The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus. Rabies is a neglected disease that primarily affects poor rural communities of the developing world. Lack of surveillance, related to limited diagnostic capabilities, contributes to the underestimation of the burden of this disease. Here we report an evaluation of the direct immunohistochemical test (dRIT) as a method for routine rabies diagnosis in southern Africa. The dRIT has potential as a practical and cost-effective test that may improve rabies diagnostic capacities where it is most needed, and with this work we hope to contribute to the advancement of the dRIT as a more generally accepted and applied method. For the first time, we have evaluated a modification of the dRIT in which a polyclonal antibody preparation was biotinylated and compared to the monoclonal antibodies used for the development of all subsequent experimental applications of the dRIT to date. We conclude that the dRIT is a superior test for rabies diagnosis that is easily adaptable to tolerate the use of different antibody preparations. We further demonstrate that the assay should be optimized with respect to the virus variants of the region where it is to be implemented. Lyssavirus, Family: Rhabdoviridae), of which the prototype is rabies virus (RABV). While RABV is most important from a global disease perspective, there are more than 12 other lyssavirus species, most of which have been associated with infrequent cases of human rabies Rabies is a neglected zoonosis that is responsible for the death of tens of thousands of people per annum In the case of resource-limited developing countries, where limited or no diagnostic confirmation is undertaken, very little rabies data are reported to relevant authorities. In some instances it has also been found that even though limited diagnosis may occur, the diagnostic results are not reported to the relevant authorities at all. This appears to be due to various logistical reasons, such as a lack of record keeping, limited communication, etc. The gold standard assay for rabies diagnosis is the direct fluorescent antibody (DFA) test To date, studies on the dRIT method have utilized monoclonal antibody (MAb) preparations  in South Africa and a number of southern African countries http://www.piercenet.com/haba/) to determine the molar ratio of biotin to the polyclonal antibody.The PAb preparation used in this study was produced at the Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI), Rabies Division, by immunizing goats with purified ribonucleoprotein (RNP) antigens obtained from two lyssavirus species ) according to the standard operating procedure To date, biotinylated MAbs have been used routinely as a cocktail of highly concentrated antibodies. During this study, however, the diagnostic efficacy of two individual MAbs was investigated, as previously described Animal rabies is a notifiable disease in South Africa. State veterinarians had submitted central nervous system (CNS) tissue samples to the OIE Rabies Reference Laboratory, the ARC-OVI, Rabies Division, for routine rabies diagnosis based on suspicion of rabies. As part of its mandate, the Reference Laboratory performs routine rabies diagnosis for disease control and management of bite victims on behalf of the Department of Agriculture Fisheries and Forestry (DAFF).All the CNS samples for the rabies-related viruses used in this study were from virus stocks that had been prepared according to the diagnostic procedures of the Rabies Laboratory at the ARC-OVI, Rabies Division . The other samples used in this study were selected from a much larger set of archived samples submitted to the ARC-OVI, Rabies Division, for routine rabies diagnosis over a period of two years (year: 2011–2012), while a sub-set of 30 archival samples (year: 1999) were included. This study thus only relied on archived CNS tissues and did not require specific ethical clearance as no live animal models were used but permission was granted by the Director of the Institute.“The animal experimental protocols, animal caging and care, as well as end point for the animal experiments performed at the ARC-OVI, Rabies Division, were approved by the Animal Ethics Committee for the use of living vertebrates for research, diagnostic procedures and product development  under 15/4/P001.”Canis familiaris; n = 132), domestic cat , black-backed jackal , bat-eared fox , yellow mongoose  and cattle   samples. Of the 255 samples, 249 were derived from the following mammalian species: domestic dog ( n = 27) . The speThe remaining CNS tissue samples (n = 6) had been derived from mouse models, each infected with one of six southern African representative lyssavirus isolates . The souPrior to performing diagnosis on the chosen samples, each of the CNS samples was placed in a sterile petri dish and small pieces of tissue were removed from multiple sites to ensure that viral antigens were obtained from representative segments of the CNS sample. The composite samples were homogenized using a mortar and pestle to facilitate an efficient supply of viral antigens throughout each of the investigated samples.All the samples included in this study (n = 255) were tested initially with the DFA test to determine the immunoreactivity scores associated with each of the samples The dRIT diagnostic assay was performed in triplicate on all the CNS samples according to the published standard operating procedure using one of the three-biotinylated antibodies , as described The total RNA extraction of any false RABV-positive CNS samples was performed using the Trizol reagent (Sigma-Aldrich) according to the manufacturer's instructions and the subsequent viral cDNA synthesis was performed according to a protocol published previously Both the forward and reverse strands of the purified PCR amplicon were sequenced using the hn-PCR primers and the BigDye Terminator v3.1 sequencing reaction kit according to the manufacturer's instructions (Applied Biosystems). The sequencing reactions were precipitated according to the manufacturer's instructions (Applied Biosystems) and subsequently sequenced using an ABI 3100 automated capillary sequencer . The sequences obtained from both the forward and reverse primers were used to create a trimmed consensus sequence of 466 nt using CLC Main Workbench  and subsequently subjected to a BLAST analysis on the GenBank website.After the assembly of the consensus sequence, an alignment was created using the ClustalW subroutine of the BioEdit software All RABV-positive tissue samples that produced apparent false-negative dRIT results with any of the biotinylated antibody preparations were subjected to antigenic typing and a molecular analysis to investigate the origin of the discrepancies in the observed immunoreactivity.All the aforementioned samples were typed as either a canid or mongoose RABV variant using a panel of sixteen MAbs supplied by the Centre of Expertise for Rabies  The total RNA extraction was performed using the Trizol reagent (Sigma-Aldrich) according to the manufacturer's instructions, and an established “one-step” quantifying real-time PCR assay The determination of the diagnostic sensitivity, specificity, Cohen's kappa value and respective confidence intervals of the dRIT diagnostic assays was determined using an exact binomial distribution .Of the 255 samples tested in the study, the PAb dRIT diagnostic assay produced one false-positive result in comparison to the DFA test. In contrast, the dRIT diagnostic reaction, relying on either of the two-biotinylated MAbs (MAb 1 and MAb 2), apparently produced several incorrect results. The dRIT diagnostic assay relying on MAb 1 produced 34 false-negative results and a single false-positive result, while the dRIT diagnostic assay relying on MAb 2 produced 19 false-negative results and two false-positive results.Two samples that were characterised as lyssavirus-negative according to the DFA test, tested positive with the dRIT diagnostic assay. The first sample . The RABV 567/04 sequence belonged to the canid RABV variant, which was isolated from the same province of South Africa  in 2004 Canis familiaris, n = 4; Felis domesticus, n = 14; Canis mesomelas, n = 1; Cynictis penicillata, n = 14; Bos taurus, n = 3) gave false-negative results with either one or both of the dRIT assays using the MAbs  and negative (n = 49) samples was used to perform the statistical analysis of the final results . The DFAIn contrast to the findings of the pilot dRIT studies released to date The theoretical diagnostic sensitivity and specificity of the dRIT assay using the MAb cocktail was also calculated based on the assumption that false-negative results would only occur when both MAbs produced false-negative results and false-positive results would occur when a single MAb produced a false-positive result . The theThe general applicability of a novel diagnostic assay, in terms of providing a reliable test that can supplement or replace the gold standard DFA test, is primarily determined by the diagnostic sensitivity and specificity of the suggested alternative. Regardless that the diagnostic effectiveness is one of the most important factors, assays must be easy to perform and interpreted in a qualitative manner. In addition, the test should rely on reagents and equipment that are inexpensive and easy to procure and maintain.Previous published applications of the dRIT Thus, an unlabelled PAb, prepared in goats at the ARC-OVI in South Africa, was conjugated to a biotin moiety and the PAb dRIT assay applied to a cohort of samples in parallel with the recommended DFA assay. The results of this study indicated that the PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was marginally higher than that of the DFA. Despite the single false-negative result associated with the DFA test, the diagnostic efficacy of the assay was still within the acceptable limits (98–100%) recommended by the WHO The two individual MAbs had a significantly lower diagnostic sensitivity, which was directly attributed to the