, the grouping, lithology and thickness of overlying strata in the study area are necessary data, but these can be queried in the basic data of the mine. Therefore, the application of OSSM is convenient and cost-effective. There are many methods to predict WPFZ height, but each method has its own characteristics. Therefore, in order to choose the most advantageous method, the data reliability, operability and practicability of these methods need to be compared and evaluated.38. Through above researches, the detection results of borehole camera method and downhole segmented water injection method are relatively close. On the premise of maximum consideration of on-site safety, in order to facilitate comparative analysis, the highest prediction data of downhole segmented water injection method is selected as the benchmark. The comparison of WPFZ height obtained by each prediction method is presented in Table The WPFZ height is predicted by three different methods in this study, we calculate the WPFZ height by empirical formula of 3301 face in this part as well. Borehole detection method is the most reliable method to determine the WPFZ height at presentThe above exploration presents that each prediction method applied in this paper can predict the WPFZ height accurately, but the operability and practicability are various for each method.The relative size characteristics of fractures in overlying strata can be accurately obtained by downhole segmented water injection method, but the development shape of fractures can´t be observed. The sealing effect of the two capsules needs to be guaranteed in the field detection, and the construction details need to be paid attention to. In addition, the water injection probe needs to be pushed into the boreholes by a drilling rig, and the high-pressure water and gas need to be connected. Therefore, the workload of this method is strenuous.The borehole camera method can visually observe the development location and shape of the fractures, and the WPFZ height detected by this method is the most accurate. However, this method can´t accurately compare the size of the fractures between the borehole walls. Additionally, there are many unavoidable problems during observation, such as the detector stuck by broken rocks or obscured by mud and so on, which needs to be detected many times.Obviously, OSSM is a theoretical method, which can predict the WPFZ height easily. Compared with other methods, although the application of OSSM requires a comprehensive grasp of the geological and mining parameters, these are the basic parameters of mine production and can be easily queried. Therefore, the application of OSSM is convenient and cost-effective.To sum up, the prediction of WPFZ height should not solely rely on conventional rules or empirical formulas. Reliable research methods should be selected and implemented only after the geological conditions and mining conditions are adequately emphasized, which provides scientific design basis for safe mining.In this study, the formation factors affecting the WPFZ height are demonstrated comprehensively by scaling model experiment. Through the experimental phenomenon, the WPFZ height is closely related to the movement characteristics of key strata and its follow-up strata. According to this, the new method of OSSM is established to predict the WPFZ height, which is based on the synchronous movement in overlying strata.According to the calculation of OSSM, a reasonable WPFZ height range in 3301 mining face of Zhujiamao Coal Mine is predicted to be 59.6 m. The WPFZ height detected by downhole segmented water injection method is 52.6 m to 55.8 m, while that detected by borehole camera method is 54.6 m. These heights are similar to that calculated by OSSM, which indicates the reliability of the new method. Based on comprehensive consideration, the WPFZ height is finally determined as 52.6– 59.6 m in 3301 mining face.Various methods for determining the WPFZ height are evaluated. Borehole detection method is the most reliable method to determine the WPFZ height at present. However, the workload of borehole detection method is strenuous. The OSSM proposed in this paper is characterized by simplification and higher accuracy.In the future, the method of OSSM should be applied in more suitable mines to further verify its applicability and accuracy. Incidentally, this method should be further improved and optimized to reduce the application requirements and be more widely applied.


