Hydraulic fracturing, as an oil-water well stimulation and injection technology, is particularly important in the production and stimulation of low-permeability oil and gas fields, and the performance of the fracturing fluid directly affects the success of the fracturing operation. Compared with traditional water-based fracturing fluids, clean fracturing fluids have the advantages of strong sand-carrying ability and easy gel breaking with no residue. Aiming at the problem of poor temperature resistance and shear resistance of the clean fracturing fluid, based on previous research, this paper selects a high-temperature-resistant clean fracturing fluid system and evaluates the performance of the system. The research results show that the system has better rheological properties, better sand-carrying performance, shorter gel-breaking time, and less damage to the reservoir. Fracturing technology is famous for the shale gas revolution in the United States, but as early as 1947, American Stanolind oil and gas company conducted the first hydraulic fracturing experiment in Hugoton oilfield in southwest Kansas. On March 17, 1949, Halliburton carried out the first commercial fracturing construction in Verma, Oklahoma, and Archer County, Texas. Since then, the technology has been widely adopted by exploration and production companies around the world to improve or extend the production capacity of oil wells . At presHydraulic fracturing is an important means of developing low-pressure and permeable oil reservoirs , 8. UsinThe fracturing fluid is the working fluid of fracturing construction. It is a fluid with a certain viscosity, which plays the role of transferring energy, forming and extending fractures, and carrying proppant. At present, there are many kinds of fracturing fluids used at home and abroad, mainly oil-based fracturing fluid, water-based fracturing fluid, acid-based fracturing fluid, emulsion fracturing fluid, and foam fracturing fluid , 12. AmoThe water-based fracturing fluid system usually contains water-insoluble substances such as polymers . After t−1, start the test at room temperature of 25°C, control the heating rate to be 3°C/min ± 0.2°C/min, continuously heat up to 150°C, and investigate the viscosity-temperature relationship of the clean fracturing fluid system.Fracturing fluid stability includes thermal stability and shear stability. That is, the viscosity of the fracturing fluid will not decrease significantly under temperature rise and mechanical shear, which plays a key role in the success or failure of construction. The temperature resistance performance of the high-temperature and low-damage clean fracturing fluid system is tested using the HAAKE Mars III rotational rheometer manufactured by the Thermo Corporation of the United States. We set the shear rate of the rheometer to 170 s−1, the temperature is set to 120°C, and the viscosity changes are observed after 60 min of constant temperature shearing.In the clean fracturing fluid system, the viscoelastic surfactant molecules are entangled to form rod-like micelles, which in turn form a spatial network structure. Due to the reversibility of the micelle formation, its apparent viscosity does not change with time, even under high shear; once the shear rate decreases, the micelles can still aggregate and rewind again, thereby restoring the viscosity of the system, which is different from traditional polymer fracturing fluids. After the traditional plant arc glue fracturing fluid is sheared, the molecular chain is permanently disconnected, and the viscosity decreases rapidly and cannot be recovered. The shear resistance of the clean fracturing fluid system with high-temperature resistance and low damage at high temperature is tested using the HAAKE Mars III rotational rheometer manufactured by the Thermo Corporation of the United States. The shear rate is set to 170 sp=0.1 mm) to measure the viscosity of the gel-breaking fluid at each time until the clean fracturing fluid system completely breaks the gel (<5 MPa·s).We should try to reduce the content of water-insoluble substances in the fracturing fluid and the gel-breaking ability before flowback, reduce its blocking of rock pores and sand-filling fractures, and increase the oil and gas conductivity. In the experiment, kerosene is used as the gel breaker for the clean fracturing fluid, and the effect of kerosene addition on the apparent viscosity and gel-breaking time of the clean fracturing fluid is investigated. At 25°C, kerosene is added according to the mass ratio of 3%, 7%, and 10% of the clean fracturing fluid, and the gel breaking of the clean fracturing fluid system is investigated under a constant rotational speed. We use a capillary viscometer . After specifying the temperature, we measure the liquid surface height (1) Preliminary Preparation of Flow Medium and Core.According to the SY/T5107-2005 test standard for evaluating the damage rate of the water-based fracturing fluid to core matrix permeability, kerosene is selected as the flow medium. The kerosene needs to be refined before the experiment. The process is as follows: We take a certain amount of kerosene, add silicon powder into it, stir evenly, and soak for a period to remove impurities and free water contained in the kerosene through the adsorption of silicon powder. Then, the kerosene is filtered by the filter funnel, and the kerosene filtrate is collected and degassed by a vacuum pump for 1 hour to complete the kerosene refining. The core is made of artificial quartz sand epoxy resin-cemented core. The core diameter is 2.503–2.504 cm, the core length is 4.09 cm, and the core is saturated with refined kerosene for 1 day.(2) Penetration Damage Determination Steps  Step 1: Pour the refined kerosene and the clean fracturing fluid after gel breaking and filtration into the high-pressure container and put the saturated core into a 25 × 80 core holder. Before the experiment starts, set the parameters, including the confining pressure value of 10 MPa, constant flow value of 2.5 ml/min, and viscosity and density values of kerosene.  Step 2: Turn on the pump to pressurize so that the refined kerosene enters the core from the opposite end of the core holder along the pipeline to implement displacement. When there is a flowing medium, at the outlet, observe the flow value. When it is close to the set flow rate of 2.5 ml/min, start to record the permeability value, that is, the permeability before damage k1. The recording method is to record once every 3 minutes until the difference between the two adjacent values does not exceed 10%.  Step 3: Stop the pump after obtaining a stable predamage permeability k1. Close the reverse line and connect the forward line and set the viscosity and density of the gel breaker filtrate on the instrument. Turn on the pump again so that the filtrate of the gel-breaking liquid enters the core from the inlet of the positive end of the core holder. When there is broken gel filtrate at the outlet, observe the flow value. When it is close to the set flow rate of 2.5 ml/min, start recording data and time. The method of recording data is the same as that of step 2, and the stabilization time during the recording period is not less than 30 minutes.  Step 4: Stop the pump, close the forward line, and connect the reverse line. After setting the viscosity and density of refined kerosene on the instrument, follow the same steps as in step 2. The permeability after the flooding can be recorded as k2. The entire injury process is always performed at room temperature.−1, the heating rate is controlled to be 3°C/min°± 0.2°C/min, and the temperature is continuously increased to 150°C. The test results are shown in On the basis that the concentration of the main agent erucamide epoxy is 4%, the salicylate (SAL) in sodium salicylate is used as the counterion to investigate the different molar ratios of sodium salicylate to erucamide epoxy  on the viscosity of the clean fracturing fluid. Each fracturing fluid is tested on a Mars III rotational rheometer, the shear rate is set to 170 sIt can be seen from Under the condition that the molar ratio of counterion to main agent is 1 : 1, the influence of different concentrations of main agent erucamide epoxy  on the viscosity of the clean fracturing fluid was investigated. The test results are shown in It can be seen from The viscosity-temperature relationship of the high-temperature clean fracturing fluid was measured using a HAAKE Mars III rotational rheometer, and the experimental results are shown in It can be seen from It can be seen from t when the small steel ball reaches the bottom of the measuring cylinder. For comparison, the same method was used to measure the suspending capacity of hydroxypropyl orphan fracturing fluids of similar viscosity. The experimental results are shown in In order to evaluate the suspending ability of the high-temperature-resistant clean fracturing fluid system and the hydroxypropyl arc glue fracturing fluid to proppant, a static sand suspension performance experiment is carried out. The experimental method is that we put the high-temperature clean fracturing fluid system into a 100 ml measuring cylinder and place it in a constant temperature water bath , and wait until the fracturing fluid reaches the specified temperature. We lightly place the small steel ball on the surface of the liquid, press the stopwatch, and record the time The breaking of the conventional vegetable gum fracturing fluid or the synthetic polymer fracturing fluid is chemical destruction; that is, the polymer chain is broken by the oxidation of the breaker so that the viscosity of the solution is rapidly reduced. The clean fracturing fluid is different; it mainly breaks the gel through contact with cinnamon or the formation of water. When the clean fracturing fluid is in contact with cinnamon, the organic matter of cinnamon enters the worm-like micelle structure, and the micelle swells, which can be decomposed into many single spherical micelles.In the experiment, kerosene was used as the gel breaker of the clean fracturing fluid, and the gel breaking of the clean fracturing fluid after adding kerosene with a mass fraction of 3%, 7%, and 10% was investigated under constant shear rate at room temperature. The gel-breaking results of the high-temperature-resistant clean fracturing fluid are shown in It can be seen from We put the high-temperature-resistant clean fracturing fluid into the centrifuge tube, take it out for drying after high-speed centrifugation, and calculate the residue content. The results show that the gel-breaking fluid of the high-temperature-resistant clean fracturing fluid has no residue, indicating that the fracturing fluid system after gel breaking has no damage to the formation. The main component of the clean fracturing fluid is surfactant, and its molecular diameter is only 1/5000 of that of guar gum. These small organic molecules are easily soluble in water, so no residue is produced.After the fracturing fluid is filtered off, the filtrate enters the formation along the fracture wall. The fracturing fluid filtrate causes the expansion of the formation of water-sensitive minerals, which reduces the porosity of the reservoir rock matrix and reduces the reservoir permeability. Therefore, the measured value of the permeability damage rate of the fracturing fluid filtrate to the reservoir matrix is also an important indicator for evaluating the quality of the fracturing fluid.As can be seen from The shear resistance of the high-temperature clean fracturing fluid system is good. In the process of the constant temperature of 120°C and shearing for 60 min, the viscosity is stable between 70 m and 90 MPa·s. Under high temperature and constant shear rate, the viscosity of the system will not decrease, indicating that the high-temperature-resistant fracturing fluid has good shear resistance performance. The high-temperature-resistant clean fracturing fluid system can automatically break the gel after encountering kerosene. With the increase in kerosene dosage, the gel-breaking time of the clean fracturing fluid is shortened. The glue-breaking fluid of the high-temperature-resistant clean fracturing fluid system has no residue.The sand-carrying performance of the high-temperature clean fracturing fluid system is better than that of the hydroxypropyl guar fracturing fluid. It can be seen from static experiments that at the same temperature, the settling rate of the small steel balls in the system is much lower than that in the hydroxypropyl guar fracturing fluid. As the temperature increases, the settling rate of the small steel balls in the fracturing fluid increases. The damage rate of the fracturing fluid system to the core is 16.35%, and the damage to the reservoir is far less than that of the hydroxypropyl guar gum fracturing fluid, which has less damage to the formation and has good reservoir protection.