high number of false-negative results produced by the MAb-based dRIT diagnostic reactions on some, but not all, of the mongoose RABV specimens. The antigenic typing of the RABV variant that produced false-negative results, as well as the remaining mongoose samples that were correctly diagnosed as RABV-positive by both MAbs, were all found to be associated with the mongoose variant of the RABV. To our knowledge, this was the first application of the dRIT assay using either of the two-biotinylated MAbs (MAb 1 or MAb 2) to the mongoose RABV variant. The inclusion of an African mongoose RABV variant thus resulted in the first demonstration whereby the classical dRIT has shown a reduced diagnostic efficacy. However, the two MAbs used in the study were supplied as “ready-to-use” solutions, and as such the working dilutions could not be optimized on the sample cohort included in this study. To investigate that the working dilution of each of the MAbs might have been over-diluted, a quantitative real-time PCR analysis of the RABV RNA concentration in each of the samples was performed. Although not indicative of the number of assembled RABV particles, the RNA concentration was used to determine whether a cut-off value could be determined whereby the inconsistency of the dRIT assay using the MAbs could be explained. Because no clear cut-off values in terms of the viral RNA copy number could be determined between the specimens that produced true-positive and false-negative results, the hypothesis that the MAbs had been over-diluted prior to shipment could not be easily addressed adequately. We also argue that it is not copy number alone that influences avidity, but the relative availability of antigenic sites recognized by a given MAb based upon variant epitopes, largely determined based on the panel of viruses used for the optimization of the assay. For the MAbs used here, the majority of the test panel had been primarily New World wildlife variants, including viruses isolated from mongoose (Caribbean), which are unlike mongoose viruses from southern Africa. With this in mind, any future application of the dRIT assay, using any biotinylated antibody preparations, should be preceded by the optimization of the antibody preparation for the given assay based upon variants of public health importance to the region. Clearly, this was an unequal advantage in the protocol evaluated here, because the MAb preparations were pre-diluted based upon other RABV variants whereas the PAb was optimized based upon local production.Another potential explanation for the observed false negative results could be that the two-biotinylated MAb preparations interact with single epitopes on antigenic sites that are less conserved in certain mongoose variants present in sub-Saharan Africa. The two-MAbs used in this study each interact with a different epitope on the nucleoprotein. A single change at the nucleotide level could theoretically result in a change in the translated amino acid sequence of the virus, resulting in altered epitopes with which the MAbs cannot interact. Despite the obvious advantages that MAb preparations have in terms of the lot-to-lot consistency, sustainable supply from immortal cell lines without the further use of animals and comparatively inexpensive costs The application of the dRIT diagnostic reaction produced no false-negative results when applied to the six representative southern African rabies-related isolates. Although not all of the currently known African lyssavirus isolates were included in this study, the successful diagnosis of the representative isolates was used as an initial proof of concept to highlight the utility of the dRIT diagnostic assay as applied to antigenically and genetically distinct lyssavirus species.Although numerous novel diagnostic assays, such as molecular amplification and lateral flow immunochromatographic assays (rapid test kits), are being developed and may show promise in terms of becoming viable options for supplementing the DFA test, they are still under development. Such diagnostic tools might have potential future applications, and the widespread implementation of the highly reliable diagnostic assays that are currently available should be encouraged. In this study, the dRIT diagnostic assay has been shown to be one such option. The dRIT test, using the biotinylated PAb preparation, has a diagnostic sensitivity and specificity that is marginally higher than that of the DFA. This fact thus justifies further evaluation of the dRIT diagnostic assay on a global scale to ensure the widespread application of this diagnostic assay, corroborate our preliminary findings and continue to compare the utility of MAbs in the dRIT when antibody concentration and cocktail optimization is not a limiting factor to experimental design.Checklist S1STARD checklist.(DOC)Click here for additional data file.Table S1Sample details and relevant immunoreactivity scores associated with the direct fluorescent antibody test and direct rapid immunohistochemical test as performed on various southern African maintenance hosts.(DOCX)Click here for additional data file.Table S2Sample details and relevant immunoreactivity scores associated with the direct fluorescent antibody test and direct rapid immunohistochemical test as performed on six representative rabies-related lyssaviruses from southern Africa.(DOCX)Click here for additional data file.


Although the microstructure of coexistence phase provides direct insights of the nucleation mechanism and their change is substantial in the phase transition, their study is limited due to the lack of suitable tools capturing the thermodynamically unstable transient states. We resolve this problem in computational study by introducing a generalized canonical ensemble simulation and investigate the morphological change of the nucleus during the water evaporation and condensation. We find that at very low pressure, where the transition is first order, classical nucleation theory holds approximately. A main nucleus is formed in the supersaturation near spinodal, and the overall shape of the nucleus is finite and compact. On increasing the pressure of the system, more nuclei are formed even before spinodal. They merge into a larger nuclei with a smaller free energy penalty to form ramified shapes. We suggest order parameters to describe the extent of fluctuation, and their relation to the free energy profile. Nucleation is a key process that initiates phase transition or structural formation in nature23456789101112131415161721315Conventional numerical simulations could not detect these unstable transient states. Instead, these researches focus on the high supersaturation regions near the spinodals (or stability limits), where transitions may occur within the simulation accessed time scale20In this work, we extend the accessible states of simulations to the whole intermediate regions between two phases. For this purpose, we develop a new simulation strategy, the generalized canonical ensemble (GCE) simulations combined with the replica exchange molecular dynamics (REMD) techniquesWe apply the GCE-REMD to water evaporation and condensation which are the most frequently encountered phase transition in daily life, but still not well understood. The result of a Lennard-Jones system is also presented as comparison. Most previous computational studies on water evaporation focused on supersaturations region, up to spinodals2324252627N is 250 for both the systems. We also simulate in larger systems (N = 1000) at a few typical pressures in order to check the finite-size effects. Sufficiently high exchange rate through the replicas ensures the equilibrium in our simulations in the whole enthalpy space, including the stable gas/liquid phases, the metastable supercooled gas and superheated liquid, as well as the gas-liquid phase coexistences, therefore the samplings satisfy the expected equilibrium distribution of the GCE, which can convert to the equilibrium distribution in the normal canonical ensemble (We perform GCE-REMD simulations under a constant pressure constraint for both the Lennard-Jones(LJ) fluid and a coarse-grained water model, the mW modelensemble . The GCEP = 0.016 to 0.104 near the critical point (dimensionless unit). We estimate the critical point as i. e., the temperatures in which the number of liquid particles equals to that of gas particles at different pressures. The snapshots are sampled without any bias inside each intermediate iso-enthalpy subspace, thus they are regarded as typical intermediates during the phase transition. It clearly shows that the micro-structure of the intermediates changes from nucleus to bicontinuous coexistence on increasing pressure. At very low pressure , the curves represent the first order phase transition. During the phase transition, the densities at two phases are significantly different. The interfacial energy is large enough to confine the minor phase within a finite volume and the interface deforms a little from the spherical surface. On increasing pressure, the interface between two phases becomes rough and the minor phase has a branched structure. At high pressure, densities at gas and liquid spinodals converge. The interfacial energy becomes negligible and the thermal fluctuations are dominant, which leads to the inter-connected continuous interfaces.In the bottom panels of To analyze this quantitatively, we divide the simulation configurations into liquid clusters and gas clusters based on the Voronoi cell analysis: a particle is identified as liquid-like if its Voronoi cell volume is less than a threshold volume, and it is gas-like otherwise. The threshold volume is determined by analyzing the distribution of Voronoi cell volume in both the liquid and gas phases . Particls, as a function of the enthalpy of per particle. HereIn A is the total area of all the interfacial Voronoi cell planes between liquid and gas clusters, and where N = 1000) for both the LJ and mW water  cluster, j is the index of the particles inside the largest