Tumour HV was moderately correlated (P < 0.001) with ρ = 0.69 between 2- and 4-h scans. Yet, in tumour subregions, the correlation of HV decreased from the centre ρ = 0.71 to the edge ρ = 0.45 (P < 0.001). SUV, TBR, and HV values were consistently higher on 4-h scans than on 2-h scans, indicating better tracer-to-background contrast. For instance, for TBRmax, the mean, median, and interquartile range were 1.9, 1.7, and 1.6–2.0 2-h p.i., and 2.6, 2.4, and 2.0–3.0 4-h p.i., respectively. Our results support that FMISO-PET scans should be performed at 4 h p.i. to evaluate tumour hypoxia in NSCLC.Positron emission tomography (PET)/computed tomography (CT) using the radiotracer 18F-Fluoromisonidazole (FMISO) has been widely employed to image tumour hypoxia and is of interest to help develop novel hypoxia modifiers and guide radiation treatment planning. Yet, the optimal post-injection (p.i.) timing of hypoxic imaging remains questionable. Therefore, we investigated the correlation between hypoxia-related quantitative values in FMISO-PET acquired at 2 and 4 h p.i. in patients with non-small cell lung cancer (NSCLC). Patients with resectable NSCLC participated in the ATOM clinical trial (NCT02628080) which investigated the hypoxia modifying effects of atovaquone. Two-hour and four-hour FMISO PET/CT images acquired at baseline and pre-surgery visits (n = 58) were compared. Cohort 1 (n = 14) received atovaquone treatment, while cohort 2 (n = 15) did not. Spearman’s rank correlation coefficients (ρ) assessed the relationship between hypoxia-related metrics, including standardised uptake value (SUV), tumour-to-blood ratio (TBR), and tumour hypoxic volume (HV) defined by voxels with TBR ≥ 1.4. As the primary imaging-related trial endpoint used to evaluate the action of atovaquone on tumour hypoxia in patients with NSCLC was change in tumour HV from baseline, this was also assessed in patients (n = 20) with sufficient baseline 2- and 4-h scan HV to reliably measure change (predefined as ≥ 1.5 mL). Tumours were divided into four subregions or distance categories: edge, outer, inner, and centre, using MATLAB. In tumours overall, strong correlation (P < 0.001) was observed for SUVTrial registration: ClinicalTrials.gov, NCT02628080. Registered 11/12/2015, https://clinicaltrials.gov/ct2/show/NCT02628080. Associated with their high metabolic demand, this causes tumour hypoxia. It is well established that tumour hypoxia induces resistance to numerous anticancer treatments, and this is particularly pertinent for radiotherapy (RT)2. There is therefore much interest in imaging tumour hypoxia to develop novel hypoxia modifiers as radiosensitisers, as well as to guide radiation treatment planning.The prognosis of patients with non-small cell lung cancer (NSCLC) is poor despite advances in the delivery of several treatment modalities. Solid tumours like NSCLC generally rely on a dysfunctional vasculature for oxygen delivery3. These exogenous and hypoxia-specific markers are reduced and re-oxidised in normoxic cells but, in hypoxic cells, their nitro radical anion is further reduced, and the compounds covalently (and irreversibly) bind to intracellular macromolecules. As pO2 values decrease, the degree of reduction augments and pO2 levels less than 10 mgHg initiate this reduction4.Chapman et al. detected for the first time in 1981 tumour hypoxia with molecular imaging and nitroimidazole compounds5, FAZA6, FMISO8, HX49, FETNIM10, and non-nitroimidazole compounds such as ATSM11. Since its development in 198912, numerous pre-clinical and clinical studies14 have identified FMISO as the most promising method for hypoxia quantification and to date, it’s the most widely studied hypoxia tracer16. Valk et al.7 and Koh et al.8 first indicated that FMISO enables hypoxia detection in human tumours. Rasey et al.14 also demonstrated the sensitivity of FMISO as a hypoxic marker, and validated the variability, presence and prevalence of tumour hypoxia in 37 patients. Importantly, Gagel et al.17 reported a correlation between FMISO uptake and Eppendorf pO2 probe measurements which suggests that the tracer is representative of intracellular pO2, and this was not observed with FDG18. Statistically significant correlations were moreover shown between FMISO uptake and HIF-1α19 as well as Pimonidazole immuno-histochemistry staining20. Evidence also showed that FMISO can detect hypoxia in the clinical setting in various tumour types including head and neck (H&N) cancer14, NSCLC21, breast cancer22, glioma23, and soft-tissue sarcoma24.PET radiotracers include nitroimidazole compounds such as EF52 levels in cells, and its uptake increases markedly as the oxygen concentration drops to values at which the oxygen enhancement ratio (OER), and thus tumour radiation response, also declines25.FMISO is a lipophilic molecule and diffuses passively across cell membranes. As a nitroimidazole compound, FMISO is reversibly reduced, re-oxidised, and diffuses outside the cell under normoxic conditions. In contrast, under hypoxic conditions, FMISO is further reduced which leads to its irreversible and covalent binding to intracellular macromolecules. FMISO binding occurs at rates inversely proportional to pO26. Kobayashi et al. showed that 4-h post-injection (p.i.) was preferred to 2-h p.i. for patients with a brain tumour27. Studies performing static imaging of lung cancer with FMISO-PET have acquired scans after at least 2 h p.i.29. Moreover, several quantitative values in FMISO-PET at 4-h p.i. were reported as reproducible30. Yet, whether these values could be equivalent at 2- and 4-h p.i. is uncertain. In H&N cancer, Abolmaali et al. indicated that scans conducted at 4-h after FMISO injection showed greater contrast than those performed at 2-h p.i.31, while recently, Kawamura et al. reported a significant correlation between the quantitative values at 2- and 4-h32. Thus, the optimal post-injection timing to image NSCLC tumour hypoxia with FMISO remains to be confirmed.Passive diffusion causes FMISO to slowly clear from normoxic cells and slowly accumulate in hypoxic cells. Imaging hypoxia at late time-points is therefore required given the low tracer-to-background contrastIn this study, we hypothesised that hypoxia-related metrics from FMISO-PET scans performed at 2-h p.i. may correlate with the values from FMISO-PET scans performed at 4-h p.i. in the context of NSCLC. This was to investigate whether similar results could be obtained at 2 h p.i. to improve the patient experience and practical aspects of FMISO scanning. The aim was therefore to assess the relationship between quantitative values in FMISO-PET obtained at 2- and 4-h p.i. in patients with NSCLC.33. The clinical characteristics of the patients are shown in Table 34.Patients with resectable NSCLC were recruited for the open-label, nonrandomized, two-cohort ATOM clinical trial  completed in accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines. Ethical approval was obtained from National Research Ethics Service Committee South Central Oxford B (16/SC/0012). All patients provided written informed consent. Patients in cohort 1 (n = 14) received oral atovaquone  twice daily. Patients were asked to take atovaquone orally together with fat-containing food to aid absorption. Patients in cohort 2 (n = 15) did not receive atovaquone. Eligible patients were aged ≥ 18 years, had a pathologic or radiological diagnosis of NSCLC, were scheduled for surgical resection, had disease > 2 cm in diameter, and had Eastern Cooperative Oncology Group (ECOG) performance status 0–2. Patients were excluded if taking known electron transport chain inhibitors. Despite a male predominance in untreated patients, the main clinical characteristics were well balanced in the two cohorts. For full details regarding trial design and patient baseline characteristics, the reader is referred to Ref.O-tetrahydropyranyl-3-O-toluenesulfonyl-propanediol, NITTP) was used from Advanced Biochemical Compounds (ABX) and the synthesis of FMISO was performed on a FASTlab synthesis module  according to the manufacturer’s instructions. The product was then sterilized by filtration through a Millex-GV 0.22 µm sterile filter (Merck Millipore)35.Single bed position image acquisition centred on the tumour was performed with GE Discovery 690 or 710 PET-CT Scanners  for 10 min at 2 and 4 h following the administration of 18F-Fluoromisonidazole (FMISO) (manufactured by University of Cambridge) with an activity of 370 MBq. Precursor (1-(2′-Nitro-1′-imidazolyl)-2-36. As with previous work, respiratory motion correction was not performed for the presented analysis38. Patients in cohort 1 had a median length of 13.5 (IQR 10.75–14) days between imaging timepoints, depending on their planned date for surgery. Patients in cohort 2 had a median length of 14 (IQR 7–14) days between imaging timepoints.The same scanner was used for the two visits of each patient with baseline scans and pre-surgery scans for atovaquone-treated and untreated patients. CT images provided attenuation correction and localization. All PET images were reconstructed with a Bayesian penalised-likelihood algorithm, Q.Clear  using a beta value of 400voxel) per voxel was first extracted for every outlined tumour volume, as previously described34.Tumours on 4-h hypoxia PET-CT images were manually outlined on the CT image by an experienced radiologist and copied to the co-registered PET image. Two-hour images were rigidly registered (CT-to-CT) to 4-h images using Hermes Hybrid Viewer , followed by manual adjustment (matching to the tumour region) when required. The tumour outlines on the 4-h images were used to delineate tumours on the respective 2-h images. A matrix containing each voxel coordinates  along with the respective radiotracer standardised uptake value (SUVmean background) was obtained by outlining blood (using a 10 mm diameter circular region of interest (ROI) on at least ten sequential axial PET slices) in the central portion of the descending aorta, with the mean value the average of all outlined voxels. To measure the hypoxic volume (HV), each tumour voxel’s SUV was divided by SUVmean background to determine the tumour-to-blood ratio (TBRvoxel) value per voxel:The background mean SUV , mean TBR (TBRmean), maximum SUV (SUVmax), mean SUV (SUVmean), and HV in tumours overall and in tumour subregions. As the primary imaging-related trial endpoint used to evaluate the action of atovaquone on tumour hypoxia in patients with NSCLC was change in tumour HV from baseline, this was also assessed in patients (n = 20) with sufficient baseline HV to reliably measure change (predefined as HV ≥ 1.5 mL)37 in 2- and 4-h images.Different metrics describing hypoxia were calculated on the 2-h and 4-h images of all 29 patients, including maximum TBR (TBR3. Voxels were then divided into four subregions or distance categories: edge (the outermost shell of voxels), outer (voxels’ centre up to 5.5 mm of the tumour outline), inner (voxels’ centre between 5.5 and 11 mm of the tumour outline), and central (voxels’ centre more than 11 mm inside the tumour outline), in line with a previous PET study about FMISO uptake in advanced NSCLC38.In-house MATLAB  code was first used to calculate the distance of every tumour voxel to the nearest edge of the outlined tumour. Voxel dimensions on PET-CT images were 2.7 × 2.7 × 3.3 mmmax, TBRmean, SUVmax, SUVmean, and tumour HV on 2- and 4-h scans. For each distance category, the chi-square test of homogeneity was employed to assess the significance of pre- to post-atovaquone changes in the proportions of voxels assigned to each region. Percentage change in tumour HV was calculated between trial visits for each patient. A P value < 0.05 was considered significant.Statistical analyses used IBM SPSS Statistics (version 27). The normality of data was inspected using Shapiro–Wilk test. Mean, median, and interquartile range (IQR) were indicated for all hypoxia metrics assessed. Spearman’s rank correlation coefficients (ρ) were reported for correlation analysis of TBREthical approval was obtained from National Research Ethics Service Committee South Central Oxford B (16/SC/0012). Trial conduct adhered to all regulatory requirements and was in full accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines.Changes in tumour hypoxia were analysed using 4-h hypoxia PET-CT, according to current standard of practice. We investigated whether similar TBR results could be obtained at 2 h p.i. to improve the patient experience and practical aspects of FMISO scanning. The relationship of hypoxia-related quantitative values in FMISO-PET obtained at 2- and 4-h post-tracer injection were therefore investigated.max, SUVmean, TBRmax, TBRmean values in tumours overall and in regions on FMISO