Various clinical studies from developed countries support the use of Casirivimab–Imdevimab Monoclonal Antibody Therapy (CIMAT) to treat outpatients with mild to moderate coronavirus disease-19 (COVID-19). However, the real-world clinical data from India, owing to the portrayed benefits, are limited. Moreover, the choice of CIMAT depends upon the willingness of the patient or his bystanders for this therapy. The study aimed to assess the impact of Casirivimab–Imdevimab Monoclonal Antibody Therapy (CIMAT) for mild to moderate COVID-19 in India.A single-center, retrospective comparative study was conducted to evaluate the impact of the CIMAT in symptomatic adult COVID-19 patients admitted to a secondary care hospital in South India, categorized as mild or moderate, and who are at high risk for progression to severe COVID-19. The data for the study were retrieved from the Electronic Health Records (EHR) of the hospital from April 2021 to March 2022, a period of 12 months. The primary outcome of the study was the length of hospitalization, and the secondary outcomes were mechanical ventilation post-infusion, readmissions after discharge, and mortality rate. Outcomes in the CIMAT cohort were compared to those of a control group of patients admitted with a diagnosis of SARS-CoV-2 during the same period who were qualified for CIMAT but were unwilling to administer it.This study included 474 patients, 48 in the CIMAT cohort and 426 in the control group. In the CIMAT cohort, the median age was 65 years (IQR: 50–73); 56.2% were ≥65 years old; 54.3% were males. Patients on CIMAT compared to the control group had a lower length of hospitalization(median: 4 [IQR 1–8]), lesser requisite for mechanical ventilation  and less frequent readmissions within 10 days post-discharge . Mortality was more among the control group, i.e., 22 (5.1%), and only one patient in the CIMAT cohort died during hospitalization (p = 0.23). The majority of the patients in the CIMAT cohort (41[89.1%]) were symptom-free within seven days of antibody cocktail administration.Patients treated with CIMAT for COVID-19 were more clinically benefitted than those treated without the antibody cocktail.All Authors: No reported disclosures.


Surgical resection is widely used to treat small tumours located in the iris and the ciliary body, due to the accessibility of these sites. By contrast, surgical removal of choroidal tumours is substantially more challenging, which is why this procedure is performed only at specialised centres. In the present article, we review the literature on surgical resection of choroidal tumours, which can be performed as endoresection (ab interno) or transscleral resection (ab externo). An important aim of this review is to describe and compare the two approaches in terms of visual outcomes, survival rates, and complications. Both approaches are indicated for the removal of large tumours (thickness > 8 mm) with small base diameters. Surgical resection of the tumour allows clinicians to obtain valuable histopathologic and cytogenetic data from the specimen and eliminates the risks associated with radiotherapy. However, both of these surgical approaches are technically challenging procedures involving the risk of severe early and late postoperative complications. Multiple options are available for the treatment of uveal melanoma, including several different forms of radiotherapy, transpupillary thermotherapy, enucleation, and surgical excision of the tumour. Consequently, the optimal treatment approach must be individualised according to tumour-related factors  and other factors, such as the patient’s general health status, age, status of the contralateral eye, and the patient’s preferences ,2.All of these treatments have advantages and disadvantages, which is why the optimal therapeutic approach remains controversial. In most centres, radiation therapy (brachytherapy and teletherapy) is the first line of treatment (and therefore the most common treatment) in patients with uveal melanoma. Nevertheless, in selected patients, surgical resection may be the treatment of choice, in order to control the intraocular tumour while preserving the eyeball and avoiding the risks of radiation and disfigurement from enucleation. Surgical resection is particularly appealing for the treatment of large uveal melanomas, due to the long-term side effects associated with brachytherapy and teletherapy ,3.In cases of uveal melanoma involving iris and ciliary body tumours, local resection is a common treatment. However, surgical resection has been little used in choroidal melanomas, due to the technical difficulty of the procedure and the risk of extrascleral or systemic dissemination. Currently, two distinct surgical techniques are performed. The first is transscleral resection (ab externo technique), also known as “exoresection” and sometimes called iridectomy, cyclectomy, or choroidectomy (and any combination of these), depending on the tumour localisation . The second surgical approach is known as endoresection (ab interno technique) . Both teSurgical resection of the tumour may be indicated as a primary or secondary treatment. In certain cases, such as tumour recurrence or toxic tumour syndrome, resection can be a valuable secondary treatment ,5.The primary aim of the present article is to review the available scientific literature to describe the main outcomes associated with primary surgical resection of choroidal melanoma (choroidectomy or cyclochoroidectomy). A second aim is to discuss the techniques and indications for the surgical treatment of choroidal melanoma.We searched the PubMed database of the U.S. Nacional Library of Medicine and the Cochrane Central Register of Controlled Trials of the Cochrane Library using the following keywords: uveal melanoma resection; exoresection; lamellar uveal melanoma resection; scleral resection; transscleral resection; and choroidectomy. The initial search yielded 307 articles.Next, we applied the following criteria to further refine the selection process for these studies: (1) English language only; (2) reports describing the surgical resection of tumours located in the choroid or the ciliary body and choroid; and (3) reports describing endoresection or exoresection techniques as primary treatments, with or without adjunctive radiotherapy.After applying these additional criteria, a total of 25 studies remained. Of these 25 articles, 12 involved studies describing exoresection (six retrospective studies and six comparative studies) and 13 were studies describing endoresection (11 retrospective studies and two comparative studies). Because our study involved a review of previously published peer-reviewed literature, no institutional approval was required.The results of the selected studies are summarised in Transscleral resection is a highly demanding and controversial procedure. It is usually performed under severe controlled systemic hypotension, an approach that entails major risks for the patient. Consequently, it is not surprising that this technique is only performed in a few referral centres. Local resection of uveal melanoma was first described in 1911 in a 25-year-old female patient with a ciliary body melanoma . In the Innovative approaches to resect larger and more posterior tumours began to emerge in the 1970s, thanks to improvements in microsurgical techniques and instrumentation. Foulds described a technique consisting of scleral lamellar dissection at the base of the tumour, removal of the deep scleral lamella attached to the tumour, and finally microsurgical excision of the tumour followed by closure of the defect through the external scleral lamella . This tePeyman et al. described a procedure ,38 that In general, transscleral resection is suitable in patients with a tumour where a high rate of complications is expected after radiotherapy, especially conditioned by the height of the tumour. Therefore, the technique is indicated in patients with the following characteristics: good general condition; potential visual possibilities; tumour height from 8 to 10 mm; a relatively small base diameter (<15–16 mm); and anterior localization . This technique can also be used as salvage therapy after brachytherapy for eyes with an active tumour or severe exudative retinal detachment, or neovascular glaucoma (toxic tumour syndrome). However, this indication  is not within the scope of the present article.Contraindications include any of the following: any systemic condition that does not allow for systemic hypotension or prolonged general anesthesia; a basal diameter >16 mm; and optic nerve involvement.The most common approach to local resection in choroidal or choroidal-ciliary body tumours is PLSU. This technique consists of making a partial thickness circular, rectangular, or polyhedral scleral flap in the area overlying the tumour, after section of the extraocular muscles (if necessary). Next, a scleral incision is made through the deep scleral layers surrounding the melanoma to expose the underlying normal uveal tissue; then, another incision is made to expose the normal retinal tissue. Next, the tumour is removed by en bloc excision along with the deep lamellar scleral flap. At this point, gentle manipulations should be performed in an attempt to remove the tumour together with the overlying inner scleral layer without disturbing the underlying retina or vitreous. Finally, the defect is closed by returning the outer scleral layer to its normal anatomic location and fixing it with multiple interrupted sutures. In most cases, to complete the procedure, adjuvant brachytherapy is performed, which may or may not be associated with complete vitrectomy, to prevent retinal detachment in the case of retinal breaks, as well as lensectomy or phacoemulsification of the lens. During the procedure, systolic blood pressure should be maintained as low as possible ≈40 mmHg), which is why patients at high cardiovascular risk are not considered suitable for this surgical intervention. Nonetheless, it is worth noting that choroidal tumour resections have been successfully performed without systemic hypotension in several studies, with good functional results 0 mmHg, w,37,40,41Numerous studies have evaluated the risk of metastasis in patients undergoing scleral resection. Unfortunately, most of those studies were small and/or had a short follow-up. Probably the largest (n = 332) and most important study of transscleral resection in this clinical setting was the study by Damato et al . Those aIn other studies, mortality rates at 5 and 10 years ranged from 5% to 8.6%, with rates of metastasis from 28% to 44%, respectively  7,8,9,1,37. DataLocal recurrence may be attributed to incomplete tumour resection or seeding caused by eyeball manipulation during surgery, especially the dissemination of tumour cells, which is promoted by the dispersion of subretinal fluid in the operative field. In most cases, the recurrence develops at the margin, but in rare cases it may be noncontiguous with the primary tumour, thus requiring more aggressive treatment . The preLocal recurrence rates in choroidal tumours range from 8.3% to 32.6%  15,16] ,16 15,16In another study by Damato et al. , 8-year Bechrakis et al.  reportedCaminal et al.  reportedEnucleation rates range from 11.1% to 32.3%, depending on the study . 