cluster, and j inside cluster. In g ≲ 125, as where H subspace, and thus the pathway of nucleation  can be constructed along the enthalpy. Therefore, the structural change at coexistence during the gas-liquid phase transition of the mW water and LJ systems at various pressures provides the mechanism of the phase transitions. At very low reduced pressure, the phase transition is followed by CNT or its slight modification, since the interfacial energy is large enough to confine the minor phase into a finite size nucleus. Here the reduced pressure means the relative pressure to the critical value. On increasing pressure, the interfacial energy is reduced. The thermal fluctuations easily overcome the deforming energy of the surface of nuclei and the nucleation gradually deviates from the CNT. At high enough reduced pressure, whose value depends on systems, the fluctuation length exceeds the periodic domain size of the system and the system develops a bicontinuous morphology. We believe that this continuously transit to the real decomposition at the critical point.Although the enthalpy may not be a perfect reaction coordinate of the phase transition, it can be a good approximation. So the sampled conformations in the GCE-REMD with successive enthalpies provide an approximate pathway of the nucleation process. In addition, the simulation is unbiased on each iso-Our findings on the morphology, beyond the spinodals and its dependence on pressure encompassing the first order to near second order phase transition, provide an integrated view in the kinetics of the nucleation process. Since the connectivity of the nucleus is finite in the first order phase transition, but diverges in second order phase transition, it also can be served as an indicator to distinguish between the first order and the second order phase transition. Because, our results prove that the free energy cost forming nucleus should strongly depend on the the microscopic structure of the nucleus, it will lead to the development of new theories of nucleation and first order phase transition that conisders the microstructure of the nucleus.H,In the GCE simulations, the number of particles, and the volume (or here pressure) of system are fixed as the canonical ensemble, but the temperature is expressed as a linear function of the enthalpy γ, and P. V. Thermodynamic stability is judged by where, H that satisfies γ in the coexistence region, which satisfiesFor Within this approach, we access the spinodals and the intermediate states.In thermodynamics, the GCE at fixed pressure is equivalent to the NPT ensemble by introducing an effective potential,Its conformational distribution isand thus we can keep the conventional MC or MD scheme with the effective potential. Technically, here the GCE is implemented as the standard NPT molecular dynamics simulation by scaling the total physical force of each particle by the factor By varying the set of parameters, the GCE can detect any enthalpy region, including the stable liquid/gas regions, the metastable supersaturation region, and the coexistence region. However, similar to the standard canonical ensemble (CE) simulations, a single GCE simulation might not be easy to be ergodic within a finite simulation time, the enhanced sampling techniques developed in the CE can be applied to the GCE to shorten the equilibrium time of simulations. Here we have incorporated the replica exchange molecular dynamics (REMD) method to form the GCE-REMD. The implementation of the GCE-REMD is directγ, is applied to constraint simulation around the center of spring γ, that satisfies The GCE may look similar to the umbrella sampling simulation, and so does the GCE-REMD to the umbrella sampling replica exchangeγ.Here x, the free energy profile at a special temperature T is obtained from the GCE-REMD simulations,For any order parameter, H in the GCE-REMD simulation, respectively, depend on the selection of parameters in the simulations, x is the H itself, we have, x is properly chosen as a real reaction coordinate, x is independent of the GCE simulation, it does not bring any possible bias in simulation to understand the nucleation as in previous methods20Here the conditional probability, 7 time steps) by 64 replicas is about 30atm for the smaller mW water system. To check the dependence on the initial conditions, the simulations start from two different initial conformations (liquid and gas), and at equilibrium we obtain the same results regardless of the initial conditions. In the larger mW system, we only simulate at two pressures 30 and 60 atm for checking the possible finite-size effects. In both cases, we slightly decrease the enthalpy range of simulations to make the enthalpy overlapping between neighbouring replicas not very small thus the simulation can reaches the equilibrium within tens million MD steps. We also have checked the possible effects of the cutoff distance of the interaction by varying In this work, we focus on the morphological change of coexistence in the mW water (and LJ) by varying the enthalpy. We simulate systems with two different number of particles, the smaller one has 250 particles, and the larger one has 1000 particles. The detailed simulation parameters are listed as  systems . As we hHow to cite this article: Jeong, S. et al. Micro-structural Change During Nucleation: From Nucleus To Bicontinuous Morphology. Sci. Rep.5, 15955; doi: 10.1038/srep15955 (2015).


Recent clinical studies confirmed the high potential of MR-guided focused ultrasound (MRgFUS) in the field of functional neurosurgery. While its ability for precise thermo-ablation within soft tissue is widely recognized, the impact of high-intensity focused ultrasound (HIFU) on larger vessels is less explored. We used a bifurcation aneurysm model in rabbits to investigate the possible effects on the walls of vascular aneurysms and to assess the risk and prospect of this procedure for managing neurovascular disorders.Experimental bifurcation aneurysms were microsurgically created in New Zealand white rabbits and sonicated using MRgFUS.A temperature of max. 54°C could be achieved close to the aneurysm, and the shape and size of the aneurysm were noticeably changed, as shown by MR angiography.The presented rabbit model proved suitable and capable of being extended to acquire data on the effect of HIFU on aneurysms and larger vessels. The fact that HIFU led to an alteration of the aneurysm without inducing rupture encourages further investigations. High-intensity ultrasound focused into a small volume is known to generate heat, which enables precise and non-invasive thermo-coagulation of tissue. WhereasThe study protocol was approved by the Swiss Animal Care and Experimentation Committee . All aneurysms were created in adult female New Zealand white rabbits (3–4 kg). A detailed description of the aneurysm creation is presented elsewhere. In brieUnder an operating microscope, a 1-cm segment of the external jugular vein was harvested and used as a venous pouch. Both common carotid arteries (CCA) were prepared, and after ligation, the stump of the right CCA was mobilized to the left CCA. After an elliptic arteriotomy, the stump of the right CCA and the venous pouch were sutured to the left CCA using an end-to-side anastomosis with 10-0 thread , resulting in an artificial bifurcation aneurysm  integrated into a clinical 3T MR system . The hemispheric phased array ultrasound transducer with 1,024 elements was driven at 650 kHz and oriented face up, filled with degassed water, and covered with a transparent film. After hair removal, the neck of the anesthetized rabbit was positioned on the film such that the aneurysm was aligned with the acoustic focus of the transducer. Tape was used to facilitate appropriate positioning and to stabilize the animal torso . As a result, the shape and size of the aneurysm were noticeably changed, as shown by MR angiography after the procedure  into the vascular system, which may enhance the efficacy of the focused ultrasound[Due to its capacity for image guidance and precise delivery of ablative heat, MRgFUS is especially interesting in the setting of brain diseases, for which a narrow area of functional anatomy as well as limited intraoperative visual orientation often impairs safe access to lesions. While recent clinical results of MRgFUS in functional neurosurgery for tremor and chronic pain are highly encouraging,3, possitrasound. By remoBy slowly and moderately increasing thermal energy using MRgFUS, we could show that—despite the heat convection through high blood flow—it is possible to reach ablative temperatures at the aneurysm and to modify its shape without inducing rupture—this being a possible risk brought on by the mechanical effect of ultrasonic beam. Even if aneurysm modification with FUS may not be viable in the near future, it is important to investigate possible interferences, e.g., for the feasibility and risk assessment of patients harboring an aneurysm near a lesion to be treated with FUS. Given the relatively few animal models for larger vascular lesions such as AVM or cavernomas, the presented model offers valuable options to acquire data on the effect of FUS on larger vascular structures.MRgFUS: MR-guided focused ultrasound; FUS: focused ultrasound; CCA: common carotid artery; VP: venous pouch; an: aneurysm; tr: trachea; sc: spinal cord.The authors declare that they have no competing interests.DC carried out the surgical procedures in animals and was responsible for planning and conduction of the experiments, data analysis, and writing of the manuscript. JF was responsible for the study concept and planning of the treatment and revision of the manuscript. SM, SE, and JK contributed to the surgical procedures in animals. EM contributed to the planning of the experiments and revision of the manuscript. BW was responsible for planning and conduction of the experiments, the overall technical setup, the data acquisition and analysis and writing of the manuscript. All authors read and approved the final manuscript.