hypoxia PET-CT scans at 2- and 4-h were analysed . Overall, the Spearman’s rank correlation coefficients (ρ) of the parameters were as follows: SUVmax (ρ = 0.87), SUVmean (ρ = 0.91), TBRmax (ρ = 0.83), and TBRmean (ρ = 0.81). Although, means of these quantitative values were lower on 2-h scans than on 4-h scans, values were highly correlated between scans (P < 0.001) . In the different tumour regions, ρ was also relatively lower, decreasing from the centre (0.71) to the edge (0.45) (P < 0.001)  of tumour HV on 2- and 4-h scans was 0.69  HV on both their baseline 2- and 4-h scans to reliably detect change were included.34. Discrepancies in meaningful changes in HV in three treated patients (27%) and four (44%) untreated patients were observed, as HV increased on 4-h scans and decreased on 2-h scans or vice versa , including three treated and six untreated patients, did not have enough baseline HV (i.e. < 1.5 mL) on 2-h scans to reliably measure change in tumour HV from baseline whereas they had sufficient HV on 4-h scans. This would be another argument for scanning at 4 h and not at 2 h p.i., especially when testing novel hypoxia modifiers.32. In addition to the evaluation of SUVmean, SUVmax, TBRmean and TBRmax values between scans, this group compared the metabolic tumour volume and total lesion hypoxia to define tumour hypoxia, instead of the common HV metric that usually includes voxels with TBR ≥ 1.4. Our study showed differences in HV between 2-h and 4-h scans. The authors also suggested that the discrepancy between their study and others, that for instance demonstrated the high reproducibility of SUVmax, TBR, and HV at 4 h p.i30. as well as the higher contrast of FMISO PET scans acquired at 4 h compared to 2 h31, could be due to a difference in analysis methodology  and to the greater sensitivity of their PET scanners. Yet, our study, which also used images from a sensitive PET scanner and Spearman’s rank correlations for the analysis, supported the acquisition of PET scans at 4 h for NSCLC due to superior image contrast, in agreement with previously published evidence31. Changes in hypoxia-related metrics in tumour subregions, not only in tumours overall, were also assessed here.A recent study reported a very strong correlation  between all hypoxia-related quantitative values assessed on 2-h and 4-h FMISO PET scans and which supported the use of FMISO PET imaging at 2 h in H&N cancer39.Moreover, differences in imaging hypoxia using tracers such as FMISO in different tumour types could be due to differences in tumour histology . Levels of hypoxia may also vary between tumour types as studies reported differences in electrode measurements of pO2 between different types of tumours41 and DiFA42 have recently been developed and could potentially enable shorter hypoxia PET acquisition times thanks to their greater hydrophilicity. Formal investigation of these tracers is however required in clinical studies.As a shorter waiting time for hypoxia PET imaging would have significant practical and logistical advantages for radiology departments and be more acceptable to patients, the tracers HX4max, SUVmean, TBRmax, TBRmean and HV metrics were true on 4-h images and we used them as a reference to compare with 2-h images. Second, patient motion may have impacted the registration of 2-h scans with 4-h ones. Image registration was therefore manually verified for each individual scan. Third, scans were analysed from a small number of patients who received FMISO for hypoxia PET imaging. However, the number of patients is relatively large for this category of imaging study, and represents one of the largest in a single tumour type to date. Collaborative initiatives, such as imaging repositories, may help standardise the methodology used.Our comparison of hypoxia-related quantitative values on FMISO-PET scans at 2 and 4 h included several limitations. First, we assumed that SUVIn this study, we compared 4-h and 2-h FMISO-PET scans to assess whether the acquisition time of hypoxia imaging could be reduced in NSCLC patients. Shorter acquisitions would ease the integration of hypoxia PET imaging within hypoxia-targeted therapy trials and potentially adoption into future routine clinical use. A good correlation for hypoxia-related metrics between 2- and 4-h scans was observed at whole tumour and subregion levels. However, there were differences in HV assessments between the two time points at whole tumour and subregion levels. Overall, given the better tracer-to-background contrast at 4 h p.i., our results support that scans should be performed at this timepoint to evaluate tumour hypoxia in NSCLC.Supplementary Information.


The numerical results indicated that the entrainment ratio of the rectangular section air ejector increased from 0.34 to 0.65 and the increment of the ER was 91.2% when the section width increased from 1 mm to 10 mm. As cW increased, the region of the turbulent kinetic energy gradually expanded. The energy exchange between the primary fluid and the secondary fluid was mainly in the form of turbulent diffusion in the mixing chamber. In addition to cW limiting the fluid flow in the rectangular section air ejector, the structure size of the rectangular section air ejector in the XOY plane also had a limiting effect on the internal fluid flow. In the rectangular section air ejector, the streamwise vortices played an important role in the mixing process. The increase of cW would increase the distribution of the streamwise vortices in the constant-area section. Meanwhile, the distribution of the spanwise vortices would gradually decrease.Due to its simple structure and lack of moving parts, the supersonic air ejector has been widely applied in the fields of machinery, aerospace, and energy-saving. The performance of the ejector is influenced by the flow channel structure and the velocity of the jet, thus the confined jet is an important limiting factor for the performance of the supersonic air ejector. In order to investigate the effect of the confined jet on the performance of the ejector, an air ejector with a rectangular section was designed. The effects of the section width ( An air ejector is a type of pneumatic device, which uses a vacuum created by the primary fluid to entrain the secondary fluid ,2,3. A lAdditionally, the cross-section shape of the ejector and the limitation degree of the restricted flow channel have an important influence on the characteristics of the internal flow inside the ejector and the performance of the ejector. In order to further study the mixing process in the restricted flow channel of the ejector, Bouheraoua et al.  conducteIn the restricted flow channel, the mixing process of the primary fluid and secondary fluid was complex . TherefocW) on entrainment ratio, velocity distribution, turbulent kinetic energy distribution, Mach number distribution, and vorticity distribution were studied.To further study the flow state of the high-speed jet in the rectangular restricted flow channel, the three-dimensional numerical simulation of the rectangular section air ejector is carried out. In this paper the effects of section width (ER) of the rectangular section air ejector was defined as follows:gm was the mass flow rate of the primary fluid, me1 was the mass flow rate of the upper secondary fluid inlet, and me2 was the mass flow rate of the lower secondary fluid inlet.The methods of ejector design include constant area mixing , constanaX), the inlet of the constant-area section (cX0), the 1/4 of the constant-area section (Xc1/4), the 1/2 of the constant-area section (Xc1/2), the 3/4 of the constant-area section (Xc3/4), and the outlet of the constant-area section (Xc1) were selected as the analytical cross-section. The structure parameters of the rectangular section air ejector are shown in cW of the rectangular section air ejector was changed, the restricted degree of high-speed jet in the rectangular confined flow channel changed, thus affecting the ER of the rectangular section air ejector and the flow characteristics inside the ejector. In this paper, the ER of the rectangular section air ejector and the flow characteristics inside the ejector were investigated when the cW of the rectangular section air ejector varied from 1 mm to 10 mm.As shown in (1)The air in the rectangular section air ejector was the ideal compressible gas;(2)The wall was a non-slip adiabatic wall;(3)Ignore the temperature change caused by the supersonic flow of gas in the whole process;(4)The mixing process was the constant pressure mixing;(5)Ignore the initial velocity of the primary fluid inlet and the secondary fluid inlet.In order to simplify the simulation, several simplifications were made as follows:The governing equation in the solving process was as follows:Continuity equation:Momentum equation:ρ was density, u was velocity, τ was viscous stress, μ was dynamic viscosity, E was total energy, P was static pressure, and δ was the Kronecker delta.Energy equation:It had been verified by experiments that the k-ω SST model in the numerical simulation was more suitable to describe the performance of the ejector and the flow characteristics in the ejector when the primary fluid was air . Therefok was the turbulent kinetic energy, ω was the specific turbulence dissipation rate, Γ was the diffusion rate, G was the turbulent kinetic energy generated by the laminar flow velocity gradient, Y was the turbulent kinetic energy generated by diffusion, and D was the orthogonal divergence term.k-ω SST turbulence model:−6.Fluent 19.2 was used to solve the rectangular section air ejector model. The primary fluid inlet, the upper secondary fluid inlet, and the lower secondary fluid inlet were all set as “pressure inlet”, while the outlet of the diffuser chamber was set as “pressure outlet”. The other walls of the ejector were set as non-slip adiabatic walls. All temperatures were set at 293 K. The coupled method was selected for the iterative solution. The second-order was adopted for the discretization scheme for pressure. The second-order upwind discretization was applied for the all solution. All residual values of the calculated results were less than 10In this paper, ICEM CFD was used to obtain the high-quality three-dimensional grid of the ejector. Since the structure of the rectangular section air ejector was symmetric along the XOZ profile, local axisymmetric meshes were selected to save calculation time. The structural mesh was adopted for meshing. In addition, the local mesh refinement approach was adopted in the region near the nozzle to improve the accuracy of the solution, as shown in cW were calculated with no less than 279,910 grids, which could reduce the calculation time, while ensuring the accuracy of the calculation.In order to ensure the accuracy of the solution and reduce the amount of computation, the grid independence of the rectangular section air ejector was verified. The number of grids increased from 148,864 to 842,258. The solutions were calculated for five different numbers of grids. Data detection points were set at the axis of the rectangular section air ejector for the validation of grid independence. The nozzle outlet (Point A) and the inlet of the constant-area section (Point B) were selected as the detection point. The velocity and pressure values of the two detection points were obtained from the calculation results. Further, the y+ of the first layer grid were kept at approximately 3 by encrypting the mesh of the wall and the nozzle, which satisfied the conditions for the k-ω SST model . The gricW. In addition, cW also affects the expansion state of the primary fluid. When cW changes, the ER and the flow characteristic of the rectangular section air ejector change accordingly. This paper focuses on the effect of cW on the performance and internal flow of the rectangular section air ejector when the primary fluid pressure (pP) is 400 kPa, the secondary fluid pressure (sP) is 100 kPa, and the XOY plane size is fixed.In the rectangular section air ejector, the mixed fluid will collide with the surrounding walls and cause a large amount of energy loss due to the limitation of cW increases from 1 mm to 10 mm, the variation of the ER of the rectangular section air ejector is shown in ER of the rectangular section air ejector increases rapidly at first and then fluctuates slightly with the increase of cW. When cW is 2 mm, the increment of the ER of the rectangular section air ejector starts to decrease. When cW is 5 mm, the incremental decrease of ER of the ejector with rectangular section is negligible, and the increment is only 0.014. When cW is 9 mm, the ER of the rectangular section air ejector reaches a maximum. When cW increases from 1 mm to 10 mm, the ER increases from 