7,15] ,15 7,15]In the studies by Puusaari et al.  and CamiThis type of surgery is associated with the risk of many different complications, both early and late. The most common early complications are vitreous hemorrhage, hyphema, subretinal haemorrhage, retinal detachment, corneal edema, and glaucoma. Late complications include cataract formation, subretinal fibrosis, posterior synechiae, retinal detachment with or without vitreoretinal proliferation, cystoid macular edema, glaucoma, scleral thinning and, very rarely, iris neovascularization. Overall, the most common complications are retinal haemorrhage, retinal detachment, preretinal or subretinal fibrosis, and cataract  7,9,37,,37,45.In a study of 156 patients, Damato et al.  found thFoulds et al.  comparedIn another study by Damato et al. , the mulDamato et al.  reportedHead-to-head trials comparing exoresection to other standard treatments for uveal melanoma are scarce. One retrospective study compared transscleral resection to enucleation  and fiveIn the study by Foulds et al.  the 5-yeAugsburger et al.  comparedPuusaari et al. , who comCaminal et al.  comparedTransscleral resection was developed due to the need to achieve better visual outcomes than those provided by brachytherapy, without compromising local control or survival. Developments in vitreoretinal surgical techniques have made it possible to avoid hypotension, which is potentially dangerous for the patient; in addition, they can help to prevent the two main complications  associated with this surgical approach. Moreover, the functional outcomes obtained with this surgical technique have substantially improved, as have the number of indications for this surgery, thus increasing the number of eligible patients. In conclusion, this surgical procedure has a high technical complexity. Candidates for this procedure include patients in good general condition with the potential to maintain vision with large tumours (8 to 10 mm in height), with a relatively small basal diameter (<15–16 mm), and an anterior location . This procedure is particularly valuable in patients with the aforementioned characteristics, due to the high complication rate associated with brachytherapy, including cataracts, neovascular glaucoma, and radiation retinopathy. Ideally, this intervention should only be performed by highly experienced surgeons in patients who are highly motivated to preserve their eyes and their vision.The endoresection approach to the treatment of uveal melanoma is controversial, due to the technical complexity of the procedure and concerns about the risk of intraocular, extraocular, and, most importantly, systemic spread. During endoresection, the tumour is fragmented into small pieces, which goes against the basic principle of cancer treatment of maintaining tumour integrity. Consequently, this technique is performed in only a few specialised referral centres.Peyman et al. were the first to describe this approach, which involves tumour resection through an internal approach and vitrectomy ,48. ThosIn general, this technique is indicated in patients with tumours that are not amenable to brachytherapy or in whom vision is unlikely to be preserved. This technique is appropriate for patients in good general condition, with large tumours (8–10 mm in height) with a relatively small basal diameter (<15–16 mm), and a posterior location (juxtapapillary), ideally in nasal quadrants. This technique has also been considered as salvage therapy after brachytherapy for eyes that still show an active tumour, and in eyes with exudative retinal detachment or neovascular glaucoma (toxic tumour syndrome). However, these latter indications are beyond the scope of this article.Contraindications for this technique include any of the following: systemic conditions that prevent prolonged general anesthesia; basal diameter >16 mm; involvement of the ciliary body; and extensive involvement of the optic nerve.The most widely used endoresection technique is vitrectomy via conventional 20–23 G pars plana, lensectomy or phacoemulsification of the lens, induction of vitreous detachment, perilesional laser endophotocoagulation, or transretinal tumour resection through the vitrectome if the retina is invaded; otherwise, the technique is carried out by means of a peripheral retinectomy, in which the retina is folded away from the tumour, when the retina is not invaded.A vitreous cutter is used to remove the tumour piecemeal; to control intraoperative bleeding during the procedure, intraocular pressure is raised significantly. The tumour is resected up to the sclera and the retina is flattened by injecting liquid perfluorocarbon. Endolaser treatment is administered to the choroidal edges of the resection and to the exposed scleral surface; photocoagulation is used to reduce bleeding and to destroy any remnants. Thereafter, direct liquid perfluorocarbon–silicone is performed. Finally, an episcleral radioactive plaque is placed at the base of the tumour to treat any tumour remnants and cryotherapy is performed over the sclerotomies. A wide-field lens and scleral indentation allow for complete removal of the tumour by the vitrectomy probe of high equatorial melanomas up to the ciliary body  Figure .When this technique was first performed, an air–silicone exchange was used to flatten the retina after the tumour had been removed; however, this maneuver can cause a potentially lethal syndrome known as presumed air by vitrectomy embolisation (PAVE). Therefore, it is strongly advised to avoid this maneuver and instead perform a direct exchange of perfluorocarbon liquid with silicone ,53,54,55Although endoresection has been widely questioned because it does not follow the classic concept of en bloc tumour removal with safety margins, it is currently widely used because studies have demonstrated good long-term results with this approach. Initially, there were some concerns regarding the risk of incomplete resection, implantation of cancer cells in the vitreous, and dissemination of tumour cells into the bloodstream , as any of these could theoretically increase systemic disease or local recurrence. However, the available data have shown that these risks are lower than originally believed. For example, Suesskind et al.  found noRetrospective case series show cancer-specific mortality rates ranging from 0% to 20%, although follow-up outcomes are highly variable ,28,29,30Only two studies, both retrospective, have compared primary endoresection to primary brachytherapy for the treatment of melanoma. Neither of those studies found significant differences in OS or disease-specific survival ,30. CamiReported rates of long-term local recurrence and enucleation are quite variable, ranging from 0% to as high as 23% ,28. SussThe use of adjuvant brachytherapy is associated with a lower rate of local recurrence ,29. In fThe main surgical complications, from most to least common, are scleral bed haemorrhage, cataracts, ocular hypertension, retinal detachment, hemovitreous, macular traction, epiretinal membranes, and bullous keratopathy followed by retinopathy and optic nerve atrophy, both of which are associated with adjuvant radiotherapy. However, the incidence of these complications varies substantially depending on the series, with some authors reporting cataracts in up to 90% of cases, retinal detachment in 33%, ocular hypertension in 34%, and vitreous haemorrhage in 80%.  18,19,2,25,26,27Recently, a pulmonary embolism syndrome associated with endoresection (presumed air vitrectomy embolisation (PAVE)) associated with air exchange has been described ,53,54,55In terms of functional outcomes, most published series have found that 20% to 30% of patients achieve a final vision above 20/200 and a mean vision between 20/40 and 20/100 ,25,26,27Head-to-head trials comparing exoresection to other standard treatments for uveal melanoma are scant. In fact, there are only two studies, both retrospective, that have compared endoresection to brachytherapy ,30 Tabl Caminal Rice et al.  comparedIn conclusion, endoresection is a highly complex surgical procedure. Despite initial concerns about the risks of local or metastatic spread, the available data show that endoresection achieves survival rates that are comparable to those obtained with brachytherapy. Nonetheless, the technical complexity of this procedure limits its use to a small number of specialised centres, mainly in cases in which primary radiotherapy is considered unsuitable. Over the past two decades, major advances have been made in ocular microsurgery, especially with the introduction of wide-field systems and new vitreoretinal devices, which have helped to improve surgical outcomes in these patients, especially visual function.Endoresection may be indicated in patients who are in good general condition and who may be able to preserve their vision, with large tumours (>8 mm in height) with a relatively small basal diameter (<15–16 mm), a postequatorial location, and preferably in the nasal quadrants. In these cases, high complication rates are expected with brachytherapy, particularly retinopathy, radiation neuropathy, and neovascular glaucoma.It is now generally accepted that adjuvant radiation after tumour resection can reduce the incidence of both recurrences and metastases ,15,16,43Choroidal tumour resection is an alternative to brachytherapy in selected cases. The available data suggest that this approach may offer better visual results and eye sparing without compromising local tumour control and survival.Resection techniques for choroidal tumours are based on modern vitreoretinal surgical techniques, which allow for the treatment of more challenging cases.The main limitations to surgical resection of these tumours are the risk of severe complications, which may include retinal detachment, proliferative vitreoretinopathy, severe intraoperative haemorrhage, air embolism, and local tumour recurrence at the resection edge.Surgical resection of uveal melanomas should be limited to centres with substantial experience in the management of intraocular tumours.KEY POINTS:


Over the course of hemodialysis, fluid and protein are restituted from the tissue compartment to the circulation compartment through the endothelia. Our previous model analysis on fluid and protein transport during hemodialysis is expanded to account for changes occurring in the tissue. The measured initial and end plasma protein concentration  for six hemodialysis studies are analyzed by this expanded model. The computation results indicate that the total driving pressure to restitute fluid from the tissue to the circulation ranges from 5.4 to 20.3 mmHg. The analysis identifies that the increase in plasma colloidal osmotic pressure (COP) contributes 78 ± 6% of the total driving pressure, the decrease in microvascular blood pressure 32 ± 4%, the increase in the COP of interstitial fluid −6 ± 3%, and the decrease in interstitial fluid pressure −5 ± 2%. Let this ratio (Cp’ − Cp)/Cp’ be termed the PPC increment. The six HDs can be divided into three groups which are to have these PPC increments 25.7%, 14.5 ± 2.6(SD)% and 8.3%. It is calculated that their correspondent filtration coefficients are 0.43, 1.29 ± 0.28 and 5.93 mL/min/mmHg and the relative reductions in plasma volume (RRPV) −22.1%, −13.1 ± 6% and −9.4%. The large variations in PPC increments and RRPV show the filtration coefficient is a key factor to regulate the hemodialysis process. Over the course of hemodialysis (HD), ultrafiltrate is being extracted out of the circulation by the dialyzer while interstitial fluid restituted from the interstitial fluid compartment (IFC) via the semipermeable endothelia to the circulation compartment. These two fluid movements lead to a reduction in plasma volume of the circulation, an increase in plasma colloidal osmotic pressure (COP) and a decrease in microvascular blood pressure. In the meantime, the fluid restitution from IFC can lead to these three changes: an increase in the COP of the interstitial fluid, a decrease in interstitial fluid pressure, and a reduction in lymphatic return. These three changes are dependent on three tissue factors: (i) The ratio of tissue volume to the plasma volume, (ii) the elasticity of the extracellular matrix and (iii) the lymphatic retention fraction. The model analysis developed previously is expanded for us to examine how the three tissue factors alter the rate of fluid restitution and the relative reduction in plasma volume (RRPV) over the time course of HD .In our previous analysis, the movement of fluid and protein across the endothelia are governed by four circulation factors: (i) the filtration coefficient of the endothelia lining the surface of the microcirculation KSo, (ii) the reflection coefficient σ, (iii) the permeability fraction γ which specifies the protein concentration of restituted fluid as a fraction of plasma protein concentration (PPC), and (iv) the autonomous constant ζ which specifies how the microvascular blood pressure is adjusted by the autonomous system and the circulation itself as the patient is taking HD. If the patient has a larger KSo, the analysis reveals that more fluid would be restituted into the plasma compartment and lesser volume reduction would be imposed to the circulation. To recognize this beneficial effect, we term the filtration coefficient as a facilitator for the patient to have more fluid restituted from the tissue to the circulation. In this paper, we will make sensitivity analyses to examine what roles are played by the circulation and tissue factors in having more fluid restitution, lesser blood volume reduction and lesser chance to the development of intradialytic hypotension (IDH) for patients with chronic kidney disease (CKD) taking HD.The equations governing the HD process form a self-contained system. This is to say when the initial conditions are given and the values of modelling parameters are assigned, one can use the equations to calculate the time course of variables such as the plasma volume, the PPC, the microvascular pressure and the COP of interstitial fluid. In the case that the PPC at the end of HD is measured, we can use the analysis to find a value of KSo. In six sets of hemodialysis studies, the initial and end PPC , initial and end hematocrit  are reported ,3,4. TheThe interstitial fluid space of the entire body is simulated as one interstitial fluid compartment (IFC) with Vt(t) as its volume at time t. It is filled by an interstitial fluid having Ct(t) as its protein concentration at time t. The plasma space in the circulation is set as the plasma compartment which has Vp(t) as its volume. The plasma flowing within has Cp(t) as its PPC. , as the initial PPC and Cp’ as the end PPC. When clarification is called for, we use Cp(0) or Cp0 instead of Cp). These two compartments are joined by endothelia , it is shown that the arterial and venous blood pressure decrease linearly with the hemorrhaged volume. LaForte et al.  found thThe interstitial fluid is embedded within an elastic extracellular matrix. A decrease in interstitial fluid volume calls for a volume reduction of the matrix and a reduction in the pressure of interstitial fluid. We simulate the pressure-volume relation as:As the concentration Ct(t) and Cp(t) are viewed as uniformly distributed throughout their corresponding compartment, the total protein mass in the interstitial and plasma compartment at time t are calculated as Ct(t)Vt(t) and Cp(t)Vp(t), respectively.The Starling’s hypothesis on fluid movement is used to define the transvascular fluid movement developed over the course of HD. Before HD, we have the initial transvascular filtration Jf(0) as:The addition of the lymphatic return Qly(t) to the fluid flux above forms the following restitution fluid flux:This term Jr(t) is depicted as the big arrow in We define the volume of fluid restituted from IFC to the plasma compartment Vr(t) and the volume of lymphatic retention Vly(t) as:Under this definition of Vr(t), we have its initial value Vr(0) as 0. We also set the end value Vr(ΔT) as ΔVr. Similarly the end value Vly(ΔT) is set as ΔVly.As fluid is being restituted from IFC, the interstitial fluid pressure is reduced. This pressure reduction may lead to a reduction in lymphatic return . Thus QlIn the above derivations, the transvascular flux Jf(t) is divided into two terms: Jr(t) and Jf(0). This division allows us to express Equation (5) in terms of pressure differences such as Pt(t) − Pt0. Since this difference is the one being calculated by the analysis, the form of Equation (5) allows us to do calculations without the need to set values for Pmic(0), Pt(0) and πt(0).Let the protein concentration of the restituted fluid be Cr(t). The protein mass being restituted from IFC over the time period  is calculated as the term on the left hand side of the following equation. Then, it is expressed as the term on the right hand side:Protein is carried out of IFC by the fluid restitution at this rate Jr(t)Cr(t). The lymphatic return carries the protein out of IFC at the rate of Ct(t)Qly(t). Let the protein concentration of the filtration Jf0 be Cf(t). The corresponding protein flux entering the IFC is given as Jf0 Cf(t). The sum of the first two items and the subtraction of the third one form the net mass flux of protein leaving the IFC at time t.In conjunction to the setting of Equation (9), the following equation  is used On the Graphic Abstract of this paper, the arteriole, capillary and venule are used to represent the plasma compartment of As ΔVr and ΔVly are the volume of fluid added to the plasma compartment and ΔVe is the volume of ultrafiltrate extracted, we can express the change in the plasma volume from Vp(0) to Vp(ΔT) as:The terms in Equations (9) and (10) are used to determine the protein mass added to the plasma compartment through the restitution and lymphatic retention. The extraction takes no protein out of the circulation. On the conservation of protein mass of the plasma compartment, we haveEquations (11) and (12) can be combined to yield these two equations:Equation (13) is termed the protein equation. The three terms on its right hand side describe how ΔVe, ΔVr and ΔVly contribute to form the PPC increment ΔCp/Cp’. If the PPC, γ, Ct0/Cp’ and ΔVly/Vp are known for a HD, the protein equation can be used to calculate the normalized restitution volume ΔVr/Vp. Then, Equation (14) is used to calculate the RRPV ΔVp/Vp.On the IFC, its volume change can be written as:The protein mass in the tissue compartment at time t is increasing at this rate: d[Vt(t) Ct(t)]/dt. The protein mass is leaving the IFC as they are carried in or out by restitution, filtration flux Jf0 and lymphatic return. The mass conservation for IFC is expressed as:φ and lymphatic retention ratio ψ to define the roles played by the IFC on fluid and protein movement:In view of the presence of Vp(0), Vt(0) and ΔVly in Equations (12)–(16), we use the following tissue volume ratio The hemodialysis process is prescribed by these nine independent variables: Vp(t), Cp(t), Vt(t), Ct(t), Vr(t), Cr(t), Cf(t), Pmic(t) − Pmic0, and Pt(t) − Pt0, define the state of patient taking HD. They are governed by nine equations: Equations (1), (2), (5), (9), (10) and (13)–(16). The colloidal osmotic pressure πp(t) and πt(t) are regarded as dependent variables as they are calculated from Cp(t) and Ct(t) through the Landis-Pappenheimer Equation .