We present a two-stage approach for the “helicopters and vehicles” intermodal transportation of medical supplies in large-scale disaster responses. In the first stage, a fuzzy-based method and its heuristic algorithm are developed to select the locations of temporary distribution centers (TDCs) and assign medial aid points (MAPs) to each TDC. In the second stage, an integer-programming model is developed to determine the delivery routes. Numerical experiments verified the effectiveness of the approach, and observed several findings: (i) More TDCs often increase the efficiency and utility of medical supplies; (ii) It is not definitely true that vehicles should load more and more medical supplies in emergency responses; (iii) The more contrasting the traveling speeds of helicopters and vehicles are, the more advantageous the intermodal transportation is. Timely relief supplies transportation takes an important role in emergency responses, directly affecting the efficiency and effectiveness of disaster relief ,2,3. FacIn this work, we focus on a kind of disaster response situation: Due to the long distance among supply nodes and demand nodes or the cut off of key roads to affected areas, helicopters are used to transport medical supplies to temporary distribution centers (TDCs) and then vehicles at TDCs are used to transit the received medical supplies to medical aid points (MAPs).Helicopters are not subject to existing transportation networks and can fly straight to affected areas, which could sharply shorten the delivery time of medical supplies.Helicopters can take off and land vertically at relatively small places. Thus, it is flexible and quick to select and clear up places as TDCs for receiving medical supplies from helicopters.In destructive disasters such as earthquakes and floods, the cut off of key roads often makes helicopters the most effective transportation mode to isolated affected areas.The above intermodal transportation of helicopters and vehicles has been gradually used in emergency practices due to its specific advantages in challenging disaster situations:However, due to the limited number of helicopters, it is difficult or impossible to transport medical supplies to every MAP by helicopters in large-scale disaster responses. In general, TDCs are set up for receiving badly needed medical supplies from helicopters and then transiting the supplies to each MAP by vehicles. Since helicopters could take off and land vertically at relatively small places, in the work we assume that any place in affected areas could be located as TDCs.Stage I: A fuzzy-based method is presented to determine the locations of TDCs and assign MAPs to each TDC. In this stage, the intermodal transportation network is constructed. Stage II: Based on the constructed intermodal transportation network, an integer-programming model is built to produce the delivery routes. Our proposed approach has potential applications in some challenging emergency situations such as vaccine delivery in large-scale infectious diseases, blood transportation after earthquakes, and antidote delivery in chemical and biological attacks.There are two subproblems that need to be solved: one is where TDCs should be located, and the other is how to arrange the delivery routes. In this work, we develop a two-stage approach for the above intermodal transportation problem. The remainder of this paper is organized as follows. In Many studies have been reported to achieve solutions for emergency logistics problems ,5,6. In et al. built a facility location model of medical supplies and developed three heuristic algorithms to solve the model m×n such that 0 ≤ iju ≤ 1 and  ;Step 3: Use  where t represents the iteration step to calculate thelocations of TDCs ;Step 4: Use  to get the value of the objective function; if , then stop the iteration and turn to Step 6 with the values of , Ut)( and Jt)); output , Ut)( and NCi(t).After selecting the locations of TDCs and assigning MAPs to each TDC in Stage I, the intermodal transportation network could be determined. Then, we need to plan the delivery routes in Stage II. In the work, we assume that each TDC is served by one helicopter in one delivery interval, so the helicopter routes are determined after we select the locations of TDCs. In this section, we develop an integer-programming model to arrange the vehicle delivery routes from TDCs to their covered MAPs.As mentioned in Thus, the optimization objective of arranging delivery routes, that is, to minimize the total duration time of all intermodal routes, is formulated as: denotes the number of used vehicles starting from the ith TDC, which is also equal to the number of vehicle routes from the ith TDC, so  denotes helicopter travel time considered in the intermodal duration time for MAPs covered by the ith TDC;  denotes vehicle travel time considered in the intermodal duration time for MAPs covered by the ith TDC.In the above formulation, The constraints of the optimization objective Equation (9) are as follows: represents the number of vehicles visiting the jth MAP.Constraint Equation (10) ensures each MAP is served only once by one of the vehicles in the delivery interval, where ith TDC  is equal to the number of vehicles returning to the ith TDC .Constraint Equation (11) ensures the number of vehicles leaving from the Constraint Equation (12) guarantees the number of used vehicles does not exceed the number of available vehicles at each TDC. represents the number of vehicles arriving at the jth MAP and  represents the number of vehicles leaving from the jth MAP.Constraint Equations (13) and (14) ensure the vehicle which arrives at one MAP should leave from the MAP, where  represents the total quantity of medical supplies allocated to MAPs served by vehicle k and  represents the total load of vehicle k when leaving from the ith TDC.Constraint Equation (15) guarantees any vehicle loads the equivalent quantity of medical supplies allocated to MAPs served by the vehicle, where k between the jth MAP (or the ith TDC) and the lth MAP (or the ith TDC) does not exceed the maximum capacity of vehicle k if traveling from the former to the later. Constraint Equations (15) and (16) together guarantee that the overall available capacity of a given vehicle does not exceed its maximum capacity.Constraint Equation (16) guarantees that the available load of vehicle Constraint Equation (17) defines the ranges of variables.Meanwhile, we also formulate two performance metrics for the intermodal transportation.(1) Average arrival time (AAT) represents the elapsed time from the ith TDC to the jth MAP. Note that the vehicle return time at TDCs is not included.The optimization objective (9) mainly reflects the total duration time, but it does not specifically consider the utility of medical supplies. The utility of received relief supplies is different for each MAP if the arrival time is different ,19,25. T(2) Biggest traveling time (BTT)In emergency practices, the deadline policy is usually adopted, that is, the needed supplies must be transported to affected areas in some regulated period  after the occurrence of disasters. Motivated by this observation, we formulate the biggest traveling time as one performance metric of the intermodal transportation:As we can see, if only in terms of BTT, the shorter it is, the better the transportation performance is.In this section, we conduct numerical experiments to verify the effectiveness of the developed approach, and observe several insights for real-world emergency responses.jR columns in Assume that some area is infected by an infectious disease, and sixty hospitals are set as medical aid points (MAPs) for providing medical service to victims. Considering the generality, we randomly generate the coordinates of the sixty MAPs from 0 to 200, as ω = 2, ε = 1 × 10-5, and the maximum number of iterations is set as 100.Using the proposed method for selecting TDCs and assigning MAPs in To show the details, we present the results when the number of TDCs is set as four. After 43 iterations, the algorithm reached its termination criterion, with the objective value (2) being 62,411.0128. The detailed results are as shown in Similarly, we obtained the detailed results with the number of TDCs ranging from two to 15, as (1) As the number of TDCs increases from two to 15, the value of objective function (2) definitely decreases. This means the sum of distances among TDCs and MAPs will be shortened with the increase in the number of TDCs.(2) The decrease of the value of objective function (2) is diminishing as more TDCs are selected. In particularly, when the number of TDCs increases to 60 (the number of MAPs), the sum of distances among TDCs and MAPs will decrease to 0, as (3) The number of termination iterations is not in obvious relevance to the number of TDCs. All the termination iterations are within 100 , and the longest running time of all the iterations is 5.61 Sec.In the following section, we will generate different transportation routes with different numbers of TDCs, and analyze the performance of the intermodal transportation with these selected TDCs.vV as 1.After selecting TDCs and assigning MAPs in vQ as 5000 and helicopter travel speed hV as 10.In order to observe the impact of the number of TDCs on the performance of the intermodal transportation, in this subsection we set the maximum capacity of each delivery vehicle In order to show the detailed effects of the number of selected TDCs on the intermodal transportation routes, we compare the results with two and four TDCs. From the results in (1) With the increase of the number of TDCs, the total duration time would be shortened with a decreasing extent. This is because more TDCs could divide MAPs into smaller groups, which will shorten the vehicle traveling distances, as (2) The average arrival time has an approximately fluctuating decrease as the MAPs are assigned to more TDCs. As the number of MAPs covered by TDCs decreases, the arrival time at each MAP tends to be shortened. The fluctuation is due to the uncertainty of the number of MAPs in the partitioning. As analyzed in (3) As shown in (4) Although the number of total MAPs is not changed, the intermodal transportation with increased TDCs will use more medical vehicles, as In above subsection, we set the maximum capacity of medical vehicles as 5000, but the setting may affect the maximum delivery number of MAPs by each medical vehicle. In this subsection, we will investigate the impact of different vehicle maximum capacities on the performance of the intermodal transportation. In order to keep the comparability, we set the number of selected TDCs as 4, and then increase the vehicle maximum capacity from 2000 to 15,000 by 1000. Results are From the results in (1) As vehicle maximum capacity is enlarged from 2000 to 15,000, the total duration time would decrease. Vehicles with bigger capacities can serve more MAPs, which will decrease the total number of used vehicles. For the given number of MAPs, the decreased in-service vehicles could reduce the total travel distance. However, the decreased extent would become small after the vehicle maximum capacity exceeds 4000, as (2) Both average arrival time and biggest traveling time have an increasing trend with the enlargement of vehicle maximum capacity, respectively changing from 62.00 to 166.57 and from 175.09 to 455.03. As analyzed above, the increased traveling distances will also lengthen the average arrival time and biggest traveling time. In details, the two curves increase relatively gently before the vehicle maximum capacity exceeds 9000, but then with sharp increases.Average arrival time has a direct impact on the availability and utility of medical supplies at MAPs, and biggest traveling time to some extent represents the completion time of the delivery task. Thus, in emergency responses, it is not definitely true that vehicles should load more and more medical supplies. In order to enhance the utility and efficiency of medical supplies, decision makers should make use of medical vehicles with small capacities or not load big vehicles to full capacity if there are available medical vehicles, especially when TDCs cover less MAPs.We further compare the performance of the intermodal transportation with different helicopter traveling speeds. For comparability, we also analyze the results with four TDCs and the vehicle maximum capacity is still set at 5000. Changing the helicopter travel speed from one to 10, the results are as The results in In developing a solution to a kind of intermodal transportation problem in emergency responses, e have proposed a fuzzy-based method to select the locations of TDCs and assign MAPs to each TDC, and an integer-programming model to determine the optimal delivery routes in the intermodal transportation network. Experimental results verified the effectiveness of the proposed approach, and observed the impacts of the number of selected TDCs, the vehicle maximum capacity and helicopter travel speed on the performance of the intermodal transportation.There are still further works we will perform. Considerations will be given to reasonably consider the loading and unloading time of medical supplies and reduce the uncertainty of the number of assigned MAPs. Meanwhile, we will improve and apply the approach into real-world disaster responses with more practical considerations such as the geographic situations.