0.34 to 0.65, and the increment of the ER is 91.2%. This indicates that cW has a great effect on the performance of the rectangular section air ejector. Therefore, in this paper the flow characteristics inside the rectangular section air ejector are studied from the aspects of velocity distribution, turbulent kinetic energy distribution, Mach number distribution, and vorticity distribution.When cW. When cW reaches 5 mm, the length of the central jet reaches a maximum, and the flow of the central jet is in a stable state. As cW continues to increase, flow separation occurs at the tail of the central jet. When cW increases to 7 mm, the central jet will expand further and produce a droplet jet at the inlet of the diffuser. This is caused by the sudden change of the structure at the inlet of the diffuser, which causes the velocity of the central jet suddenly increasing at the end of the constant-area section. Furthermore, due to the restrictions of the wall, the central jet will have a violent collision with the wall in the process of flow when cW is 1 mm. The friction between the central jet and the wall leads to the decrease of the central jet velocity, which is more obvious at the axis of the central jet. cW. Due to the limitation of the wall structure, the central jet cannot expand freely and the velocity gradient of the central jet also changes. When cW is 1 mm or 2 mm, the velocity of the central jet in the constant-area section is lower than that of other structures. With the increase of cW, the velocity distribution area of the central jet expands gradually.The rectangular section air ejector does not have rotational symmetry. Therefore, the analysis method of the rectangular section air ejector is different from that of the conventional circular section air ejector. The XOY plane and the XOZ plane of the rectangular section air ejector were selected to analyze the velocity distribution, as shown in TKE) distribution of the rectangular section air ejector in the XOY plane under different cW. As demonstrated in cW, the region of the TKE gradually enlarges. In the mixing chamber, the energy exchange between the primary fluid and the secondary fluid is mainly in the form of turbulent diffusion. As cW increases, more energy is exchanged. When cW increases to 5 mm, the TKE in the constant-area section will no longer increase. The energy transfer process of the two fluids is stable. At this time, the energy exchange between the two fluids reaches a stable stage.TKE distribution of the rectangular section air ejector in the XOZ plane with different cW. TKE near the nozzle outlet fluctuates strongly when cW is 1 mm. When cW increases to 2 mm, the fluctuation region of the turbulent kinetic energy moves downstream, but the fluctuation of the TKE is still higher than that of other structures. Meanwhile, the mixing of the two fluids occurs closer to the inlet of the constant-area section. With the increase of cW, the mixing area of the two fluids gradually expands and moves to the outlet of the constant-area section. When cW increases to 5 mm, the fluctuation of the TKE occurs close to Xc1/2, and the fluctuation only occurs near the wall. As the cW continues to increase, the primary fluid entrains the secondary fluid to the downstream of the constant-area section, where the mixing of the two fluids is gradually enhanced. At the same time, the fluctuation region of the TKE in the constant-area section will decrease until it disappears completely.TKE distribution of the rectangular section air ejector along the X axis under different cW. TKE gradually weakens with the development of the fluids in the ejector. With the increase of cW, the region with the strongest turbulent kinetic energy gradually moves to cX0, and the shape of the fluctuation region of the TKE gradually tends to be consistent. It should be noted that the wall constraint on the fluid flow in the rectangular air ejector will be weakened with the increase of cW, but the dimension of the rectangular section air ejector in the XOY plane is fixed, which also plays a limiting role on the fluid flow in the rectangular section air ejector.cW was studied in this paper.When the primary fluid leaves the nozzle, the central jet expands and accelerates into the mixing chamber. In the mixing chamber, the central jet forms a shock train alternating with expansion and compression. As far as we know, the structure of the shock wave, depending on the distribution of Mach number, has a direct effect on the performance of rectangular section air ejector. Therefore, the Mach number distribution of the rectangular section air ejectors under different cW. As illustrated in cW is 1 mm. As cW increases, the area of the central jet gradually increases, and the length of the shock train also gradually increases. When cW increases to 5 mm, the length of the central jet reaches a maximum, but the length of the shock train continues to increase. When cW is 9 mm, the length of the shock train reaches a maximum, and the ER of the air ejector also reaches its maximum. In addition, the shock train also forms a symmetric structure with the XOZ plane as the axis, which is called the double shock wave structure. This has not been observed in the conventional circular section air ejector. With the increase of cW, the double shock wave structure gradually moves closer to the XOZ plane. As cW increases to 7 mm, it begins to coincide at the front of the shock train. However, when cW increases to 10 mm, the dual shock structure moves away from the XOZ plane. For further analysis, the Mach number distribution of the rectangular section air ejector in the XOZ plane under different cW was obtained, as shown in cW is 1 mm. With the increase of cW, the shock train gradually appears in the ejector, and the length of the shock train gradually becomes longer. This is caused by the gradual approach of the double shock wave structure to the XOZ plane. When cW increases to 10 mm, the length of the shock train in the ejector becomes shorter again, indicating that the dual shock wave structure is further away from the XOZ plane.cW, the high-speed central jet impinges on the wall in the rectangular section air ejector. The vortex in the ejector gradually twists and stretches, expands to all directions in space, and forms a vorticity field. The vorticity field includes streamwise vortices and spanwise vortices. The streamwise vortices play an important role in the entrainment process between the primary fluid and the secondary fluid. The spanwise vortices are generated by the velocity gradient between the primary fluid and the secondary fluid, which is mainly distributed near the nozzle outlet. The streamwise vortices (Ωs) and the spanwise vortices (Ωn) are defined as follows [D0 is the diameter of the nozzle outlet, and U0 is the mean velocity of the primary fluid the inlet of the nozzle.Vorticity is an important parameter to characterize the degree of turbulence. Under the restriction of  follows :(10)Ωs=DcW on the streamwise vortices in the rectangular section air ejector was studied in this paper. cW. In the figure, the red region is the positive vorticity, rotating in a counterclockwise direction. Meanwhile, the blue region is the negative vorticity, which rotates in a clockwise direction. In cW is 1 mm, the streamwise vortices at cX0 almost fill the entire section, and the wall attachment phenomenon of the streamwise vortices is the most obvious due to the limitation of rectangular flow channel. This wall attachment phenomenon is not conducive to the mixing of the two fluids, resulting in the lowest ER of the rectangular section air ejector under this structure. With the increase of cW, the streamwise vortices at cX0 begin to weaken. Simultaneously, the streamwise vortices develop gradually along the XOY plane, promoting the mixing of the two fluids.The rotational motion of the streamwise vortices causes a relative flow between the primary fluid and the secondary fluid, which increases the mixing effect of the two fluids. Therefore, the influence of cW. In cW is 1 mm. With the increase of cW, the spanwise vortices structure near the nozzle outlet began to expand towards the wall and gradually weakened. When cW increases to 5 mm, the constraint of the wall on the spanwise vortices is weakened. As cW continues to increase, the variation of the spanwise vortices in the constant-area section is no longer obvious.Driven by viscous shear forces, the spanwise vortices are generated by the velocity gradient of the primary fluid and the secondary fluid along the edge of the geometric structure. The spanwise vortices are the vortex component perpendicular to the streamwise vortices. During the development of fluid flow, the spanwise vortices interact with the streamwise vortices. At the initial stage of the mixing process, the velocity gradient between the primary fluid and the secondary fluid is the largest, and the vortex structure near the nozzle is mainly the spanwise vortices. With the development of fluid flow, the spanwise vortices near the nozzle outlet begins to expand toward the wall and gradually weakens. cW on the ER and the fluid flow characteristics of the rectangular section air ejector when the primary fluid pressure was 400 kPa and the secondary fluid pressure was 100 kPa, the velocity distribution, the turbulent kinetic energy distribution, the Mach number distribution, and the vorticity distribution in the rectangular section air ejector were studied by adopting the three-dimensional numerical simulation method. The following conclusions were obtained:(1)cW, the ER of the rectangular section air ejector first increases rapidly and then fluctuates slightly. When cW increases from 1 mm to 10 mm, the minimum ER is 0.34, the maximum ER is 0.65, and the increment of the ER is 91.2%.With the increase of (2)cW, the distribution of the TKE gradually expands. In the mixing chamber, the energy exchange between the primary fluid and the secondary fluid is mainly in the form of turbulent diffusion. When cW increases to 5 mm, the TKE in the constant-area section no longer increases. Currently, the energy exchange between the two fluids reaches a stable stage. As cW continues to increase, the primary fluid entrains the secondary fluid to the downstream of the constant-area section, and the mixing of the two fluids gradually increases in the downstream. In addition to cW limiting the fluid flow in the rectangular section air ejector, the dimension of the rectangular section air ejector in the XOY plane also has a limiting effect on the fluid flow in the rectangular section air ejector.With the increase of (3)cW, the region of the central jet gradually increases, as does the length of the shock train. When cW increases to 5 mm, the length of the central jet reaches a maximum, but the length of the shock train continues to increase. When cW is 9 mm, the length of the shock train reaches a maximum, and the ER of the rectangular section air ejector also reaches a maximum.With the increase of (4)cW, the mixing effect caused by the streamwise vortex is weakened, and the loss of the two fluids increases in the energy exchange process. Increasing cW will increase the distribution of the streamwise vortices in the constant-area section, and simultaneously, the distribution of the spanwise vortices will gradually decrease.In the rectangular section air ejector, the streamwise vortices play a primary role in the mixing process. Due to the limitation of In order to investigate the influence of