The aim is to evaluate the diagnostic value of Activin A levels in serum and pleural fluid on Parapneumonic Pleural Effusion (PPE).The authors collected serum and pleural fluid from 86 PPE and 37 Non-PPE (NPPE) patients. Including Activin A, levels of biomarkers such as Lactate Dehydrogenase (LDH), Procalcitonin (PCT), and C-Reactive Protein (CRP) were measured. All factors were calculated for association with days after admission. The diagnostic potential of biomarkers on PPE was considered by Receiver Operating Characteristic (ROC) curve analysis.Levels of Activin A in serum and pleural fluid of PPE patients were significantly higher than those of the NPPE patients. Moreover, concentrations of Activin A in pleural fluid showed a more obvious relevant days after admission. ROC curve analysis found that Activin A in pleural fluid had AUCs of 0.899 with 93% sensitivity and 84% specificity for PPE diagnosis.Activin A in pleural fluid correlated with disease severity could act to diagnose PPE. Approximately, pleural fluid concomitantly appeared in 40% of patients with pneumonia.Traditionally, it's necessary to distinguish exudate and transudate of pleural fluid via the quantifying ratio of Lactate Dehydrogenase (LDH) and album in the pleural fluid to serum for Light's Criteria Rule.,,As a foremost member of the Transforming Growth Factor-β (TGF-β) superfamily, activin A was recognized in several physiological functions including inflammation and tissue repair.In the present study, the authors aimed to evaluate the clinical value of Activin A level in serum and pleural fluid on the diagnostic potency of PPE.This retrospective study was carried out between Jan 2018 and Feb 2021 at Yixing people's hospital affiliated Jiangsu University. This study was approved by the Ethics Committee of Yixing People's hospital (20200022) and complied with the ethical standards of the Declaration of Helsinki. And all participants consent to participate in the study with their written names. 86 patients diagnosed with PPE aged ≥ 18 years were enrolled. And enrolled subjects were excluded from the following criteria, (1) Age < 18 years; (2) Pregnancy; (3) Malignant tumor; (4) Serious renal or heart or liver diseases. 37 patients with Non-Parapneumonic Effusion (NPPE) were also selected for the present study. All participators were categorized as PPE and NPPE groups.The authors recorded the related clinical characteristics and laboratory parameters from peripheral venous blood and pleural fluid within 24h after admission. Basic parameters including White Blood Cell Counts (WBC), CRP, PCT and LDH were collected.PPE as a type of pleural lesion was diagnosed according to the criteria.The peripheral venous blood between 6 am and 7 am and pleural fluid within 24h after admission was prepared to check the concentrations of Activin A by using Enzyme-Linked Immunoassays (ELISA) kits  according to the manufacturer's recommendations.U test.Categorical variables between groups were compared using by The authors enrolled 86 patients diagnosed with PPE and 37 with NPPE. Among the NPPE group, 16 were confirmed as malignancy pleural fluid with pleural cytology report, 17 were diagnosed with transudate pleural fluid and 4 were linked to other pathogenesis. The authors investigated the common biomarkers including WBC counts, CRP, PCT, and LDH in a blood sample, and found that there were differences between PPE and NPPE patients. Moreover, inflammatory factors including WBC counts and LDH in the pleural fluid were also significantly different between the two groups . FurtherIt's necessary to earlier definite PPE associated with clinical options and prognosis. The authors evaluated the efficacy of Activin A in predicting PPE by assessing the ROC curve. To the best of our knowledge, it's firstly to assess concentrations of Activin A both in serum and in pleural fluid for diagnosis of PPE. In the present study, elevated levels of serum and pleural fluid Activin A appeared in PPE compared with NPPE patients. Furthermore, increased levels of Activin A in the pleural fluid were determined to be associated with days after admission. Pleural fluid Activin A had acceptable sensitivity and specificity to diagnose PPE.,As a consequence of pressure, transudative fluid outcome in the pleural space. Coupled with increased inflammatory cells in the pleura, pleural fluid as an exudative fluid developed with complex etiological factors.,Activin A was proven to participate in the development of pulmonary inflammation.Once diagnosed, a therapeutic strategy including enough antibiotics and proper drainage is necessary. Patients with severe cases of PPE always suffered from longer days after admission with standard treatment regimens. Therefore, it's essential to proactively determine the severity of their condition, in order to identify patients who would need aggressive interventions earlier. In this study, all PPE patients were made in drainage as standard therapy. In order to evaluate the association between Activin A and disease severity, the authors used the number of days after admission to represent the severity of PPE. The data of the present study confirmed that Activin A in pleural fluid correlating with days of admission is just an appropriate indicator of severity in PPE.As a retrospective study, the present study faced some limitations. First, the numbers of NPPE patients are fewer, and it's difficult to classify subgroup analyses with the different pathogen of pleural fluid. Second, meanwhile, the authors have not compared the levels of Activin A in BALF, serum, and pleural fluid. Third, levels of Activin A in pleural fluid at different time points after admission were unknown.The present study revealed that Activin A concentration in pleural fluid exhibited a capacity for diagnosis of PPE and for gauging the severity of the disease. The present data supported a close relationship between levels of Activin A and local inflammation in the pleura. Whether it is suitable to distinguish PPE from multiple etiologies of pleural fluid needs further investigation.Liu K, Zhou G and Ji X prepared the samples and carried out the data analysis. Fen Y collected samples. Gu Y and Ding H designed the project. Ding H wrote the manuscript.The authors declare no conflicts of interest.