The success of rehabilitation will not depend on just clinical procedures. A proper dental technique (ceramist) is required as well as the respect for some biomimetic principles to obtain the desired final result. This study has the purpose of describing a prosthetic rehabilitation with laminate veneers and all-ceramic crowns of a patient unsatisfied with a previous esthetic treatment because of the negligence of some biomimetic principles. A 45-year-old female patient was admitted to the dental clinic complaining about the lifelike appearance of her all-ceramic restorations. Before the fabrication of new restorations, a mock-up was conducted to verify the patient's satisfaction. A ceramist conducted all the fabrication process so that surface characterizations could be visually verified and the lifelike appearance of natural tooth could be reproduced. After the cementation procedure, the patient reported being satisfied with the lifelike appearance of the new restorations. Based on the clinical findings of the present case report, it can be concluded that the reproduction of the lifelike esthetic appearance of natural teeth and the visualization of the final results before definitive procedures are essential to obtain the clinical success. Since their introduction on clinical routine, the porcelain laminate veneers and all-ceramic crowns have proven to have satisfactory long-term esthetic results superior to other restorative materials as long as they are properly planned and fabricated –9. This To date, there are several ceramic systems available on dentistry market that may reproduce the teeth with great naturally . HoweverNevertheless, the success of rehabilitation will not depend on just clinical procedures. A proper dental technique (ceramist) is required and should have special skills , 15 and A 45-year-old female patient was admitted to the dental clinic  reporting dissatisfaction to a previous treatment and complaining about the lifelike esthetic appearance of her anterior maxillary teeth, in addition to the grayish discoloration of the gingival margin adjacent to the left central incisor (number 21).The intraoral examination revealed the presence of all-ceramic crowns on the anterior maxillary incisors (from number 12 to number 22) and porcelain laminate veneers on canines of the same arch (number 13 and number 23). All those elements showed unacceptable lifelike esthetic appearance, high opacity (mainly on canines), color mismatch, and lack of texture . The conIt was decided to replace the all-ceramic restorations through discussion of treatment possibilities. After anamnesis and intraoral exam, diagnostic impressions were performed with irreversible hydrocolloid ; then the casts were mounted in a semiadjustable articulator. The all-ceramic restorations were removed with a diamond bur . It was observed that the metal cores of the anterior maxillary incisors were properly adapted and all the restored teeth had a satisfactory preparation design . The diaThe soft tissue displacement was performed with gingival retraction cords  because the patient gingival biotype showed a satisfactory thickness , minimizA special dental stone type IV  was poured on the definitive impressions and then conducted to the dental laboratory phase which was performed carefully to avoid the same mistakes observed on the previous all-ceramic restorations. Lithium disilicate ceramic ingots  on medium opacity shade (MO 0) were used to fabricate the laminate veneer coppings and high opacity ingots (HO 0) to all-ceramic crowns. Those ingots are necessary to mask the dark subsurface of the teeth with metal cores and the grayish appearance observed in the right central incisor finish line (number 11). A lithium disilicate layering ceramic was used on A2 color shade for restorations veneering; then their characterization was performed with A2, white and pink  stains, respecting the color nuances necessary to reproduce a lifelike esthetic appearance.The restorations were divided into thirds associated with emergency profile guidelines allowing the gradual ceramic layering and whitening the restoration from cervical to incisal plane . GraphitAfter the all-ceramic restorations were finalized , their iBased on the try-in, a colorless luting agent was chosen. A transparent dual-curing luting agent  was mixed and applied to the inner surface of the restorations. Then they were placed on the prepared teeth and held in place. Their excesses were removed  and the After the cementation, the color of the all-ceramic restorations matched with the patient's natural teeth . BesidesNowadays, a progressive demand of patients for a high level of esthetic treatments is observed on clinical routine . In the Despite the previous esthetic treatment with all-ceramic restorations, the patient was unsatisfied with the lifelike esthetic result obtained. One of the reasons that led to patient's dissatisfaction can be observed on To avoid those clinical complications, the literature recommends that the final results are necessary to be visualized before a definitive procedure . An alteNevertheless, the success of the prosthetic treatment depends on the adequate application of laboratory techniques , 16. In Besides, the selection of the cement type and color as well as the restoration placement is essential to obtain clinical success . In the Within the limitations of this study, it can be concluded that the success of all-ceramic restorations involves excellent esthetic results with a satisfactory integration of them to the gingival margin. However, the reproduction of the lifelike esthetic appearance of natural teeth and the visualization of the final results before definitive procedures are essential to obtain the clinical success.


Stimulated reservoir volume (SRV) fracturing in tight oil reservoirs often induces complex fracture-network growth, which has a fundamentally different formation mechanism from traditional planar bi-winged fracturing. To reveal the mechanism of fracture network propagation, this paper employs a modified displacement discontinuity method (DDM), mechanical mechanism analysis and initiation and propagation criteria for the theoretical model of fracture network propagation and its derivation. A reasonable solution of the theoretical model for a tight oil reservoir is obtained and verified by a numerical discrete method. Through theoretical calculation and computer programming, the variation rules of formation stress fields, hydraulic fracture propagation patterns (FPP) and branch fracture propagation angles and pressures are analyzed. The results show that during the process of fracture propagation, the initial orientation of the principal stress deflects, and the stress fields at the fracture tips change dramatically in the region surrounding the fracture. Whether the ideal fracture network can be produced depends on the geological conditions and on the engineering treatments. This study has both theoretical significance and practical application value by contributing to a better understanding of fracture network propagation mechanisms in unconventional oil/gas reservoirs and to the improvement of the science and design efficiency of reservoir fracturing. Stimulated reservoir volume (SRV) fracturing has become an important technique to improve well production and the recovery of unconventional reservoirs in oil/gas field development. With the development of the SRV fracturing technique, studying the mechanism of fracture network propagation with SRV fracturing has been an academic focus, including the variation rules of stress fields, mechanical mechanisms of fracture propagations and the corresponding geological and engineering factors.Fracture propagation is strongly influenced by the mechanical interaction between neighboring fractures throughout the fracture growth history. This mechanical interaction, called the stress shadowing effect, is quantified using a boundary-element numerical model originally developed for natural-fracture propagation simulation. Erdogan and Sih  calculatThe methods of microseismic monitoring, numerical simulation and rock physics experiments have typically been used to analyze the mechanisms of fracture propagation patterns (FPP) with SRV fracturing in unconventional reservoirs. Complex hydraulic fracture geometry is regarded as a common place occurrence in naturally fractured reservoirs largely based on the interpretation of microseismic data by Fisher et al. , MaxwellGriffith  first poPrevious studies have investigated theoretical models based on the crack mechanics theory or rock stress experiments, but all the above approaches may be considered to be less rigorous because the branch-fracture propagation description is not comprehensive. The branch-fracture propagation description assumes the stress field and propagation angle to be constant. In fact, the interference of the combined stress field would result in a continuous, mutative propagation angle at the fracture tip. This study presents a theoretical model of fracture network propagation with SRV fracturing in tight oil reservoirs. Based on the modified DDM, mechanical mechanism analysis and initiation and propagation criteria, a reasonable solution of the theoretical model is obtained and verified with the numerical discrete method. Using theoretical arithmetic and computer programming, the variation in the formation of the stress field, FPP and branch fracture propagation angle and pressure are analyzed.SRV fracturing is essential in the development of tight oil reservoirs, and the formation of fracture networks is key in increasing production. Several important geological factors affect the formation of fracture networks: rock mineral composition, rock mechanical properties, natural fractures and horizontal stress field.Mineral Composition. Mineral composition is key to rock brittleness . The briRock Mechanical Properties. To a large extent, rock mechanical properties are affected by the mineral composition (or rock brittleness). The concept of rock brittleness combines both Poisson’s ratio and Young’s modulus. Poisson’s ratio is used to reflect the ability of the rock to fail under stress; as values of Poisson’s ratio decrease, the brittleness of the rock increases. Young’s modulus reflects the ability of the rock to maintain a fracture . A higheNatural Fractures. The orientation of the natural fracture will affect the propagation of the hydraulic fracture and the formation of fracture network . The resHorizontal Stress Field. Under the condition of a low horizontal stress difference, the hydraulic fracture would propagate along natural fractures, and a radial fracture network would be induced. Hence, the stress anisotropy in fractured formations with low horizontal stress difference is weak, and as fractures propagate along different directions, the resulting net pressure difference is less  and 31]31]. TherIn view of the problems of stress concentration in the fracture tips, this section uses a modified displacement discontinuity method (DDM) by introducing the shape function as a Lagrange interpolation cardinal function to improve the precision of discontinuous displacement for the discrete state. The computational model of the combined stress field is constructed by the stress shadow effect and stress boundary condition. This model considers the influence of the fracture length on the stress field distribution during the fracturing treatments.x, y plane of an infinite