Physician-scientists have long been in high demand owing to their role as key drivers of biomedical innovation, but their dwindling prevalence in research and medical communities threatens ongoing progress. As the principal avenue for physician-scientist development, combined MD–PhD training programs and NIH-funded Medical Scientist Training Programs (MSTPs) must address all aspects of career development, including grant writing skills.The NIH F-series grants – the F30 grant in particular – model the NIH format of federal funding, and are thus ideal opportunities to acquire biomedical research grant preparation experience. Therefore, in this report, we describe a curricular model through which predoctoral MSTP students obtain exposure to – and training for – F-series grant conceptualization, writing, and evaluation.Since the development of these longitudinal courses, we observed trending improvements in student funding success rates, particularly among original submissions, and perceived benefits among participating students. Physician-scientists deliver irreplaceable value to both clinical and scientific communities by bridging efforts to treat both human disease and innovate healthcare delivery. A central “pipeline” for training physician-scientists is the dual-degree (MD–PhD) training program, which provides an integrated predoctoral training framework, linking MD and PhD training for trainees with potential as leaders in both biomedical research and clinical practice . Among tBarriers to grant funding success are far reaching and include lengthy periods of clinical training, and reduced research time, inadequate mentorship, and lack of institutional support . While mTherefore, the current document outlines a two-tiered curricular approach to grant writing preparedness for MD–PhD students. We propose that predoctoral grant writing training in grant writing would likely improve long-term retention and success of physician-scientists, bolstering this dwindling workforce in the academic sector.The NIH-funded Medical Scientist Training Program (MSTP) at the University of Alabama at Birmingham (UAB) has taken multiple steps to encourage and develop grant writing skills in MSTP students. Beginning with the entering class of 2013, the MSTP instituted the formal requirement for all students to submit an F-series grant (F30/F31). In accordance with NIH guidelines, students were required to submit within 4 years of matriculation (year 2 of graduate research).Survival Skills for Physician Scientists (STP2043) was created in the summer between the first and second year of preclinical medical training, a time at which time all trainees complete their second research laboratory rotations. The overall goal of this course is to introduce students to the grant writing process, give them a more involved and deeper understanding of their rotation projects, and facilitate general career development skills. To accomplish this, students are introduced to all components of an NIH training grant, and they practice writing scientific “specific aims” and training plans based on their summer research project; this is done in close collaboration with their summer research supervisors. Weekly meetings last 60–90 min consist of an overview of funding basics, career development, interpersonal interactions, time and data management, overview and completion of a Specific Aims page and Research Training Plan. Assigned resources given to all students include Making the Right Moves: A Practical Guide to Scientific Management for Postdocs and New Faculty [Mastering Your PhD: Survival and Success in the Doctoral Years and Beyond [Grant-writing Instructions, A Practical Guide to Writing a Ruth L. Kirchstein NRSA Grant [The Grant Application Writer’s Workbook [In 2014, a course entitled  Faculty , MasteriSA Grant , and TheWorkbook . The lasSurvival Skills for Physician Scientists course was reorganized in 2019 around topics including an introduction to fellowship grants, review of example F30 grant applications, authorship, a literature review with a UAB-affiliated librarian, an overview of the Background and Goals section, Specific Aims, and Research Training plan, a mentorship lecture with the graduate school dean, introduction to Individual Development Plan, and a Mock Study Section for peer and faculty review of Specific Aims pages at the end of the course. These changes were designed to provide students with a broad understanding of grant writing, the review process, and to focus on those aspects of grant writing that do not necessarily require a fundable project. The writing assignments were targeted to those sections describing their scientific history and career goals  and on the Aims page. Key career development principles relevant to grant writing, such as literature review and mentorship, were also added, while topics such as time and data management and interpersonal interactions were limited to reading assignments and brief discussions.The Grants & Grubs, was constructed to help trainees prepare sections of their grants during the process of their F-series grant-writing. As such, the goal of this course was to assist with the creation and submission of an F30 or equivalent grant application. The monthly series consists of 7 lectures (1.5 h/session) offered during the first and second years of PhD graduate research. Alongside MSTP faculty/administration, all sessions are co-led by MSTP students who had already previously submitted – and subsequently been awarded – an F-series grant. Lectures provide an overview of the different grant components as well as presentation of the other more technical components of the grant including forms, approvals, and letters of recommendation and support. Students are not required to write the components of the grant as part of the course, but can bring their grants for review prior to submission deadlines for real-time feedback. Resources included Making the Right Moves [The Best Kept Secrets to Winning Grants [Grant Application Writer’s Workbook [A Practical Guide to Writing a Ruth L. Kirchstein NRSA Grant [A Practical Guide to Writing a Ruth Kirschstein NRSA Grant [The Grant Application Writer’s Workbook [An additional series of lunch lectures, entitled ht Moves , The Besg Grants , Grant AWorkbook , A PractSA Grant , numerouSA Grant , along wWorkbook . Based oSurvival Skills for Physician Scientists and Grants & Grubs courses were collected from students via online forms, given anonymously. A 5-point Likert agreeability scale ranging from "Strongly Disagree” to "Strongly Agree” was used to determine student satisfaction with different aspects of the course. Responses from two to nine students were analyzed for each course per year from 2014 to 2019 for Survival Skills for Physician Scientists and from 2019 to 2020 for Grants & Grubs. The total number of responses collected and pooled for each course ranged from 7 to 40. Data were reported as frequency of responses . Ethical approval was provided by the UAB Office of the IRB.Evaluations for the Data on F30/F31 funding rates for MD–PhD students and other graduate-level students at UAB from 2013 to 2021 were provided by the UAB Office of Sponsored Programs. The start year used was defined as the actual or anticipated year that funding would begin, rather than the year the grant was submitted. Data used for analyses included student program  doctoral program), activity description (F30 or F31), sponsor within NIH, grant status , type of submission , and dates of award. Withdrawn applications or those transferred from another institution were excluded from analysis, and all award information was de-identified. National success rates were obtained from NIH RePORT (report.nih.gov). It should be noted that for UAB and GBS we collected data on all submissions (whether reviewed or not), while for NIH the only data reported were for applications that were reviewed. Data on the number of grant submissions at UAB prior to 2013 could not be obtained so success rates for years prior to 2013 are not reported. We calculated the number of grant submissions, number of grants awarded, and the percent funded/success rate (number of grants awarded/number of submissions × 100) for each group from 2013 to 2019. Data for the MSTP F-series grants were pooled due to the comparatively low number of submissions compared to GBS and national submissions.p-value less than 0.05. All statistical calculations were performed using GraphPad Prism v 9.1.0.Success rates were compared between different groups using Student’s unpaired t test for comparisons of two groups, and False Discovery Rate two-stage step-up method of Benjamini, Krieger, and Yekutieli was used when multiple t-tests were performed. Success rates over time by year were analyzed using simple linear regression, with statistics computed relative to a horizontal slope. Significance was defined as Survival Skills for Physician Scientists and Grants & Grubs courses. Overall, we found that students were satisfied with the course structure and would recommend these courses for other students  and Making the Right Moves (60%) printed materials receiving positive feedback, the proportion of funded grants remained unchanged  grant funding rates from both the GBS program and national F-series funding rates. In accordance with the new requirement, MSTP student F30/F31 submissions sharply increased threefold and remained stable until the end of reporting period  Fig. . We founIn the current report, we provide a novel framework for developing MD–PhD student grant writing skills, using F-series grants as mechanisms of both financial support and growth through a necessary career skill. Implementing similar training support for MSTP trainees will likely improve trainee preparedness in other domains, bolstering a healthcare workforce that is essential for medical innovation. Overall, we believe that predoctoral grant writing coursework may improve success in both retention and success of academic physician-scientists academia.Despite its benefit to trainees, the incorporation of grant writing training is largely lacking among current MD–PhD – including NIH MSTP – programs. According to the information provided on national MSTP websites, only 44% (22/50) of current NIH-funded MSTP programs provide grant writing preparation/training, and fewer  offer dedicated courses and/or mock study sessions. Although 44% of programs endorse student applications for extramural funding, only 7/50 programs require grant submission as a component of training. Therefore, although several institutions provide longitudinal grant writing support to trainees, our observations suggest that the grant writing preparation varies widely across institutions.Grants & Grubs course from 2017 to 2019, suggesting that students are submitting fundable applications more consistently. Additionally, it should be noted that seeing the effect of recent course changes will take up to 2 years to be apparent. We also looked at the distribution of funded grants by institute for both UAB programs and compared this to the national distribution of funded grants by institute. While we expected the number of institutes applied to for UAB MSTP and GBS students to be lower than that for national F-series submissions, the number of institutes for funded grants from UAB students was much shorter, with only 7/13 total institutes funding UAB MSTP awards. Further, the top NIH institute for each program funded 30% of UAB MSTP awards (NHLBI), whereas the top national rate of 26.8% originated from the NCI.However, we did observe a more consistent funding rate and number of awards with reduced variability for F30 grants, particularly original submissions, by our UAB MSTP students following initiation of the Grants & Grubs. Students enjoy participating in a less-intensive course during the preclinical stage, or the summer spanning the first 2 years of medical school. Similarly, trainees felt that beginning the Grants & Grubs course in spring of their first year was early enough in the PhD to give adequate time prior to grant submission, while still allowing sufficient time to conceptualize their projects.Aside from increasing success rates for F-series grants, a more intangible desire of the course was to foster students’ confidence in grant writing and submissions. We consistently noted from subjective evaluations that students were challenged by our courses, improving knowledge through hands-on practice. Specifically, our students directed most positive feedback to hands-on activities within the course where they could receive feedback on their projects and writing style via peer reviews. Students also noted that access to a database of recently funded F30/F31 grants serves as a central resource during their drafting process. Course timing was also identified as an important indicator of usefulness, especially for Despite the benefits observed among trainees in response to our course offerings, a few limitations in our analysis should be considered. Firstly, our single-center cohort study design precludes us from making broad conclusions regarding course outcomes. Furthermore, measuring the effectiveness of curricular support is based on federal funding rates, which vary widely both across NIH institutes and over time. As an inherent challenge to biomedical research funding, it is important to candidly discuss the subjective and fluctuating ecosystem of federal funding. Doing so would likely encourage trainees to distinguish between grant writing skill development from the ultimate funding decisions.In conclusion, we believe that a structured curriculum of grant writing preparation is key to the long-term success of all MD–PhD students as they develop into independently funded physician-scientists. Regardless of whether the extramural grants they write during training are funded, the process of engaging them in grant drafting and simulated reviews offers needed insight into their academic careers.