φ, ψ and ξ are termed the tissue factors. Among the latter two groups, KSo, σ, γ and φ are recognized as passive factors while ζ, ξ and Ψ as active factors. The term “active” means their value can be changed by the autonomous, circulatory and lymphatic system.From the governing equations, we find that the fluid restitution and plasma volume reduction are governed by three groups of parameters. The first group is made up of these two HD factors ΔVe (or ΔVe/Vp) and ΔT. They define how the HD is done. The circulatory factors are KSo, σ, γ and ζ. These parameters The reference HD is set to have ΔT as 240 min and ΔVe as 2167 mL. As the initial conditions, we set Cp0 as 7.12 g/dL, Vp0 as 2785 mL and Ct0 as 2.16 g/dL. On the governing factors of the reference HD, the filtration coefficient KSo is set as 0.82 mL/(min mmHg), σ as 1, γ as 0.09, ζ as 36.7 mmHg, For HD that the initial and end PPC are reported, one can use Equations (13) and (14) to calculate ΔVr/Vp and ΔVp/Vp0. The calculations are made with γ set 0.09 and The HD experiments done by Minutolo et al.  to patieThe relative extraction in reference to the plasma volume, measured initial and final PPC, the calculated PPC increment, the initial and final arterial hematocrit Ha and Ha’, and the hematocrit increment are listed in Calculations of the time dependent variables of HD1-6, such as plasma volume reduction and PPC increase, are done with these parameter settings: σ as 1, γ as 0.09, ζ as 36.7 mmHg, For HD1 and HD2, 48% and 49% of the blood volume is extracted out by the dialyzer over a HD time of 240 min. On the other hand, 18.4% of the blood volume is extracted out of patients in the HD3 group over a period of 20 min. The HD of simulation S3 is set to extract 50% of the blood volume out of patients of HD3 over a period of 240 min. The HD characteristics, initial PPC and calculated final PPC of patients in S3 group are listed in the last row of p < 0.05), the plasma volumes of HD1, HD2 and HD3 groups are also different from each other  for the reference HD now take 92.5% and 75% as their respective values. In reference to the protein equation, we see that the sum of these increments ΔVe/Vp0, −0.925ΔVr/Vp0 and 0.75ΔVly/Vp forms the actual PPC increment generated by the three fluid movements. It is noted that a negative increment means that ΔCp is negative, i.e., the protein concentration is being reduced or diluted. The PPC increment (ΔCp/Cp’) defines how the PPC is being concentrated or diluted by the HD process. The protein equation (Equation (13)) identifies that the PPC increment is determined by these three fluid movements: extraction by the dialyzer ΔVe, restitution of fluid from the IFC ΔVr and lymphatic retention ΔVly. The values of ΔVe/Vp, −ΔVr/Vp and ΔVly/Vp are the PPC increments generated by the three movements when the protein concentration of the latter two fluids is set as zero. The reference HD has γ set as 0.09. These two quantities (1 − γCp/Cp’) and (1 − γThe effect of changing HD time (ΔT) on the PPC, the driving pressure Δπp − ΔPmic and RRPV is shown in Comparisons of the three curves reveal that the higher is KSo, the lower are the PPC increment and the driving pressure. The results shown in The following special feature is shown for patients with high KSo. When the HD is run at a HD period of 240 min, the restitution rate after the first 30 min becomes larger than the extraction rate. As a result, the patient experiences an plasma volume increase of 2.5% Vp0 at the end of HD. For patients taking the 60 min HD, the significant increase in Jr(t) as shown in The permeability fraction γ sets the PPC of restituted fluid Cr as γCp. As γ takes a larger value, the restituted fluid is to have a larger protein concentration. Its addition to the plasma compartment dilutes the PPC at a lesser degree so that the plasma COP is to stay at a larger value. This increase in plasma COP leads to a higher restitution flux. Thus γ can be considered as a facilitator to enhance the restitution flux. The increasing trend in RRPV for patients having larger γ is indicated by the three curves depicted in How the RRPV changes for patients with a larger autonomous constant ζ is shown in φ. The first setting is the HD with zero tissue factors (TF0). The other three settings are identified as ξ15φ5, ξ15φ2 and ξ30φ2. Here, ξ15 means that the value of ξ is set as 15 mmHg and φ2 means that φ is set as 2, i.e., the volume of IFC is set as 2 times of the plasma volume Vp. The conditional change from TFO to ξ15φ5, ξ15φ2 and ξ30φ2 means the patient is changed to one having a more rigid extracellular matrix and/or a smaller IFC volume. The results shown in The time courses on PPC increment, the driving pressure on the vascular side, that on the side of IFC, and RRPV are shown in To assess how the lymphatic retention ratio affects the HD process, we calculate the case that ψ is set as −5%, φ as 4 and ξ as 4 mmHg. The initial and end PPC remain to have the same value. It is calculated that KSo takes 0.95 mL/min/mmHg as its value, ΔVr/Vp 69.2%, ΔVp/Vp −12.2% and DP’ 8.7 mmHg. The correspondent values calculated for the condition TF0 are 0.88 mL/min/mmHg, 66.1%, −11.7% and 8.9 mmHg respective. The value comparison on ΔVp/Vp indicates that the lymphatic retention acts as an inhibitor to the development of more fluid restitution.In this “paired” analysis, we set the value of γ as 0, 0.2, 0.3 and 0.4 and then iterate the corresponding value of KSo that makes Cp’ the same as that of the reference HD. Five pairs , , ,  and  are derived. In If the PPC at 2 h is measured, then we can select the right pair of  such that the predicted PPC can match the initial, midway and end PPC. Previously, the initial and end PPC are used to determine a value for KSo as the value of γ is already set as 0.09.Suppose that the HD is done over a shorter period . Then, the PPC calculated from the analysis will rise in the way depicted in The time courses of restitution flux Jr(t), the driving pressure Δπp(t) − ΔPmic(t), and the protein flux Cr(t) Jr(t) are plotted in For the six HDs, the computed values of KSo, ΔVe/Vp, Δπp, ΔPmic, Δπt and ΔPt are listed in The relative restitution volume ΔVr/Vp, the driving pressure DP’ (=Δπp − ΔPmic + Δπt − ΔPt) restituting fluid to the plasma compartment, and the four individual pressures normalized by DP’ are given in From the values given in Let ΔVp1 be the blood volume change calculated by the van Beaumont Hematocrit Equation . The valThe time course of PPC increment, driving pressure and plasma volume for HD1, S2A and S3A are plotted in For HD1, S2B and S3B, the time courses of ΔCp(t)/Cp(t), Δπp(t) − ΔPmic(t) and Vp(t)/Vp0 are depicted in In our previous analysis we examine primarily on how fluid is restituted from the tissue to the plasma compartment and how the volume of plasma compartment and the PPC are altered by the fluid restitution and ultrafiltrate extraction. The computed results indicate that the larger is the filtration coefficient, the more is the volume of fluid being restituted and the lesser is the volume reduction of the plasma compartment. Accordingly we regard the filtration coefficient works as a facilitator that can lead to more fluid being restituted from the tissue to compensate for the blood volume reduction induced by hemodialysis. The current modelling analysis indicates that these governing parameters: the permeability fraction, the reflection coefficient and the autonomous constant can also be termed as a facilitator to the development of more fluid restitution. On the other hand, a smaller volume for IFC, a more rigid extracellular matrix that contains interstitial fluid and more lymphatic retention by the IFC will cause lesser fluid restitution from the IFC to the plasma compartment. Thus it is appropriate to term these three tissue factors as an inhibitor to the generation of more fluid restitution.The role of filtration coefficient in getting the PPC to be more concentrated and in preventing the development of hypovolemia is prominently displayed by the analytic results derived for HD1, S2A and S3A and that derived for HD1, S2 and S3. For the first group, the relative extraction drops from 75% to 68.5% and then to 65.5% while the patients have the same filtration coefficient. The correspondent PPC increment drops from 26% to 24% and 22% while RRPV from 22% to 21% and then to 18%. For the second group, the filtration coefficient drops from 0.43 to 0.88 and then 5.93 mL/min/mmHg while the relative extraction stays at 75%. The correspondent PPC increments drops from 26% to 17% and then to 7% while the RRPV from 22% to 12% and then 1%.For the first group, the relative extractions ΔVe/Vp are selected such that their relative extraction ΔVe/Vb is about 50%. The projections of different Cp(t) and Vp(t) for this group means that ΔVe/Vp is a more direct index to characterize the effect of extraction on the HD process. This is consistent with the recognition that seven of the eight governing equations do not explicitly contain these two parameters Ha and Vb. If HD1-3 were done at the same ΔVe/Vp and ΔT ,4, then By setting the hematocrit in the microcirculation as the arterial hematocrit and the splenic RBC release as zero, van Beaumont developed a hematocrit equation to show that this RRPV ΔVp1/Vb is equal to the hematocrit increment −ΔHa/Ha’. The protein equation derived from current analysis is used to calculate ΔVp/Vb from the measured PPC increments of HD1-6. The calculations are done under the setting: γ as 0.09 and the lymphatic retention ψ as zero. As shown in As reported by Fogh-Andersen et al. , the proφ set as 4 and ξ set as 4 mmHg.By using the isogravimetric technique and changing the plasma COP, Gamble et al.  identifyMinutolo et al.  reportedThe analysis identifies seven modelling parameters or factors that control or regulate the fluid restitution and plasma volume reduction occurring over the course of HD. By analyzing the PPC data obtained from patients having normal, anemic and more anemic hematocrit, we identify that their filtration coefficient takes these values: 0.43 to 0.88 and 5.93 mL/min/mmHg while the volume of fluid restituted from tissue is 71%, 83% and 60% of the extraction volume. More PPC measurements with HD done on various protocols are called for. Their data analysis will provide more information on how the modelling parameters regulate or govern the HD process.The data analysis of HD1 reveals that the plasma volume to the end of HD is reduced by 22%. This large reduction suggests that the patients in the HD1 group are likely to develop severe hypovolemia and subsequently IDH. Patients with CKD have large drop in arterial blood pressure during HD and a large blood pressure variability (BPV) is associated with high mortality . The ana