elastic solid. The basic solutions proposed by DDM to the problem of conventional boundary and crack boundary are discontinuous displacements [DDM is based on the analytical solution to the problem of constant discontinuity in displacement over a finite line segment in the acements .a. SON is the local coordinate system. Ds and Dn are the tangential and normal discontinuous displacements, respectively (m = 1 ∼ 3) is introduced as the Lagrange-interpolation cardinal function. Consequently, the discontinuous displacement of arbitrary point can be rewritten as follows:k ≠ m) is the shape function; and ξ1 = −2a, ξ2 = 0, ξ3 = 2a. Therefore, To improve the accuracy of the discontinuous displacement between one fracture element and the other two adjacent elements, the shape function us and un), stress fields  and discontinuous displacements (Ds and Dn) of the fracture element in the local coordinate system are given by the following:fm that is only related to the fracture element coordinates; Based on the theory of elastic mechanics, assuming that the fracture element surface produces symmetrical tangential and normal discontinuous displacements, the relational expression among the displacements (i = 0~2), the analytical formula can be given as follows:s, n) to  and , respectively; s, n) to  and , respectively.For the function SO’N, the stress field  of the fracture element i under an in-situ stress field can be defined as follows:i in the N direction; i; i in the X and Y directions, respectively; and i.In the local coordinate system i becomes the following:j on fracture element i in the X direction, respectively; j on fracture element i in the Y direction, respectively; β is the angle between the N direction of element i and the X axis.For a fracture element, assuming the surface displays a symmetrical net pressure, the normal and shear stresses on the surface of the element are generated from the superposition of the stress shadows produced by other elements  produced by stress shadow effects must be recalculated at every time step. The stress field must be superimposed at the final time step to determine the new composite stress field distribution in the global coordinate system Hydraulic and natural fracture interactions occurred in three forms in laboratory experiments : (1) hydBecause natural fractures develop in tight oil reservoirs, SRV fracturing often induces complex fracture network growth. However, to form the complex network, the natural fractures must initiate and produce open rupture or shear slippage, which can effectively link natural fractures to form branch fractures. When a hydraulic fracture propagates along a natural fracture tip after intersecting with the natural fracture, branching of the hydraulic fracture to form a complex fracture network is possible. However, the fluid pressure at the intersection point must exceed the fluid pressure drop from the intersection point of the natural fracture tip and satisfy the initiation and propagation criteria at the natural fracture tip . Based oσn and σt are the normal stresses perpendicular and parallel, respectively, to the natural fracture surface, expressed in MPa; T0 is the rock tensile strength, MPa; pi is the fluid pressure at the intersection point, MPa; Δps is the pressure drop between the intersection point and fracture tips, expressed in MPa.The hydraulic fracture does not pass through the natural fracture but induces the diversion of fracture propagation. The propagation of the hydraulic fracture diverts when the fluid pressure of the intersection point cannot cause the hydraulic fracture to pass through the natural fracture. Rather, the hydraulic fracture induces natural fracture slippage, initiates at the tips, and diverts propagation along the direction of maximum principal stress. The mechanical condition is given by the following:θ is the contact angle, knf is the natural fracture permeability, expressed in mD; μ is the fluid viscosity in the reservoir, expressed in mPa.s; ϕnf is the dimensionless natural fracture porosity; Ct is the natural fracture comprehensive compression coefficient, expressed in 1/MPa; p0 is the original fluid pressure in the reservoir, expressed in MPa; Lnf is the natural fracture length, expressed in m; and pi = pnet(i) + σyy, where pnet(i) is the net pressure at the intersection point, expressed in MPa.According to the theory of two-dimensional linear elastic fracture mechanics, the following equations can be obtained: Thus, the formula for the net pressure at the intersection point becomes the following:he hydraulic fracture passes through natural fracture, inducing a partial opening of the natural fracture. The hydraulic fracture passes directly through the natural fracture and propagates along the initial direction . According to the Blanton criteria , the fluTherefore, the following formula for the net pressure at the intersection point is obtained:The hydraulic fracture passes through the natural fracture, inducing the diversion of natural fracture propagation. Simultaneously, the net pressure at the intersection point cannot only exceed the rock tensile strength and normal stress to pass through the natural fracture but also must induce natural fracture slipping.In this section, the mathematical model of the branch fracture initiation and propagation criteria is constructed based on the maximum circumferential stress theory and the stress intensity factor theory of the composite type I-II fracture.Based on the maximum circumferential stress criterion, we can assume that the crack extends along the direction of the circumferential stress, reaching a maximum value; the crack initiates in this direction when the circumferential stress reaches a critical value.(1) The stress field equations for a composite type I-II fracture at a fracture tip in polar coordinates (as shown in (2) The propagation angle equations of a composite type I-II fracture  can be written as follows:The equation can be rewritten as follows:θ0 is constant when the shear stress changes, Eq (E and ν.Because M8+M29+M2where M=(3) Maximum Circumferential StressIn the function M is positive and negative, the conditions at the fracture tips achieve maximum circumferential stress values of -70.53° ≤ θ ≤ 0 and 0 ≤ θ ≤ 70.53°, respectively.For the composite type I-II fracture in σθθ)cr is the critical value of the maximum circumferential stress and depends on the fracture toughness of the type I fracture, which is also a constant related only to the material properties and independent of the composite fracture state. The fracture criterion must describe the type I fracture, and the material properties must be invariant.(4) Fracture Initiation Criteriaθ0 equals zero. The value of KI equals KIC when initiating the fracture. Therefore, the critical stress equation of fracture initiation becomes the following:For the pure type I fracture, Keq and KIC are the equivalent fracture strength and the fracture toughness at the fracture tips, respectively.Consequently, the composite type I-II fracture initiation criteria becomes the following:a, the natural fracture is in a state of two-axial compression stress, and the surface produces a shear stress τxy under the combined stress field . We construct a local rectangular coordinate system, SON, by using the center of the fracture surface as the origin and the S axis as the long axis. In the global coordinate system, XOY, the angle between the normal direction of the crack surface and the ox axis is α  is the net pressure inside the fracture at the intersection point between the natural fracture and hydraulic fracture, expressed in MPa.According to the 2-D linear elastic theory of fracture mechanics, natural fractures are an independent system unit. Therefore, when hydraulic fractures interact with natural fractures in the direction perpendicular to the normal stress caused by the combined stress field, the pressure component of the fracturing fluid can be expressed as the following:Therefore, under the multi-fracture combined stress field, the type I SIF at the branch fracture tips that is produced by the normal stress can be defined as follows:The shear stress parallel to the natural fracture surface under the stress field can be obtained as follows:Consequently, under a multi-fracture combined stress field, the type II SIF at the natural fracture tips produced by the shear stress can be defined as follows:We use the basic parameters in a subjected to a pressure p was analyzed. The analytical solution of the normal discontinuous displacement can be given by the following:To compare the superiority of different model solution methods, a line fracture of length 6a. Considering the solutions of analytical method, DDM and modified DDM, the calculated results including the normal discontinuous displacement and normal stress of fracture in the N direction can be obtained  fracture, in which the hydraulic fracture passes through the natural fracture without deflection. When the contact angle is 90°, shear and slip failure occur, causing pure type II (slip type) fractures; the deflection angle of the branch fracture then reaches a maximum value of 70.53°.Greater net pressure favors the formation of a complex fracture network. When the stress difference is constant and the net pressure inside the fracture increases, the branch fracture propagation deflection angle decreases. Additionally, the radius of curvature of the deflecting propagation trajectory increases. Therefore, the branch fracture can encounter more natural fractures in the vertical direction, increasing its bandwidth. This process is conducive to the production of a more complex fracture network. During hydraulic fracturing, the net pressure is dynamic with fracture propagation and is a controllable factor; thus, the net pressure can be increased by increasing the flow rate of the fracturing fluid in a tight oil reservoir.Smaller stress differences favor the formation of the complex fracture network. When the net pressure inside the fracture is constant and the stress difference increases, on the one hand, the branch fracture deflecting propagation angle increases and the radius of curvature of the deflecting propagation trajectory decreases. Additionally, the branch fracture converges towards the direction of principal stress over a small range because of the higher pressure drop within the fracture, allowing the branch fracture to continue to propagate more difficultly. On the other hand, when the stress difference are too low, the hydraulic fracture propagates more easily along the natural fracture surface that has a weak strength. This situation is not conducive to increasing of band length of the fracture network because it reduces the chance to encounter more natural fractures in the horizontal direction.Therefore, the SRV fracture treatment requires not only the hydraulic fracture to propagate farther along the direction of maximum principal stress to encounter more natural fractures but also a smaller propagation angle to produce a larger fracture-network band width and to encounter more natural fractures. Combining both of these conditions, we can achieve the formation of a complex-fracture network system.Based on the fracture initiation and propagation criteria of mixed-mode I and II, the variation rule of branch fracture propagation pressure with contact angle, natural fracture half-length, horizontal stress difference and rock fracture toughness can be calculated, as seen in The