ORF7) of VZV, as well as the development of a skin- and neuro-attenuated live varicella vaccine comprising an ORF7-deficient mutant, v7D. This work could provide insights into the research of novel virus vaccines based on functional genomics and reverse genetics.Primary varicella-zoster virus (VZV) infection causes varicella, which remains a prominent public health concern in children. Current varicella vaccines adopt the live-attenuated Oka strain, vOka, which retains the ability to infect neurons, establish latency and reactivate, leading to vaccine-associated zoster in some vaccinees. Therefore, it is necessary to develop a safer next-generation varicella vaccine to help reduce vaccine hesitancy. This paper reviews the discovery and identification of the skin- and neuro-tropic factor, the open reading frame 7 ( Varicella zoster virus (VZV) is a neurotropic human α-herpesvirus that causes varicella (or chickenpox) during the primary infection, and then establishes lifelong latency in neurons of the dorsal root, cranial, enteric and autonomic ganglia . Years lVaccination is a cost-effective way to prevent varicella. Currently, only live-attenuated vaccines are available for protection against varicella, and the most widely used varicella vaccines employ the Oka vaccine strain of VZV (vOka). vOka was developed by Michiaki Takahashi in 1974 through serial passages of the wild-type parent Oka (pOka) strain of VZV in cell cultures . This meHowever, there have been concerns raised about the use of vOka varicella vaccines following the analysis of adverse events (AEs) reported in post-market surveillance ,20,21,22To date, concerns over the safety of vOka have led to many studies that have evaluated the use of inactivated viruses or recombinant viral subunits as vaccines against VZV infection ,28. VaccVZV induces pathological abnormalities, mainly in human skin and nerve tissues, during infection. Characterizing the crucial genes related to the skin- and neuro-tropisms of VZV will aid in the understanding of VZV pathogenesis, and it will also facilitate the development of improved vaccines. However, VZV is strictly human-specific, and has a highly cell-associated nature in cultured cells. These qualities make it challenging to study VZV gene functions and distinguish VZV from the other α-herpesviruses, which can infect a wide range of host species and generate large amounts of cell-free particles, leading to a much earlier and clearer understanding of their replication and pathogenesis compared to VZV. Recently, thanks to advances in molecular virology and reverse genetics, VZV gene function in specific cells and tissues has been characterized extensively by mutagenesis of the VZV genome, through the use of human tissues cultured ex vivo or implanted in severe combined immunodeficient (SCID) mice in vivo  32,33,3,333,34.galK gene), via homologous recombination in E. coli [ORF10 [ORF14 [ORF47 [ORF7 as a novel gene essential for VZV replication and spread in human skin [ORF7 deletion mutant showed a severe growth defect in these neuronal cell models, and its neuro-attenuated phenotype was further confirmed in human fetal dorsal root ganglia (DRGs), both ex vivo and in vivo [ORF7 as the first, and currently only, known full-length neurotropic gene of VZV. Altogether, our work identified ORF7 as a skin- and neurotropic factor of VZV, and we found that while retaining replication competence in other cell lines, an ORF7-deficient mutant that is defective in spread in human skin and neurons would be a potential live-attenuated vaccine candidate against VZV infection.We aimed to identify the key viral virulence and tissue-tropic factor(s) for the rational design of a next-generation, safer, live-attenuated VZV vaccine. For this purpose, we used bacterial artificial chromosome (BAC) technology to develop an efficient method for generating recombinant VZV mutants ,35. To b E. coli ,32. The i [ORF10 , ORF14 [0 [ORF14  and ORF44 [ORF47 , we idenman skin . Among t in vivo . TherefoORF7-deficient VZV vaccine virus, we employed a stop-codon mutation strategy to minimize changes to the original genome sequence of the wild-type virus [ORF7 was replaced with a three-frame stop-codon cassette in the BAC clone of the VZV wild-type pOka strain, for the abrogation of ORF7 expression. Then, the loxP-flanked BAC vector containing the GFP expression cassette was excised from the viral genome by co-transfecting the purified BAC clone with a Cre expression vector into human fibroblasts, thus reconstituting the ORF7-deficient candidate vaccine virus, v7D. Compared with the original wild-type strain of VZV-pOka, there was no difference in the genome sequence of v7D, except for the stop-codon mutation within ORF7 and a single 34 bp loxP site in the noncoding region between ORF60 and ORF61. Whole-genome sequencing of serially passaged v7D showed that its genome was stable for at least 25 passages in cell culture.To construct the pe virus . FirstlyORF7 deletion mutant [ORF7 expression prevents the replication and cell-to-cell spread of v7D in human skin and neuronal cells, both in vitro and in SCID-hu mice in vivo. In contrast, wild-type VZV and vOka established a robust lytic infection in these cells. In addition, v7D failed to infect DRGs in the guinea pig and cotton rat models of VZV infection, and showed no intrathalamic neurovirulence or repeated-dose systemic toxicity in non-human primates. These preclinical toxicity evaluations show that v7D is highly skin- and neuro-attenuated, and should have a reduced risk of vaccine-associated complications, thus meeting the critical safety criteria for a next-generation live-attenuated varicella vaccine.To thoroughly confirm the skin- and neuro-attenuation phenotype of v7D, a variety of cell and animal models currently available for evaluation of VZV infection and tissue tropism were used . Similarn mutant ,33, the Despite the attenuation phenotype, v7D exhibited wild-type growth in MRC-5 human fetal lung fibroblasts, allowing for the production of v7D as a live vaccine . FurtherVZV only infects humans, and thus VZV vaccine studies are limited to the use of common experimental animal models, which lack virus–host interactions under natural infection scenarios, to obtain immunogenicity data as indirect clues to VZV vaccine efficacy in humans. For preclinical immunogenicity evaluation of v7D, small animal models, including mice, rats, guinea pigs and rabbits, as well as non-human primate models, were used . Three sOur preclinical data suggest that v7D is a promising, safer, live vaccine candidate against varicella. Nevertheless, many questions remain and should be addressed in future research.Through future clinical observations, the v7D vaccine is expected to show several safety advantages compared to the vOka vaccine, including lower frequency of both varicella-like rashes and vaccine-associated HZ cases. However, fully confirming the long-term safety of v7D will require decades of observation for possible reversion to wild-type virulence, latency and reactivation of the vaccine virus, and revealing all AEs associated with v7D vaccination.ORF61 mutant with stop-codon mutation reverted to wild-type virus in cell culture, but this virus retained the coding sequence of a RING finger domain of pORF61, which could have contributed to its wild-type reversion [ORF7, which could be beneficial to the stability of the virus genome and its attenuation phenotype. Nevertheless, it is necessary to continue to evaluate the risk of wild-type reversion of v7D in subsequent studies.The possibility of reversion to a wild-type phenotype always raises concerns over the use of live-attenuated vaccines. However, like all herpesviruses, the VZV DNA genome is highly stable, and we have not observed the occurrence of wild-type reversion of v7D during serial cell passaging and long-term in vivo experiments. An early study showed that an eversion . For v7DIn addition, we do not know whether v7D would establish neuronal latency, although v7D cannot establish an initial lytic infection in human neurons and is much less likely to reactivate from possible latency, if at all. Therefore, it will be an important research question to determine whether v7D establishes latency and expresses VZV latency-associated RNA transcripts (VLT) in human neurons, based on the in vitro and in vivo models that are available to study VZV latency.v7D has distinct properties of attenuation that may enable it to induce different patterns of immune response, compared to that of vOka, in humans. Therefore, with reference to an early study of vOka , the dynORF7 expression. ORF7 encodes a virion tegument protein that localizes to the Golgi apparatus in the infected cells [ORF7 does not prevent viral entry, viral genome replication, viral protein expression or retrograde transport of virus particles from axon terminals to somata, but substantially affects syncytia formation, secondary envelopment and subsequent cell-to-cell spreading of progeny viruses in differentiated skin and neuronal cells in vitro [v7D is a pure and genetically defined VZV strain that has a replication defect in both human skin and neuronal cells due to the abrogation of ed cells , the secin vitro ,44,45. Tin vitro . Most rein vitro . FurtherIn addition, to overcome the limitation of VZV’s strict human specificity, future research on the mechanisms of v7D attenuation and protective immunity induced by v7D vaccine could be complemented by work in non-human primate models of simian varicella virus, which recapitulates the clinical features of VZV infection in humans .ORF7 as a skin- and neuro-tropic factor of VZV, and we generated an ORF7-deficient, pOka-derived live virus vaccine candidate, v7D. Compared to vOka, v7D has a reduced risk of vaccine-associated AE in humans, and shows similar immunogenicity in animal models. Future research should focus on testing the safety and effectiveness of the v7D vaccine, determining its immune procedure and dosage through clinical trials, and understanding the mechanism of v7D attenuation and protective immune response induced by this vaccine virus. Together, this knowledge could help guide the future use of v7D as a safe viral vector for the prevention and possible treatment of infectious diseases.Varicella remains an important public health concern worldwide. The vaccine virus vOka retains neurovirulence and may cause vaccine-associated HZ in some vaccinees. Thus, a safer neuro-attenuated vaccine is needed to improve vaccine acceptance and aid in establishing herd immunity against varicella, together with vOka vaccines. Through a genome-wide mutagenesis screen, we identified