Malignant pleural mesothelioma is a cancer of the lung lining, normally caused by asbestos, that develops decades after exposure. Chemotherapy, and recently more targeted drugs, show some benefit although only a minority of patients respond and invariably the cancer eventually escapes control. Several key genetic changes in mesothelioma differ from patient to patient, which may influence how their cancer responds to treatments. We have developed a new preclinical model using fertilised hen’s eggs as an alternative to laboratory rodents. Mesothelioma cells are labelled to allow monitoring of tumour growth and/or regression using fluorescence and longitudinal bioluminescence imaging in addition to histology. All cell lines tested efficiently form tumour nodules within seven days, supported by a blood supply and stromal chick cells. The model is rapid, cost effective, scalable, and adaptable with multiple potential endpoints, to enable evaluation of drug targets against the range of common mesothelioma genetic backgrounds.BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess drug targets using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, potentially offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors  Malignant pleural mesothelioma (MPM) is an aggressive cancer of the lung lining, mainly caused by environmental exposure to asbestos. Despite strict asbestos regulation in most high-income countries, the global incidence of MPM has not yet peaked, and it is predicted to remain a significant cause of morbidity and mortality for decades . MPM is BAP1, P16 and NF2 ) 1:200 and pH9; and mouse anti-Ki-67  1:200 and pH9. Antibody binding was visualised with diaminobenzidine (DAB), samples were counterstained with haematoxylin to assist in distinguishing between human tumour and chick cell nuclei, and sections were mounted with DPX mountant . Images of the slides were acquired using digital slide scanners .Immediately after dissection tumour samples were placed in 1 mL 10% neutral buffered formalin  for 16 h and then transferred to 70% ethanol. Following automated tissue processing, samples were embedded in paraffin blocks and sectioned at 4 µm onto SuperFrost Plus slides. Immunohistochemical staining was performed on the fully automated Leica BOND RXDigitalised whole slide images of tumour nodules stained for Ki-67 were scored using open-source, digital pathology software QuPath version 0.2.0-m7 . Brieflyin vitro, total RNA was extracted as previously described [GAPDH , ACTB , MMP9  and VEGF . Relative transcript expression is reported for genes of interest normalised to the mean values for ACTB and GAPDH (2ΔCq).Tumours harvested from the CAM were rinsed in ice-cold PBS and transferred to RNAlater . Total RNA was extracted as previously described  using a escribed  using RNMicroscopy RGB images of tumour nodules on top of the CAM were cropped to an area of approximately 1000 µm around the tumour nodule. A region of interest excluding the tumour was selected for analysis. The IKOSA CAM Assay  was used to determine the mean vessel thickness, as well as the total vessel area, total vessel length, and number of branching points normalised to the area analysed.MRI was performed using a horizontal bore 9.4 T Bruker Biospec 94/20 USR system . Following 10 min incubation at 4 °C, the egg was placed in a custom-built cradle, and an actively decoupled 3 cm diameter surface coil was affixed on the eggshell above the site of the tumour. An 86 mm volume coil was used for signal transmission, while the surface coil was used for signal detection. Initially scout images were acquired using a three-plane gradient echo sequence to localise the tumour. High resolution 3D images were subsequently acquired using a 3D TurboRARE  pulse sequence with the following parameters: field of view 40 mm × 40 mm, matrix size 400 × 400 × 20, TR/TE = 1800/6.8 ms, effective TE: 80.74 ms, echo train length 8, slab thickness = 2 mm, averages 1, flip angle 90, scan time of approximately 8 min.p values less than 0.05 were considered significant.Statistical analyses were performed in GraphPad Prism version 9 for Mac . Distribution of data was assessed by Shapiro–Wilk test and data were analysed by parametric or non-parametric tests as appropriate. The number of independent samples, definition of error bars, and the statistical test employed are described in relevant figure legends. Zoanthus sp. green fluorescent protein with bright fluorescence and high expression in mammalian cells. Labelling efficiency was around 90% in each cell line as assessed by fluorescence microscopy  typically formed nodules above the CAM whilst MESO-8T  nodules were often found beneath the CAM . We staiInteraction of MPM cells with their microenvironment is crucial to progression of the human disease, where the cancer cells can instigate fibroblast infiltration . We therThe chick vasculature is also immunoreactive for αSMA, revealing tumour-adjacent blood vessels within the CAM . Internain ovo as a 3D CAM xenograft. The Cq values for ACTB and GAPDH housekeeping genes were unaffected by the switch from 2D culture to 3D CAM . This relationship appeared more robust in some cell lines than others , reinforcing the utility of BLI as a live measurement for CAM xenografts that reflects not only tumour size but also tumour cell content and proliferative capacity. Considering the higher sensitivity of the BLI assay it may have utility even in less proliferative nodules where Ki-67 is hard to score.Immunohistological staining for Ki-67 is widely used to estimate the proliferative index for FFPE tissue . CAM nodGiven the variability in both tumour weight and BLI signal for xenografts established from 2 million cells of any given cell line A,B, we iMPM remains an area of critical unmet clinical need, despite a growing understanding of the processes driving its development and spread. Capitalising on this is unfortunately slow and costly, with drug development often taking 10 years or more from concept to clinical application, and with an estimated cost of around a billion dollars in research costs for each drug entering practice. Research performed using cultured cell lines, or rodent models, has historically corresponded poorly to outcome in eventual clinical trials. Thus, a continuum of preclinical models is required to embody different aspects of a human disease to screen and validate drug responses. Here, we describe protocols and analysis for a novel 3Rs-compliant CAM model for MPM that is rapid, economical, scalable, and adaptable, and which covers the spectrum of MPM histopathological types and common genetic alterations. It provides a wide range of in vivo readouts of tumour biology and viability within days rather than months lending itself to development as a useful preclinical model.The CAM proved to be a conducive environment for MPM, as all 10 cell lines that we tested engrafted very effectively, particularly the epithelioid MPM where, on average, viable nodules formed in 80% of cases. As the CAM model is very economical compared to in vivo approaches in rodents, this facilitates use of a sufficient number and diversity of low passage cell lines to capture some of the heterogeneity of MPM and encompass the common genetic changes. For future evaluation of therapeutics, this could facilitate identification of relatively small subgroups that are likely to respond and may prevent discard of viable compounds. The diverse readouts available for CAM xenografts can be tuned depending on the experimental question, enabling specific hallmarks of cancer to be monitored, for example when assessing anti-proliferative, anti-invasive or anti-angiogenic therapeutic compounds.in vitro. For example, CAFs in MPM produce CTGF, which promotes MPM growth and correlates with survival outcomes, providing a potential therapeutic target [Xenografts are less able to recapitulate the histology of the original tumour when derived from cell lines established in 2D culture, compared to fresh patient derived samples, whatever the host organism. However, the CAM model enables MPM cell lines to adopt a 3D architecture, which is characteristic for each cell line and partly addresses the importance of the tumour microenvironment. We observed pronounced interaction of MPM tumour nodules with chick fibroblasts as well as the chick vasculature. Comparing MSTO-211H xenografts in SCID mice  with MSTc target ,54. The c target  and, altc target .Like immunodeficient mouse models, at early stages of embryonic development the chick lacks a fully functioning immune system which facilitates engraftment of human tumour cells on the CAM. The chick immune system develops during the period when tumour nodules grow, and by E18 fully immunocompetent chick lymphocytes can be detected . HoweverOne challenge in fully realising the potential of CAM models in assessing drug responses is selecting the best methodology to evaluate tumour growth or regression. Tumour dimensions may be estimated, or excised nodules weighed, however these methods have limitations. Using dual-labelled MPM cell lines allowed tumours to be monitored by both fluorescence and bioluminescence, and we found the latter invaluable in distinguishing viable tumour nodules that were fully engrafted and vascularised during the experimental timeline. Although the utility of BLI could potentially be limited by the transduction efficiency, our experience is that many cell types transduce with high efficiency, as was the case with all the MPM cell lines tested. The reliance of BLI on ATP makes it superior to fluorescence imaging as BLI only detects metabolically active tumour cells, whilst the reliance on transduced luciferase ensures that only tumour cells and not chick cells are quantified. The latter is particularly important for MPM CAM xenografts, where we saw substantial infiltration of fibroblast-like chick cells. Importantly, whilst the BLI signal showed moderate correlation with tumour weight, we found a high correlation of BLI with Ki-67 staining as an endpoint measure of viable tumour cells. BLI methods have been published for other CAM xenografted cancer cell types, for example urothelial carcinoma  and pancIn other studies, CAM xenografts have been successfully treated with drugs administered by topical application , intraveWhile the MPM-CAM xenograft model cannot fully replace rodent or other higher organismal model systems, it provides a complementary 3Rs compliant model to study tumour biology that we believe will in future allow more efficient screening of targets and help identification of subgroups more likely to benefit from therapy. Our studies suggest that certain MPM cell lines with highly responsive BLI signals may be most amenable for testing anti-proliferative therapies, including MESO-7T, MESO12-T and MSTO-211H. In contrast, for anti-invasive therapies histological analysis of MESO-8T nodules would be preferable, whilst many cell lines appear appropriate for evaluating anti-angiogenic therapies using IKOSA CAM analysis combined with immunohistochemistry. Furthermore, these initial MPM-CAM xenograft models for cultured MPM cell lines open the door to ongoing development that can incorporate further aspects of the MPM tumour microenvironment and architecture, for example by co-engrafting MPM cell lines together with human fibroblasts and/or immune cells, and engrafting patient-derived cells or tissue explants coupled with alternative preclinical imaging methods.MPM-CAM xenografts can be efficiently established using MPM cell lines derived from tumours with a range of histopathological sub-types and tumour suppressor inactivation. These xenografts can mimic stromal and vascular interactions of clinical MPM lacking in 2D and most 3D cell cultures. Bioluminescence imaging can be readily used to evaluate MPM tumour burden over time, and is readily combined with multimodal imaging, transcriptional and histopathological analysis to determine tumour size, vascularisation, invasion, stromal composition, and proliferative capacity. The MPM-CAM model could therefore provide an invaluable component of the drug development pathway for MPM, and the methods for combined readouts could be extrapolated for use in multiple cancer types.