contact angle has a large effect on the propagation pressure required to divert the propagation of the natural fracture. When the contact angle is 0°, pure type I open fractures are produced. These fractures are produced simply by overcoming the minimum principal stress and rock fracture toughness. The path of the hydraulic fracture coincides with the natural fracture and passes through it. When the contact angle increases further, the effective net pressure at the intersection of the hydraulic fracture and natural fracture decreases, and the required propagation pressure required, hindering the formation of a fracture network. However, when the contact angle approaches 90°, pure type II fractures occur, in which steering the natural fracture requires maximum net pressure. Therefore, producing a fracture network is difficult.The natural fracture half-length has some influence on the propagation pressure required to induce a branch fracture. On the one hand, with increases in the natural fracture length, the generated equivalent-stress intensity factor increases, reducing the magnitude of the net pressure that can induce the extension of a branch fracture; this reduction in the magnitude of net pressure occurs when the natural fracture is shorter. On the other hand, longer natural fractures correspond to a greater loss of net pressure at the tip when the hydraulic fractures encounter the natural fractures, resulting in the need for higher pressure to form a fracture network.Stress differences have a greater effect on the propagation pressure required for the deviation of the natural fracture. When the stress difference increases, the net pressured required to steer the natural fractures to extend along the branch increases markedly. It becomes more difficult to expand or initiate a branch; this difficulty occurs because of the increases in the branch propagation angle. The increased angle reduces the radius of curvature to a smaller trajectory that reaches the main branch in the direction of the maximum principle horizontal stress. This intersection decreases the net pressure rapidly, resulting in the need for a higher pressure to induce a branch fracture.The rock fracture toughness has a smaller effect on the propagation pressure required for the deviation of the natural fracture. With increases in the rock fracture toughness, the natural fracture propagation resistance increases. Therefore, natural fractures require greater pressure to produce a net branch fracture, resulting in increased difficulty to form a fracture network.The simulation results show that the natural fracture not only steering extends with the in-situ differential stress and rock fracture toughness but is also influenced by the length and orientation of the natural fractures. Therefore, during the SRV fracturing in the tight oil reservoir, more mature and dense natural fractures are suggested to be selected in the block, and the influence of the natural fracture length and orientation of the fracture network morphology should be considered.Varied lithologic compaction, poor physical property and high flow resistance are inherent characteristic for tight reservoir. Hydraulic fracturing technology are widely used to change the flowing regime near the wellbore, and increase the channel of oil flowing to improve the well performance. To reveal the influence of SRV on oil well performance, considering the situation of without fracturing (SRV=0), conventional fracturing (Bi-wing & Symmetric fracture) and SRV fracturing, the production performance including daily production and cumulative production curve of oil well can be obtained by numerical reservoir simulator, as shown in Results show that without fracturing, oil production is extremely low in tight oil reservoir and it’s hard to achieve economic rates, so hydraulic fracturing is a necessary process to accelerate production. Conventional fracturing could create bi-wing & symmetric fracture, providing significantly more fracture-surface contact with the reservoir, thus accerates oil production obvisously. However, SRV fracturing produces higher rates with the following mechanism.SRV fracturing could communicate hydraulic fracture with natural fracture and create 3D fracture network with certain length, width and height, that will increase the stimulated reservoir volume and enhance the overall system permeability. In tight oil reservoir, diffusivity-related pore-pressure changes cannot move very far from the fracture, so little fluid is produced outside the extent of the SRV. Within the SRV, SRV maximize the contact area between fracture-surface and matrix, so oil could flow from the matrix to the created fracture network through the shortest path easily. SRV could maximize producing ratio and recovery percent of reservoir, having significant effect on oilfield production.So it’s necessary to study the mechanism of fracture network propagation with SRV fracturing in tight oil reservoirs. The relevant results studied including geological conditions and engineering treatments have both theoretical significance and practical application value by contributing to a better understanding of fracture network propagation mechanisms in tight oil reservoirs and to the improvement of the science and design efficiency of reservoir fracturing.SRV fracturing has emerged as an important technology to exploit oil/gas from unconventional reservoirs, in which fluids are injected at high rate and pressure to crack the reservoir rock in order to create pathways with higher permeability to accelerate production. This paper analyzes the mechanism of fracture network propagation with SRV fracturing in tight oil reservoirs from both geological and engineering perspectives, the following conclusions can be drawn:During the process of fracture propagation, the stress field produces local stress concentration at the fracture tips, and the initial orientation of maximum horizontal principal stress deflects into the area around the fracture.Propagation pattern 3  is the most conducive to forming a network fracture system by combining a principle fracture and a branch fracture.A specific set of reservoir geological attributes , lower tensile strength and fracture toughness of rock, mature natural fractures , and lower stress anisotropy ) are more conducive to the propagation of hydraulic fractures into a complex fracture network. According to the engineering conditions of fracturing operations, higher net treatment pressure (propagation pressure), reasonable fracturing azimuth direction (contact angle) and larger fracturing scale (hydraulic fracture length) will assist the formation of complex fracture networks with SRV fracturing in tight oil reservoirs.


In this article, we describe the use of Python for large-scale automated server-based bio-image analysis in FARSIGHT, a free and open-source toolkit of image analysis methods for quantitative studies of complex and dynamic tissue microenvironments imaged by modern optical microscopes, including confocal, multi-spectral, multi-photon, and time-lapse systems. The core FARSIGHT modules for image segmentation, feature extraction, tracking, and machine learning are written in C++, leveraging widely used libraries including ITK, VTK, Boost, and Qt. For solving complex image analysis tasks, these modules must be combined into scripts using Python. As a concrete example, we consider the problem of analyzing 3-D multi-spectral images of brain tissue surrounding implanted neuroprosthetic devices, acquired using high-throughput multi-spectral spinning disk step-and-repeat confocal microscopy. The resulting images typically contain 5 fluorescent channels. Each channel consists of 6000 × 10,000 × 500 voxels with 16 bits/voxel, implying image sizes exceeding 250 GB. These images must be mosaicked, pre-processed to overcome imaging artifacts, and segmented to enable cellular-scale feature extraction. The features are used to identify cell types, and perform large-scale analysis for identifying spatial distributions of specific cell types relative to the device. Python was used to build a server-based script  capable of routinely handling image datasets at this scale and performing all these processing steps in a collaborative multi-user multi-platform environment. Our Python script enables efficient data storage and movement between computers and storage servers, logs all the processing steps, and performs full multi-threaded execution of all codes, including open and closed-source third party libraries. Our goal is to quantify tissue perturbations inflicted by implanted neural recording devices, since their performance depends upon the state of the surrounding tissue. Our current understanding of microglia is largely based on qualitative visual analysis of two-dimensional (2-D) micrographs. There is a compelling need for an objective, quantitative, and fully 3-D analysis of microglia arbors over extended (multi-millimeter) tissue regions large enough to encompass the implanted device. Toward this goal, we present a method combining 3-D confocal imaging of extended tissue regions, large-scale computational image analysis, quantitative neuromorphology, and bio-informatics. The created processing pipeline was developed using python as the building block to join all the required modules together.The images consist of coronal sections of 4% paraformaldehyde fixed rat brain motor cortices, some with electrodes implanted for 30 days , which were cut into 100-μm thick slices, and labeled . A Rolera EM-C2 camera  on an Olympus spinning-disk confocal microscope was used to record images . Overlapping image tiles were combined into a 3-D montage of extended fields. Figure Solving the problem requires integration of multiple software systems, because no one particular toolkit offers all the required algorithms to process these images. In general, to routinely process these big images, a fully automatic pipeline is required that is capable of integrating all software tools (open and close source tools), developed in different programming languages , into a one-click single solution, which allows a non programming expert to process these images. In particular, FARSIGHT  is a public domain Java-based image processing program that can be used to display, edit, analyze, process, and save many image formats including TIFF, GIF, JPEG, BMP, DICOM, FITS and “raw.” In particular, for the explained pipeline we have used the preprocessing algorithms offered by ImageJ/Fiji which are integrated into the proposed pipeline.The proposed solution to analyze the huge amount of data by this study consists of a number of core steps: a registration and mosaicing step, followed by a preprocessing step to denoise the images, extraction of meaningful features, which are primarily based on segmentation/classification of cell nuclei, and tracing of microglia arbors. Although each of these steps can be performed manually for a given dataset, integrations of the results of each algorithm are labor intensive and prone to human errors. Furthermore, the study of these complex biological micro-environments requires collaborative work between interdisciplinary groups that requires careful maintenance of record files to keep track of the steps performed in each particular dataset. In addition, one must often process legacy datasets with certain change in parameters. One efficient way to obtain consistent and reproducible results is through the use of a pipeline like the