Secondary lymphoid organs (SLOs) are important initiators and regulators of immunity. To carry out this function, the blood vasculature must deliver oxygen and nutrients and recruit circulating lymphocytes into the SLO parenchyma, where they encounter cognate antigen. High endothelial venules (HEVs) are specialised postcapillary venules that specifically serve this function and are found in all SLOs except spleen. It is becoming clear that alterations to HEV network density and/or morphology can result in immune activation or, as recently implicated, in providing an exit route for tumour cell dissemination and metastases. In this review, the structural plasticity of HEVs, the regulatory pathways underpinning this plasticity, and the relevance of these pathways to cancer progression will be discussed. High endothelial venules (HEVs) are specialised blood vessels, found in secondary lymph nodes, where they play an important role in initiating and regulating immunity.Recent studies indicate that HEVs can also provide an exit route for tumour cell dissemination and the establishment of metastases.The dual roles of HEVs imply that they can both limit and accelerate progression of cancer.It is important to address gaps in our understanding of HEV remodelling in order to uncover routes to promoting immune activation via HEV, whilst simultaneously limiting their ability to disseminate cancer cells. HEVs comprise high endothelial cells (HECs) that are readily distinguished from other blood endothelial cells by their characteristic plump, cuboidal morphology, first described in the late 19th century , HECs prBox 1X epitope, which decorate PNAd, including the fucosyltransferase FucT-VII (Fut7) and core 2 branching enzyme Core2 GlcNAcT (Gcnt1), were found to require higher levels of LTβR-dependent signalling for expression than the sulfotransferase GlcNAc6ST-2 (Chst4) . In. In74].  (Chst4) . Such di (Chst4) .Figure IAlt-text: Box 1Since the development, structure, and function of HEVs have been described in detail in previous reviews, a summary of the key features of HEV is described in + dendritic cells (DC)  has been extensively studied and is a well-regulated process that occurs in three distinct phases , 17.. Thlls (DC) . Upregulonocytes ,16. FRCsonocytes . During [+ cells , whilst [+ cells . In addi[+ cells  and last[+ cells . Recentl[+ cells . In addifunction , resultifunction .hi DCs [The vascular-stromal quiescence phase can last for several weeks and is mediated by CD11chi DCs . This phhi DCs . HEVs alhi DCs . LN vaschi DCs . Whilst hi DCs .mesoscopic imaging techniques  and light sheet fluorescence microscopy (LSFM), has allowed for the global analysis of HEV network remodelling in response to infection [Box 21β2-expressing B cells and not by VEGF-A stimulation [Lymphocytic choriomeningitis virus (LCMV) is a widely used mouse model for examining immune responses during viral infection due to it being noncytolytic and capable of inducing a wide range of immune responses, depending upon which LCMV strain is used . LCMV hamulation . Therapemulation . Whethermulation . MSC admmulation . UnderstAlt-text: Box 2The development of ques see , such asnfection , immunisnfection . Such stnfection ,24. IncoRegional LNs, which drain established tumours and are the first to receive metastatic cells (referred to as sentinel LN), are known to undergo tumour-reactive lymphadenopathy accompanied by both lymphatic and vascular expansion , 27.. SeThe histological findings described earlier have been confirmed by electron microscopy imaging of metastatic sentinel LNs from oral and pharyngeal squamous cell carcinoma (OPSCC) patients, revealing the presence of remodelled HEVs engorged with RBCs displaying thin and dilated lumens with loose structure and noncontinuous basement membrane . Real-tiSince alterations to sentinel LN HEVs are observed before tumour cells become detectable, it is conceivable that tumours may ‘prime’ sentinel LN vessels via lymph-borne tumour-specific or inflammatory mediators to prepare for the arrival of malignant cells. Indeed, there is evidence to suggest that lymph-borne factors as opposed to blood-borne factors stimulate HEV remodelling, as injecting dead tumour cells and plasma from tumour-bearing mice does not induce systemic LN HEV remodelling . FurtherIn a murine model of VEGF-D-driven tumour metastasis, reduced expression of bone morphogenetic protein-4 (BMP-4) in HEVs was associated with a remodelling of HEVs towards a flat, thin-walled phenotype . Whilst Lymph-borne factors that drive sentinel LN remodelling may be distinct from those involved in immune reactive remodelling. In support of this, it has been shown that nude mice are susceptible to sentinel LN remodelling but resistant to endotoxin-induced LN remodelling, suggesting a lack of T cell involvement in the former . In thisFurther studies are, however, required before it can be concluded that sentinel LN and immune reactive LN remodelling are distinct processes. Indeed, it is possible that immune cell activation in sentinel LNs  driven bThe involvement of LNs as a gateway for further dissemination of tumour cells is supported by correlative evidence from mouse models of breast and prostate cancer , 40. as + luciferase+ 4T1 cells, it was possible to detect metastasis in the lungs through the use of whole-animal in vivo bioluminescence imaging [Tumour cell dissemination via HEVs was confirmed recently by two studies. The first study demonstrated that the intralymphatic microinfusion of 4T1 mammary carcinoma tumour cells leads to the accumulation of 4T1 cells within the subcapsular sinus of the draining LN . Three d imaging . Lung me imaging . Further imaging , confirm imaging . Dendra2 imaging . Tumour  imaging . These tInterestingly, tumour establishment was not required for HEVs to disseminate intralymphatic-infused 4T1 cells . HEVs maWhat is currently lacking is a global analysis of sentinel LN HEV remodelling using the aforementioned 3D imaging techniques. Characterising the tumour-driven progression of HEV remodelling, and comparing this to previously characterised changes to HEV networks in immune reactive LNs, may reveal shared features that could be therapeutically targeted if such changes are indeed found to be important mediators of tumour metastasis.Box 3In a retrospective cohort of 146 invasive breast cancer patients, HEVs were found to correlate with disease-free, metastasis-free, and overall survival . In addi+ splenocytes can be recruited to spontaneously induced TLS in a model of inflammation-driven carcinogenesis [–/– mice, which lack all peripheral LNs, leads to the recruitment and induction of specific T cell responses, suggesting in situ priming at TLS sites [In keeping with their function in SLOs , severalogenesis . FurtherLS sites . Even inLS sites ,58,68.Alt-text: Box 3The presence of HEVs in primary human solid tumours and its association with lymphocyte infiltration has been shown in several tumour types, including breast, lung, ovarian, colon, and melanoma . ImportaUnlike in secondary lymphoid organs (SLOs), however , the fun+ tumour-infiltrating T cells, leading to a reduction in tumour growth burden [In spite of the aforementioned caveats, the correlation between HEV formation, increased lymphocyte infiltration, and more favourable prognosis in various cancer types ,61,62 hah burden . Normalih burden .HEVs can also be exploited to effectively deliver chemotherapeutic drugs. Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease characterised by a dense stroma and extensive desmoplastic reaction, which limits efficient drug delivery . TargetiThe dual role of HEVs as promoters of tumour cell dissemination in naïve/remodelled sentinel LNs and as drivers of effective antitumour immunity in cancer  raises sThe signalling pathways involved in SLO formation and maintenance are largely overlapping and selective targeting of HEV neogenesis is therefore an important factor to consider . HoweverWhether intratumoural HEVs can support tumour cell dissemination is also currently not known. The functional consequence of intratumoural HEVs as both immune cell carriers and exit routes for tumour cells should be explored. In support of a tumour cell disseminating role, the presence of HEV-like vessels in OPSCC primary tumours was associated with sentinel LN metastasis . AlternaOutstanding QuestionsWhat are the factors that drive sentinel lymph node (SLN) HEV remodelling?Does SLN HEV remodelling lead to impaired immune activation?Are SLN HEVs more efficient at disseminating tumour cells than basal LN HEVs?What impact do HEV-inducing therapies have on LN HEV networks and how might this affect their role as both regulators of immunity and sites for tumour cell dissemination?Are intratumoural HEVs capable of disseminating tumour cells and how might this be further affected by HEV-inducing therapies?Alt-text: Outstanding QuestionsLastly, do the progressive changes of HEVs during sentinel remodelling mark the loss of immune cell recruiting functionality, leading to an impairment in immune activation? If so, then how might it be possible to drive remodelling towards an immune cell carrier instead of tumour cell carrier? There is evidence that resection of sentinel LNs leads to a significant reduction in metastatic spread but evidence also points to an important role for sentinel LNs in the induction of effective antitumour immune responses . A bette


It uses a liquid CO2 system containing a small amount of water-based fracturing fluid to carry sand, and it is characterized by sand blending at normal pressure, convenient preparation, the integrated application of resistance reduction and sand carrying, and no dedicated closed sand blender requirement. We developed a self-crosslinking emulsion-type water-based fracturing fluid (ZJL-1), which contained ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic associations, for quasi-dry CO2 fracturing, and the comprehensive properties of the ZJL-1 fracturing fluid were evaluated. The results showed that the ZJL-1 fracturing fluid had obvious viscoelastic characteristics, a heat loss rate of less than 10% at 200 °C, a good thermal stability, sufficient rheology under high temperature and high shear conditions, and a good thermal stability. The resistance reduction rate reached 70%, which demonstrates a good resistance reduction performance. Compared with conventional guar fracturing fluid, ZJL-1 can carry more sand and has a lower core damage rate. The on-site use of quasi-dry fracturing showed that optimizing the mixing ratio of liquid CO2 fracturing fluid and ZJL-1 fracturing fluid effectively enhanced oil and gas recovery. This can be used to optimize quasi-dry fracturing and can be used as a reference.Quasi-dry CO In regard to oil and gas production, unconventional resources, such as shale gas and tight oil, have attracted much attention in the past decade and have become the focus in the development of energy resources by the global oil industry. According to the U.S. Energy Information Administration, global tight oil production will increase from 33% of the total land oil production to 51% in 2040 to alleviate the world’s growing energy demand and will account for nearly 10% of the global oil production. Low-porosity and low-permeability