Increasing evidence has shown that epigenetic modifications, such as DNA methylation, histone modification, RNA modification, and non-coding RNAs, can regulate the head and neck TME and thereby influence tumour development. Epigenetic modifications can regulate the expression of different genes and subsequently alter the TME to affect the progression of head and neck tumours. In addition, the cell components in the TME are regulated by epigenetic modifications, which, in turn, affect the behaviour of head and neck tumour cells. In this review, we have discussed the functions of epigenetic modifications in the head and neck TME. We have further examined the roles of such modifications in the malignancy and metastasis of head and neck tumours.Head and neck tumours are common malignancies that are associated with high mortality. The low rate of early diagnosis and the high rates of local recurrence and distant metastasis are the main reasons for treatment failure. Recent studies have established that the tumour microenvironment (TME) can affect the proliferation and metastasis of head and neck tumours  Globally, head and neck tumours are the most common malignant tumours . More thvia multiple mechanisms, such as Notch and STAT3 signalling pathways  of HNSCC via the RIG-I/IFNα signalling pathway  . It affe1/Bcl-xL . In head1/Bcl-xL . Recent 1/Bcl-xL . In naso1/Bcl-xL . Moreovetastasis . Based oThe role of the immune system in tumour development is widely accepted, and therapeutic modalities targeting tumour immunity have been frequently reported . To faci+ T cells, CD4+ T cells, natural killer cells (NKs), and dendritic cells (DCs), which act as anti-tumour agents. The TME also contains myeloid suppressor cells (MDSCs), regulatory T cells (Tregs), and tumour-associated macrophages (TAMs), which act as immunosuppressive agents . In HNSCC, the expressions of PD1 and PD-L1 are upregulated, which represents one of the key immune checkpoints in HNSCC –53. PD-1hylation , 58. Thi+ T cells and leads to immunity evasion , microRNAs (miRNAs), and lncRNAs, interact with the components of the TME, thereby affecting tumorigenesis and progression –88. Thespression . Low levpression . TherefoH19 is upregulated in CAF and participates in the glycolytic pathway of CAF via the miR-675-5p/PFKFB3 axis, which promotes the progression of oral cancer , the Changchun Scientific and Technological Development Program (grant no. 21ZY26) and the Jilin Province Scientific and Technological Development Program .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coinfections, both bacterial and viral, occur with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but prevalence, risk factors, and associated clinical outcomes are not fully understood.We used the Coronavirus Disease 2019-Associated Hospitalization Surveillance Network (COVID-NET), a population-based surveillance platform to investigate the occurrence of viral and bacterial coinfections among hospitalized adults with laboratory-confirmed SARS-CoV-2 infection during March 2020 and February 2022. Patients receiving additional standard of care (SOC) molecular testing for viral pathogens (14 days prior to admission or 7 days after), including respiratory syncytial virus, rhinovirus/enterovirus (RV/EV), influenza, adenovirus, human metapneumovirus, parainfluenza viruses, and endemic coronaviruses, were included. SOC testing for clinically relevant bacterial pathogens (7 days before admission or 7 days after) from sputum, deep respiratory, and sterile sites were included. The demographic and clinical features of those with and without bacterial infections were compared.Staphylococcus aureus was the most common isolate overall; Pseudomonas aeruginosa was the second most common respiratory isolate .Among 2,654 adults hospitalized with COVID-19 and tested for all 7 virus groups, another virus was identified in 3.1% of patients. RV/EV (1.2%) and influenza (0.4%) were the most commonly detected viruses. Half  of hospitalized adults with COVID-19 had bacterial cultures taken within 7 days of admission, and 1,092 (6.1%) of these had a clinically relevant bacterial pathogen. A higher percentage of those with a positive culture died compared to those with negative cultures . This figure includes 1,408 bacterial cultures from 1,066 individuals. Deep respiratory sites include endotracheal aspirate, bronchoalveolar lavage fluid, bronchial washings, pleural fluid, and lung tissue. Commensal organisms were excluded.Consistent with previous studies, a relatively low proportion of adults hospitalized with COVID-19 had concomitantly identified viral or bacterial infections. Identification of a bacterial infection within 7 days of admission is associated with increased mortality among adults hospitalized with COVID-19. Conclusions about the clinical relevance of bacterial infections is limited by the retrospective nature of this study.Evan J. Anderson, MD, GSK: Advisor/Consultant|GSK: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Kentucky Bioprocessing, Inc: Data Safety Monitoring Board|MedImmune: Grant/Research Support|Medscape: Advisor/Consultant|Merck: Grant/Research Support|Micron: Grant/Research Support|NIH: Funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines|PaxVax: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant|Sanofi Pasteur: Grant/Research Support|Sanofi Pasteur: Data Adjudication and Data Safety Monitoring Boards|WCG and ACI Clinical: Data Adjudication Board.


In this study, impact of second order slip for Maxwell fluid at vertical exponential stretching sheet is deliberated. Dufour and Soret impact for vertical exponential stretching sheet under nonlinear radiation are deliberated. Thermal and concentration slips with viscous dissipation are taken into account under the Buongiorno’s model. Under the above assumptions, the differential model constructed using the boundary layer approximations using the governing equations. The similarities transformations are introduced which applied the differential model  and developed the dimensionless differential equations . The dimensionless differential equations are cracked by numerical scheme. The impact of physical parameters are presented by tables and graphs. The curves of fluid velocity enhanced due to increasing the values of velocity slip. Velocity slip is a fluid-boundary interaction in physics. If the velocity slip increased, the fluid velocity profile would eventually become increasing. Temperature curves declined by improving values of  The preduction of Maxwell fluid model got more attention be several researchers due to lot of applications in fields engineering and science. The impact of upper convicted Maxwell fluid at moving plate presented by Sadeghy et al.2. The results of Deborah’s number and fraction factors have opposite behavior. The impact of nonlinear radiation and time dependent flow of Maxwell fluid discussed by Mukhopadhyay et al.3. The unsteady and Maxwell fluid parameters and skin friction have same behavior of increasing found in their investigations and also their results used in fabulous in the polymer industry fields because this phenomena exist due to heat transfer between the fluid and surface covering it. Nadeem et al.4 discussed the nanomaterial flow of Maxwell fluid at moving surface with MHD effect. Sharma et al.5 deliberated Maxwell fluid model with nanomaterial flow at stretching sheet. Nadeem et al.6 highlighted the impact of Maxwell micropolar fluid with stagnation region at Riga sheet. Kumar et al.7 studied the magnetic dipole using the Maxwell fluid at stretching sheet. Gowda et al.8 debated about Maxwell liquid model using the Casson nanomaterial fluid by stretching disks. The different fluid models have been studied using the non-Newtonian fluid and Newtonian fluid over stretching surface (see Refs.15).The non-Newtonian fluid have been achieved key role in various fields of real life like as plastic polymers, drilling muds, optical fibers, metal spinning, cooling of metallic plates in cooling baths, hot rolling paper production and so on. According to past literature, a single model have no capability to predict the all features of the non-Newtonian fluids. Every non Newtonian fluids have its own properties and their significant role. Further, these models divided into three kinds namely: rate-, integral-and differential-type of fluids. The Maxwell fluid known as rate type fluid because this type of fluid predict the impact of relaxation time which cannot be predicted by other type of fluid. The Maxwell fluid model was predicted by early time known as Maxwell16. This presentation was quite effective. High heat transfer efficiency can be attained when conventional liquids scatter these crystals, despite the fact that most solid particles have poorer thermal conductivities than typical heat transfer liquids. Chamkha17 studied the solar radiative effects for natural convection over a vertical sheet. Chamkha et al.18 studied the heat generation of nanofluid flow at porous surface. Magyari and Chamkha19 analyzed the impact of micropolar nanomaterial fluid flow under chemical reaction and heat generation. The heat transfer of microplar nanomaterial fluid at Riga sheet highlighted by Nadeem at el.20. The modified nanomaterial fluid under thermal slip is deliberated by Nadeem et al.21. The nonlinear stretching sheet for nanomaterial fluid by Alblawi et al.22. The numerical results of nanomaterial fluid is studied by Awan et al.23. Awan et al.24 investigated the Jeffrey nanofluid at a stretching sheet. Awan et al.25 investigated the impact of non-Newtonian fluid flow over oscillatory stretching sheet. Nadeem et al.26 premeditated the influence of nanomaterial fluid flow at curved surface. Nadeem et al.27 premeditated the flow of non Newtonian in the presence of stagnation point region. Abbas et al.28 discussed the phase flow model of nanofluid at vertical wedge. Kumar et al.29 explored the results of nanomaterial fluid under MHD effect at stretching sheet. Punith et al.30 highlighted effects of Dufour and Soret with convective effects at stretching sheet. Kavya et al.31 explored the influence of MHD nanomaterial fluid flow at shrinking cylinder. Upadhya et al.5 explored the phase flow of casson micropolar fluid flow under entropy generation. Sharma et al.32 studied the Maxwell fluid flow at stretching sheet. Recently, numerous investigators have discussed the flow behavior under the different assumptions see in Refs.46.In the field of the thermal system, conventional heat transfers of base fluid, such as engine oil, ethylene glycol, and water, have been crucial. These liquids' limited ability to transport heat results from their poor thermal performance. The thermal characteristics of conventional fluids can be developed by suspending metallic and non-metallic solid particles in