one developed here.Our approach addresses these problems in the following ways. First, since this pipeline is based on a pluggable architecture, each of these modules can be turned on/off, or a new module can be plugged in based on the need of a given problem. Second, given that this pipeline is designed to be used on a routine basis by biologists and other people who might know little of image processing, the careful design and organization of the modules are of utmost importance to allow re-processing of data for occasions when the default pipeline does not work. This failure to cope with a particular dataset can be due to changes in the imaging protocol, experimental condition, new artifacts introduced in the data, etc. The maintenance of the record files at each stage will help the image processing expert to fix encountered problem in a more efficient way.In this paper, we present a processing pipeline targeted toward a biologist who can extract the relevant features for a given dataset without knowing the intricate details of the image processing algorithms. This pipeline is based on the idea of one-click processing automation: to accomplish this goal, the parameters used to tune the algorithm are maintained in a separate file that can be changed according to the requirements of each dataset. We illustrate the different steps of our method  by running the pipeline on one of the datasets and present the results as well as limitations of the proposed solution. Also, improvements over the used algorithms are described, which were required in order for our pipeline to work in a realistic amount of time in such large scale.The current pipeline was developed to create a modular architecture that integrates different algorithms written in distinct programming languages capable of analyzing high-content confocal images of brain tissue, with the goal of studying the immune system reaction to the implantation of neuro-prosthetic devices. The architecture of the pipeline is presented in Figure This application requires the correct registration and mosaicing of high-resolution three-dimensional (3-D) images of extended tissue regions that are much wider than the lateral field of view of the microscope. To accomplish that, a series of partial views of the overall region of interest are acquired, and then combined to form a synthetic image . Tsai et al.  developeFor our application we have found the combination of different algorithm which works best for us. We have used the Insight Toolkit  and scale (the frequency domain), but also in orientation. In our work, we use the fast discrete curvelet transform implementation to enhance the arbors of microglia and compute their local orientation at each pixel. The curvelet coefficients are simply the inner product of the input with each of the basis of curvelet waveforms. For example, given a digital pixel array  ∈ L2, 0 < t1, t2 < n, the digital curvelet coefficient CD can be computed asDi,l,k is the digital (D) curvelet wave form, j is the scale parameter, l refers to the orientation parameter and k =  refers to the spatial location parameter of the curvelet waveform. One approach to image enhancement is to use a threshold to eliminate small curvelet coefficients and retain only the large ones. If CDt denotes the coefficients after enhancements and T the threshold, thenCurvelets are two dimensional waveforms that can be used for sparse representation of curvilinear structures in images. In space, a curvelet ΨCDt. Due to the memory requirement and compatibility with the FARSIGHT framework, we have used a curvelet tiling approach which we have developed using c++, and integrated in the main pipeline by using python.Then, the enhanced image can be obtained by taking the inverse curvelet transform of In order to perform the analysis of an image, the first and most important step is to identify regions corresponding to individual cells (cell segmentation) to extract meaningful features, which can subsequently be used by analytical tools to gather information required by the proposed study. Many algorithms have been proposed in the literature and the most common ones depend on the watershed transform, level-set theory, template matching, and wavelet analysis. For our pipeline the building block is the algorithm described in Al-Kofahi et al.  which isThe above outlined algorithm becomes impractical if it is applied directly to images of the size required by the described study, since time and memory requirements grow exponentially. In addition, we need to quantify the presence of other bio-markers around the cells using secondary channels; this increases the memory requirements by a factor equal to the number of additional channels. For this reason, in our pipeline we have developed a divide-and-conquer method to correctly segment a montage of any size, using a selected tile size according to the processing capabilities of the system being used. The image montage is split into overlapping regions by using a big enough padding according to the maximum expected object size. Among the cells that lie on the border, some of them belong to the current tile and some others belong to the adjacent tile. When the adjacent tile is processed, these overlapping regions are segmented again. To merge the results and avoid object duplication, only the cells lying within the border are retained. All other cells on the border whose centers lie outside the actual tile are rejected. This approach was implemented in C++ using the Insight Toolkit, and it was parallelized using the OpenMP library to efficiently process multiple tiles simultaneously. The feature computation was also performed in parallel for each tile. These improvements in the algorithm implementation makes the use of the described algorithm practical for the problem at hand, something which was not possible before this pipeline was built. These features are subsequently used by the classification algorithm to distinguish between the different cell types present in the image. The most important cell type have been the microglia cells, since they are the driving hypothesis behind the failure of implanted neuro-prosthetic devices.One of the goals of this pipeline is to correctly identify cell types in multi-spectral images of brain tissue. This brain tissue comprises of cells of different types such as Neurons, microglia, Astrocytes, and Endothelial cells. An important issue concerning the electrode performance and the effects of device geometry is the proximity of these different cells to electrode sites. Thus, classification of these cells is a fundamental step in the analysis before characterizing their spatial distribution. In both these examples, the scale of the data being analyzed is extremely large. A typical dataset consists of hundreds of thousand of cells. This calls for a robust and efficient cell classification algorithm that is scalable to our needs, and which can be easily trained by a biologist. Human annotation is tedious, expensive and subjective. There will be intra- and inter-observer variance with respect to the selection of the most informative samples for the training of the classifier. In general, humans are biased at picking the most informative examples. For this reason, a mathematical tool which reduces this bias is required.We have used a semi-supervised machine learning algorithm to train the classifier, which minimizes the amount of human effort. A special case of this kind of algorithms makes use of the active learning framework which essentially solves the problem of objectively picking the most informative set of examples from a large unlabeled pool; it is based on the assumption that not all training samples are equally informative. Active learning is a paradigm that helps in classifier learning with minimal effort from the user by concentrating on informative examples only. By querying the most informative examples at every iteration, we can build the classifier at a lower cost. Active learning methods are iterative and the algorithm updates its knowledge about the problem after obtaining the labels for the queried examples at each iteration. The active learning approach used in this pipeline is based on the logistic regression classifier as described by Padmanabhan . As partAfter segmenting and classifying the cell nuclei of Microglia, the next step is to digitally reconstruct or trace the arbors of these tree like structures to extract meaningful information. These reconstructions form the basis for quantifying arbor morphologies for statistical analysis. Tracing algorithms can be classified based on the method used to digitally reconstruct these arbors. To our knowledge these algorithms can be classified into (i) Active Contour  represents the squared reconstruction error. The second term in (3) represents the classification error for a weight matrix. The dictionary learned in this manner has excellent representational power, and enforces strong discrimination between the two classes . After learning the D, L, Γ; given a new image, the sparse representation Γ in D can be obtained using the sparse coding algorithms  to model each microglia as described in Megjhani et al., (submitted); an MST like any graph consists of nodes and edges. In our case, each node is the location of pixels detected as seed points. Each edge is the cost of considering that a voxel belongs to the microglia process. The cost was defined by computing the geodesic distance between the two nodes. The MSTs were constructed using an adaptation of Prim algorithm Prim . The results are then merged together to create a final microglia morphology reconstruction of the whole image montage. The dice size is selected according to the maximum expected length of the microglia processes.We have developed a new tracing algorithm by integrating a widely used technique in the image processing literature, which consists of using appropriate dictionary learning of the image features. To the best of our knowledge, this is the first kind of tracing algorithm which uses this powerful mathematical technique in the field of bio-image analysis. Large-scale tracing of microglia arbors using over complete dictionaries  learns the curvilinear structure surrounding the putative seed points and classifies the structure based on the learned dictionary thm Prim . StartinThe developed pipeline generates the results in a format which can be understood by the FARSIGHT system, an open source and cross-platform toolkit for analyzing multi-dimensional and multi-spectral biological images. Of particular interest for our project is to correctly visualize, edit, and analyze the segmentation and tracing results. Even the best available automated systems today have a non-zero error rate, implying the continued need for visual proofreading and corrective editing systems for which the FARSIGHT Trace Editor was used .In the future we will add the vessel channel to our pipeline. Vessels will help us add another layer of information to our analysis. This will increase the amount of information which can be used by our clustering and progression discovery algorithms in order to relate the underlying patterns when comparing normal with disturbed brain tissue. Finally, it is also important to highlight that all the code developed by this project is open-source and available trough the FARSIGHT repository at (This work was supported by DARPA Grant N66001-11-1-4015.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.