reservoirs, such as tight oil, gas, and shale gas reservoirs, are generally characterized by poor reservoir physical properties, a permeability generally lower than 0.1 mD, and natural productivity lower than industrial standards [2 fracturing has been developed at home and abroad. This technology uses 100% liquid CO2 as the fracturing fluid, thereby avoiding water sensitivity and water locking damage to reservoirs, and CO2 is discharged as gas during flowback without residue and damage [For unconventional oil and gas resources, the exploitation method necessarily has its particularities, and conventional hydraulic fracturing easily results in water sensitivity and water locking damages to reservoirs, which impacts the increases sought in oil production ,4. In red damage ,6,7.2 dry fracturing test. The test results showed that liquid CO2 dry fracturing was 1.9 times more effective than nitrogen fracturing and 4.9 times more effective than nitrogen foam fracturing [2 to improve its sand carrying capacity, but increasing the flow rate of CO2 increases the friction loss in the pipeline [2 thickeners. The results showed that 1.5~2.0% concentration of TNJ-3 type thickeners increased the CO2 viscosity by 240~490 times and enhanced the performance of carbon dioxide fracturing fluids in TNJ-3 test evaluations [2 by 17–184 times, and the thickener has been successfully applied to construction sites in the Changqing gas field in the Ordos Basin for liquid CO2 fracturing, but because it is a nonpolar solvent with poor water solubility and low degradability, the thickener remains in the formation after compression and can cause reservoir damage [2/N2 foam fracturing fluid system by mixing liquid N2 into liquid CO2 and used methoxy nonafluorobutane (C4F9OCH3) as a foaming agent, which retained the liquid CO2 fracturing fluid. However, the introduction of N2 greatly reduced the static pressure of the fracturing fluid system, which establishes higher requirements for the fracturing pump truck, thus limiting its application range [2 dry fracturing and have achieved certain results [2 fracturing technology was applied in the Yanchang Oilfield 167 times for the production of tight oil, tight gas, and continental shale gas, and the production stimulation effect was remarkable [2 fracturing also has many problems, including low viscosity, poor sand-carrying and fluid-loss control capabilities, high frictional resistance, and difficulty in effectively creating fractures; moreover, special closed sand-mixing equipment is used, and the scale of sand addition is limited. As a result, the thickening, drag reduction, and sand-carrying properties of liquid CO2 are poor, and the ideal stimulation effect cannot be achieved; the fracturing equipment costs, material costs, and construction costs are too high for this method to be popularized and applied on a large scale [For the Devonian water-sensitive shale formation in eastern Kentucky, the US Department of Energy (DOE) funded a liquid COacturing . Some repipeline . Wang etluations . The fatr damage . The BJ on range ,14,15. W results ,17,18. Cmarkable . Howeverge scale ,21.2 fracturing, which is a combination of liquid CO2 dry fracturing and conventional water-based fracturing, is currently being studied [2 fracturing technology proceeds as follows: CO2 thickener, water-based thickener, and proppant are added into water to form a water-based mixture with high viscosity and high sand ratio (water-based fracturing fluid accounts for 10–30%), the water-based mixture is blended with 90–70% liquid CO2 at the high pressure tee manifold of wellhead to form a mixed-phase liquid with some viscosity. Liquid CO2 is a weak solvent that is immiscible with clean water. The quasi-dry CO2 fracturing process requires the use of a liquid CO2 system containing a small amount of water-based fracturing fluid to carry sand. Moreover, the mixing ratio of liquid CO2 and water base is optimized. Under the condition of ensuring the stable performance of fracturing fluid, the dosage of the water-based fracturing fluid is required to be as low as possible and the damage to be minimal. This technology is characterized by low damage, no requirement for a dedicated closed sand blender, relatively simple operation flow and control, and suitability for large-scale sand addition and fracturing, etc., [2 fracturing technology and meets the requirements for fracturing construction technology with the high sand ratio.Based on the above reasons, quasi-dry CO studied . The quag, etc., , and it Traditional water-based fracturing fluid thickeners are mainly in the form of solid powders, and the solution dissolves slowly when used, which not only increases the workload but also increases the cost and causes the product to shear and degrade, affecting the use effect. It is also necessary to use a cross-linking agent for cross-linking to carry sand. The cross-linking effect is unstable and poorly controllable, resulting in a low sand ratio and insufficient sand-carrying capacity, resulting in unsatisfactory or even poor production enhancement effects. The integrated operation of low-concentration drag reduction and high-concentration sand-carrying cannot be well satisfied. Emulsion-type fracturing fluid thickeners, such as conventional polyacrylamide emulsions, are usually used in the preparation of low-concentration slick water. The amount of sand is less than that of low-sand, the cross-linking conditions are harsh, and it is difficult to prepare sand-carrying fluid. Therefore, this research develops a self-crosslinking, emulsion-based, water-based fracturing fluid system (ZJL-1) that satisfies the integration of drag reduction and sand-carrying and does not require a crosslinking agent for crosslinking. The fracturing fluid dissolves and self-crosslinks, and a strong spatial network structure is formed through ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic associations, so it has excellent shear resistance. It can still achieve a high sand ratio and sand carrying at high temperatures. A series of performance evaluations of the ZJL-1 fracturing fluid included studies of the temperature-resistance capacity, viscoelasticity, shear-resistance capacity, frictional properties, proppant suspension ability, gel breaking properties, and core damage properties. This fracturing fluid system can provide technical support for oilfield fracturing sites. (1) Reagents: The self-crosslinking emulsion thickener was prepared by inverse emulsion polymerization. In a three-necked flask protected by nitrogen gas, add 140 g of silicone oil, 4 g of Span80 and 1 g of OP-10, and stir at high speed for 30 min. Dissolve 40 g of acrylamide, 10 g of 2-acrylamido-2-methylpropanesulfonic acid, and 5 g of dimethyl diallyl ammonium chloride in 140 g of deionized water. After the monomers are completely dissolved, slowly drop them into a three-necked flask, stir while adding dropwise, control the drop rate, finish the drop within 25–30 min, control the stirring speed, use an emulsifier to emulsify for 1.5 h, and keep the state of the water-in-oil emulsion stable. Add 0.05 g of benzoyl peroxide, 0.05 g of azobisisobutyronitrile, and 0.05 g of tert-butyl peroxide to the emulsion at 45 °C for initiation, raise the temperature of the reaction system to 85 °C, and control the reaction at this temperature 14 h after the reaction, the jacket of the reaction vessel was circulated through cooling water, and the temperature was lowered to 25 °C. The white emulsion obtained in the reaction vessel was a self-crosslinking emulsion type fracturing fluid thickener. After the reaction was completed and purified, the conversion rate was 95.6%. The structural formulas of AMPS and the self-crosslinking emulsion-based thickener are shown in −3 mg/L, the relative molecular mass M is 8.14 × 106 [The intrinsic viscosity [14 × 106 . The selAcrylamide, 2-acrylamido-2-methylpropane sulfonic acid (AMPS), dimethyl diallyl ammonium chloride (DMDAAC), and silicone oil were all purchased from Macklin’s Reagent Co., Ltd., Shanghai, China. Span80, OP-10, benzoyl peroxide, azobisisobutyronitrile, and tert-butyl peroxide were purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai, China.Nitrogen (purity 99.95%) was provided by Xinxing Gas Co., Ltd., Wuhan, China. China. All of the above reagents were of industrial purity.3.(2) The proppant sample was low-density ceramsite, with particle sizes of 0.2–0.4 mm and densities of 1.0–1.3 g/cm(3) Conventional guar fracturing fluid was provided by Yanchang Oilfield.(4) Synthesized brine was used in the present study, and the ionic composition is shown in (5) Core for experiments: a long core spliced with natural cores was provided by S Oilfield through artificial single seam splicing; the basic physical properties of the natural cores are shown in Using a Bruker Tensor 27  Fourier Transform Infrared Spectrometer, the liquid to be measured was characterized in the infrared by applying drops of the liquid to a pure potassium bromide pressed tablet .The surface morphology of ZJL-1 was investigated after self-crosslinking using a Thermo Nicolet 380 Scanning Electron Microscope . ZJL-1 was frozen at −20 °C and then freeze-dried for 24 h. Before SEM observation, the freeze-dried samples were cut open to expose the internal structure of the cross-section, sprayed with gold and then observed .The temperature was raised from 25 °C to 500 °C with a Perkin-Elmer Pyris Diamond TG thermal analyzer  to analyze the thermal stability of ZJL-1. First, glass slides were wiped clean, and then appropriate amounts of sample was applied to them. Then, the glass slides were baked in a drying oven at 60 °C for a period of time to remove the moisture in the samples. Nitrogen was used as the protective gas, and the heating rate was 10 °C/min.Tests of viscoelasticity and rheological properties were carried out with a Haake RS6000 rheometer . The storage modulus (G′) and loss modulus (G″) of ZJL-1 were determined at different concentrations, with the temperature set to 25 °C, the 35 mm cone-and-plate test system and oscillating measurement mode selected, the oscillating angular frequency set to 1 Hz, and the yield stress set to 0.5 Pa .−1 for 2 h. The relationship between the apparent viscosity of the fracturing fluid and the time curve was measured [The experiment used a HaakeMars60 rheometer . The reactor inside the rheometer was a steel cylinder, and the pressure of the reactor was 40 MPa, which met the requirements of this experiment. ZJL-1 was poured into the reaction kettle, the temperature control line and the pressure sensing line were connected, the kettle was heated to 160 °C with the external circulation of the water bath, nitrogen was injected into the reaction kettle, the pressure was adjusted to 20 MPa, and the shear speed was set to 170 smeasured ,29. Fracmeasured ,31,32.The ZJL-1 fracturing fluid was sealed in a container and placed in a constant temperature water bath at 90 °C, and kerosene was added to stir and break the gel. A ZNN-D6B six-speed rotational viscometer  was used to measure the viscosity of the fracturing fluid before and after breaking the gel and record the break time. A DT-102A automatic interfacial tension meter  was used to measure the surface interfacial tension of the breaking fluid, and the breaking performance of the ZJL-1 fracturing fluid was investigated .1 is the differential pressure when water flows through the pipeline, and ∆P2 is the differential pressure when the ZJL-1 fracturing fluid with added thickener flows through the long pipe. The method employed a pipe with an internal diameter of 10 mm and a test pipeline with a length of 2 m, and the drag reduction efficiency values were tested with different concentrations of thickener added at different flow rates at a temperature of 25 °C and a pressure of 15 Mpa [Frictional drag property tests were carried out with a large-sized high-temperature and high-pressure fluid circulation device, as shown in f 15 Mpa ,35.3, and the rotational speed of the stirrer was 1000 rpm [A static sand suspension visible reactor was used. ZJL-1 was fully dissolved in water with stirring. Settling of the proppant in the ZJL-1 fracturing fluid with different thickener concentrations was observed using the indoor static observation method , and the1000 rpm .1 and K2 are the permeability values before and after core damage, respectively, used to evaluate the extent of damage to the formation caused by the fracturing fluid [Evaluations of core damage were carried out with a fracturing fluid-caused damage test device , as showng fluid .−1, a C=C stretching vibration peak at 1666.03 cm−1, and secondary vibrational peaks at 1614.03 cm−1 and 1552.10 cm−1. The characteristic amide II band and amide I absorption bands appeared at 1373.05 cm−1 and 1398 cm−1 for the branched -CH, C-H in-plane bending vibrations, and the peak at 629.09 cm−1 was a characteristic absorption peak for a sulfonic acid group. In the infrared spectrum of ZJL-1, there were two primary amide peaks at 3199.29 cm−1 and 3339.03 cm−1, a peak at 3035.78 cm−1 for the stretching vibration of the vinyl =C-H, a peak at 1670.59 cm−1 for C=C stretching vibration, a sulfonic acid symmetrical stretching vibration peak at 1438.84 cm−1, a secondary amide II stretching vibrational peak at 1261.04 cm−1, a tertiary ammonium vibrational peak at 1195.37 cm−1, and the peak at 618.88 cm−1 1 is characteristic of the sulfonic acid group; the absorption peaks of the AMPS monomer functional groups appeared in the infrared spectrum of ZJL-1, which confirmed that ZJL-1 was chemically modified by AMPS.After ZJL-1 was dissolved in water, the carboxyl, sulfonic, and quaternary ammonium salt groups in and between the molecular chains of the copolymer underwent self-crosslinking through ionic bonds, the quaternary ammonium groups in the molecular chains and the sulfonic groups of the anionic emulsifier dissolved in water to form secondary self-crosslinks through ionic bonding, and the long-chain hydrophobic groups in the molecular chains and the nonionic groups of the nonionic emulsifier underwent tertiary self-crosslinking through van der Waals forces and hydrophobic association. In addition, there were strongly polar hydrogen bonds, including H-O bonds and H-N bonds, in the molecular chains, which enhanced the self-crosslinking ,41,42. T3H in ZJL-1, which is the largest weight loss observed. As the concentration of ZJL-1 was increased, the total amount of mass lost decreased, with the smallest total mass loss for 1.5 wt% ZJL-1, 7.71%, occurring between 25 and 200 °C. A thermal weight loss of less than 10% for the polymer indicates that the substance is able to withstand the corresponding temperature. The apparent viscosity of ZJL-1 is closely related to the density of the spatial mesh structure, the bond length between molecules, and the entanglement strength of the spatial mesh structure. With the increase of temperature, the intermolecular chemical bonds are gradually broken and this formed reticular structure is gradually destroyed due to the increase of molecular activity. Therefore, the results of the thermogravimetric analyses indicated that ZJL-1 can withstand temperatures up to 200 °C. On the one hand, the existence of the network structure delays the thermal degradation of ZJL-1, and on the other hand, the sulfonic acid group increases the temperature resistance of ZJL-1. The better temperature resistance of ZJL-1 is conducive to maintaining better rheological properties in high temperature environments [As shown in ronments .G′ reflects the elastic behavior of a polymer caused by its reticular structure. Decreases in G′ indicate that the elastic behavior of the polymer is weakening. When G′ > G″, it indicates that the polymer exhibits relatively strong elastic behavior. The hydrophobic groups introduced into the molecular structure produce hydrophobic associations, which causes the molecular chains to intertwine, the spatial structure becomes more complex, and the elasticity of the solution is enhanced . With inAs shown in The gel breaking time was 30 min, and the gel breaker exhibited a milky white color without suspended solid matter. The supernatant of the gel breaker was taken for testing. The viscosity of the gel breaker was determined at 90 °C as 1.7 mPa·s, the surface tension of the gel breaker was 22.7 mN/m, and the interfacial tension between the gel breaker and dehydrated kerosene was 1.06 mN/m. After gel breaking, the fracturing fluid exhibited low viscosity, surface tension, and interfacial tension, which favored rapid and thorough flowback after construction .3/min, the drag reduction efficiency of the fracturing fluid system reached 72%. The trends for changes in drag reduction efficiency shown in the figure indicate that the ZJL-1 fracturing fluid system meets the requirement for low frictional drag in field construction and is an excellent drag-reduction and thickening fracturing fluid system. Research has shown that a simple polymer long-chain structure is easy to break under high shear and cannot achieve a long-lasting lowering effect, so thickening agent has side groups introduced. The molecular chain forms a spiral structure that can be fully extended in water and has a certain elasticity, and its solutions form elastic bottom layers in the pipeline to lower the resistance.As shown in The falling times and settling speeds were determined at 90 °C and 15 MPa with a proppant settling test device for the single-particle proppant, the proppant with a sand ratio of 20% in the ZJL-1 fracturing fluid, and conventional guar fracturing fluid. Calculation performed with the experimental results yielded permeabilities and core damage ratios, as shown in 2 fracturing technology was used with this well. ZJL-1, hydrophobic long-chain ester-based thickener, proppant, gel breaker, etc., were added to clean water to form the water-based system with high viscosity and high sand ratio (the proportion of water-based fracturing fluid was 30%), which was blended with liquid CO2 (proportion of 70%). The designed total construction displacement was 6.1–7.3 m3/min, the liquid CO2 injection displacement was 4.3–5.2 m3/min, the ZJL-4 fracturing fluid system injection displacement was 1.8–2.1 m3/min, and the selected proppant was 40–70 mesh ceramsite.The fracturing target layer in Well XX-Xie 1 has a burial depth of 3446 m, a porosity range of 7.19–21.84%, and a permeability range of 0.03–50 mD with an average of 2.4 mD. It is dominated by fine sandstone, has a clay mineral content of 14.2%, is characterized by low porosity and low permeability overall, and is a reservoir with moderate to strong water sensitivity. The flowback rate after conventional fracturing is 50–60%, so the postfracturing effect is not satisfactory. To reduce the extent of reservoir damage, quasi-dry CO2 fracturing construction was carried out smoothly at S Oilfield Well XX-Xie 1. The process flow was divided into two parts: one part involved pressurizing liquid CO2 and pumping it to the wellhead, and the other part involved blending clean water, ZJL-1, a hydrophobic long-chain ester-based thickener, proppant and gel breaker with a fracturing blender truck to form the proppant mortar with high viscoelasticity, which was pumped to the wellhead. These two pumped liquids converged at the tee of the wellhead, formed a mixed liquid phase with sufficient viscosity and structure under turbulent conditions to carry out fracturing construction with integrated drag reduction and sand carrying. In this construction, the amount of liquid CO2 fracturing fluid consumed was 350 m3 in total, the amount of ZJL-1 fracturing fluid consumed was 150 m3, the amount of sand added was 40 m3, the construction pressure was 25–60 MPa, the fracturing pressure was 47.5 Mpa, the liquid CO2 fracturing fluid injection displacement was 4.5 m3/min, the ZJL-1 fracturing fluid injection displacement was 2.0 m3/min, the shut-in pressure was 29 Mpa, the mean sand ratio was 18%, and the maximum sand ratio was 25%. The actual construction parameters and the design parameters were relatively highly matched, and the fracturing construction parameters and construction curves are shown in 3, the daily oil production was 10.2 m3, the daily gas production was 2.5 × 104 m3, the flowback period was 10 days, and the flowback rate was 120%. The field application showed that the production rate of this well was increased with this fracturing mode as compared with the use of the previous fracturing mode. The daily oil production per single well doubled, and the daily gas production increased by a factor of 6.5 with the evident oil production increase.In July 2020, quasi-dry CO2 fracturing, which formed a strong spatial network structure involving ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic associations, so self-crosslinking was enhanced without the need for additional crosslinkers.In this study, we developed a self-crosslinking emulsion-type ZJL-1 fracturing fluid for quasi-dry CO−1, the viscosity of the ZJL-1 fracturing fluid was still higher than 70 mPa s, indicating good shear resistance. The drag reduction rate reached 70%, indicating good drag reduction performance. Compared with the conventional guar fracturing fluid, the ZJL-1 fracturing fluid showed better sand-carrying performance and caused less damage to the core permeability. Field tests showed that upon optimizing the mixing ratio for the liquid CO2 fracturing fluid and ZJL-1 fracturing fluid, the increases in gas and oil production were obvious. This was beneficial for environmental protection and reservoir protection and met the requirements of integrated application for resistance reduction and sand carrying, and it has broad application prospects.The heat loss rate of the ZJL-1 fracturing fluid was less than 10% at 200 °C, and it showed good thermal stability and obvious viscoelasticity. After shearing for 2 hours at 160 °C with a shear rate of 170 s