them. Nanofluids are those fluids that have suspended base fluid and nanomaterial. Nanofluid was invented by Choi and Eastman47 studied the natural convection of hydro magneticin porous medium. Chamkha48 analyzed the MHD and Hall effects with free convection at porous surface. Takhar et al.49 highlighted the influence of MHD for time depend flow in semi-infinite plate. Chamkha50 highlighted the impact of MHD three dimensional flow of free convection at permeable sheet. Chamkha51 discussed the MHD thermal radiative impacts on permeable surface. Takhar et al.52 studied the influence of MHD rotating flow at moving surface under the Hall and free stream effects. Chamkha and Ben53 highlighted the influence of mixed convection MHD flow at porous surface with Soret and Dufour’s impacts. Modather et al.54 discussed the flow of MHD oscillatory flow with micropolar fluid at vertical permeable plate. VeeraKrishna et al.55 investigated the impacts of MHD Hall impacts for second grade fluid flow at porous surface. Krishna and Chamkha56 analyzed the flow of MHD rotating nanofluid in porous medium under the Hall effects. Kumar et al.57 initiated the Reiner–Philippoff fluid under the MHD and Cattaneo–Christov heat flux. Krishna et al.58 deliberated the time dependent MJHD flow through porous surface. Awan et al.59 highlighted the influence of MHD radiative flow of nanomaterial fluid under solar energy. Few investigators have been studied about MHD flow under several effects and fluid models see Refs.66.Investigation about magnetic field has been much attracted by several authors due to physically importance as well as engineering and chemistry namely: pumps, generators (MHD), bearings and so on. ChamkhaImpact of second order slip for Maxwell fluid at vertical exponential stretching sheet is deliberated. Dufour and Soret impact for vertical exponential stretching sheet under nonlinear radiation are deliberated. Thermal and concentration slips with viscous dissipation are taken into account under the Buongiorno’s model. Under the above assumptions, the differential model constructed using the boundary layer approximations using the governing equations. The similarities transformations are introduced which applied the differential model  and developed the dimensionless differential equations . The dimensionless differential equations are cracked by numerical scheme. The impact of physical parameters are presented by tables and graphs. No one emphasized the Soret effects and second-grade slip at a vertically stretching sheet with Maxwell nanomaterial fluid flow. These findings could be applied to the industrial sector, which has shown to be more effective Fig. .Figure 144):The mathematical model of Maxwell fluid model is considered over vertical exponentially stretching sheet. The impact of second order slip with thermal and concentration slip are considered at the vertical exponential stretching sheet. The influence of Viscous dissipation and Soret effect with Buongiorno’s model to analysis the Brownian and thermophoresis. The differential system developed by using the boundary layer approximation after applying on governor models equations. The governing equations are presented as below:The caushy stress tensor of the second grade fluid is defined asThese tensors are defined as below:Here, material derivative , nanoparticle mass density The related boundary conditionsHere, derivative denoted as prime with respect to The main physical expression of the fluid model is local Nusselt number and Sherwood number which are most vital role in this field. The expression of the following properties are presented as).In Eq.  \documenIn the dimensionless form,The local Reynolds number is 44):Solving the dimensionless system of differential equations are reduced in first order using the bvp4c technique. The procedure of the following method is as  and developed the dimensionless differential equations . The dimensionless differential equations are cracked by numerical scheme. The main results of the physical parameters are presented as below:


Kaempferol, a natural flavonoid abundantly found in plants, is known to have pharmacological properties, such as anti-inflammatory and anti-cancer effects. In this study, we investigated the antiviral effects of kaempferol against a varicella-zoster virus (VZV) clinical isolate in vitro. We found that kaempferol significantly inhibited VZV replication without exhibiting cytotoxicity. Kaempferol exerted its antiviral effect at a similar stage of the VZV life cycle as acyclovir, which inhibits VZV DNA replication. Taken together, our results suggest that kaempferol inhibits VZV infection by blocking the DNA replication stage in the viral life cycle. Varicella-zoster virus (VZV), a member of the alpha herpesvirus subfamily, is spherically shaped with a diameter of 150–200 nm and consists of a nucleocapsid layer containing a double-stranded DNA (dsDNA) genome of ~125 kb, a tegument layer, and an envelope layer embedded with viral glycoproteins. Primary VZV infection through the respiratory mucosa causes varicella (chickenpox) and establishes latency in cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia .More than 90% of the world’s population is infected with VZV, and one-third suffers from VZV reactivation . The maiThe development of antiviral therapies against VZV is important because VZV diseases continue to occur even in countries where VZV vaccines are routinely available  and becaFlavonoids such as kaempferol, which is known to have pharmacological properties—including anti-inflammatory and anticancer effects ,18 are aThe effect of kaempferol on VZV replication was determined by conducting plaque reduction assays. To this end, human foreskin fibroblasts (HFFs) were inoculated with the cell-associated clinical VZV isolate, YC01 (VZV-YC01), at a multiplicity of infection (MOI) of 0.1 and then treated with kaempferol at concentrations of 5, 7, 10, and 15 µg/mL or with acyclovir at concentrations of 0.5, 1, 5, and 10 µM; dimethyl sulfoxide (DMSO) was used as a vehicle control. Cells were re-treated with kaempferol, acyclovir or DMSO on day 3 after inoculation and the number of plaques was counted on day 6. Concentration-response studies showed that kaempferol at a concentration of 7 µg/mL or higher suppressed VZV replication by more than 50% compared with cells treated with DMSO and yielded an estimated 50% inhibitory concentration (IC50) value of 6.36 ± 0.73 µg/mL . The IC5To assess the cytotoxicity of kaempferol, we treated HFFs with kaempferol  or DMSO for 72 h, then determined the cytotoxicity using Thiazolyl Blue Tetrazolium Bromide (MTT) assays. Kaempferol caused no cytotoxicity at any concentration tested . CollectDuring VZV replication, immediate early (IE), early (E), and late (L) genes are expressed in order, starting with the expression of IE62, a major transactivator protein . We firsTo determine the stage of the VZV life cycle inhibited by kaempferol, we performed a time-of-drug-addition assay using acyclovir as a control A. HFFs wTo determine whether kaempferol targets the DNA synthesis stage of the VZV life cycle, we first determined the time point at which VZV DNA synthesis starts. Since one VZV life cycle takes 18–22 h , we harvEugenia jambolana Lam (1.3 mg/kg), Acacia nilotica L. (21.7 mg/kg), Azadirachta indica A. Juss. (0.5 mg/kg), Terminalia arjuna (Roxb.) Wight & Arn. (8.9 mg/kg), Ficus religiosa L. (160.8 mg/kg), Aloe barbadensis Miller (257.7 mg/kg), Rosmarinus officinalis L. (1.2 mg/kg), and Euonymus alatus Thunb. (5 mg/kg) [Kaempferol is abundant in medicinal plants such as 5 mg/kg) ,26. Furt5 mg/kg) .We found that kaempferol possesses potent antiviral activity against VZV without exhibiting cytotoxicity . Thus, wPrevious studies have reported antiviral activities of kaempferol, showing that kaempferol functions as a neuraminidase inhibitor for influenza viruses  and a re2 environment. The maintenance and propagation of the clinical VZV isolate, YC01 (VZV-YC01), have been described previously [Human foreskin fibroblasts (HFFs) and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum and 1X penicillin-streptomycin at 37 °C in a humidified 5% COeviously . KaempfeHFFs in a 12-well plate were inoculated with serially diluted cell-associated VZV-YC01. At 1 h after inoculation, cells were treated with kaempferol , acyclovir , or DMSO (vehicle control), and subsequently re-treated with kaempferol or acyclovir at the same concentration (or DMSO) after 3 days. At 6 days after inoculation, cells were fixed by incubating with 10% formaldehyde for 10 min at room temperature and then stained with 0.3% crystal violet. The next day, the number of plaques was counted and the half-maximal inhibitory concentration (IC50) value was calculated using GraphPad Prism 7 .HFFs in a 96-well plate were treated with kaempferol at 5, 10, 15, and 20 µg/mL, or with DMSO. After 48 h, cell viability was measured using MTT assays as described previously . MTT wasHindIII  [HindIII sequence at the end. The primer sequences used for amplification were 5′-CCC AAG CTT ATC GTC TGT AGA CAC ACG ATG-3′ (forward) and 5′-CCC AAG CTT CGC ACT GGG GTG AAT TTA G-3′ (reverse). The PCR product was digested with HindIII and ligated using T4 ligase (Enzynomics) into a pJHA324 vector in which the HCMV MIE enhancer/promoter sequences had been removed. The orientation of the VZV IE promoter insert was confirmed by DNA sequencing . Transient transfections and luciferase assays were performed using Omicsfect™  and Dual-Luciferase Reporter Assay System , respectively, according to the manufacturers’ protocols as described previously [HCMV MIE enhancer/promoter sequences were removed from the vector pJHA324 using the restriction enzyme , Korea) . The VZVeviously .For quantitative analysis of viral DNA, nuclei of VZV-infected cells were prepared as described previously , and tott-test. p-values were determined by unpaired two-tailed Student’s t-tests. p-values <0.05 were considered statistically significant.Data are presented as means ± standard deviations (SD). The significance of differences between means was determined with the Student’s


Aims: We present a 5-case series of low-grade oncocytic tumour of the kidney to further discuss their clinicopathological characteristics.Methods and results: Five patients were included in this study. There were three females and two males aged 45–66 years, with a median age of 65 years. Four tumours were located in the right kidney, and one was located in the left kidney. Most of the tumour sections were yellow-brown in colour. Tumour sizes ranged from 2.5 to 4.5 cm, with a median size of 3 cm. Microscopically, the tumours were well-circumscribed but lacked a fibrous capsule; the tumours consisted of monomorphous oncocytic cells arranged mainly in solid and nested architectural patterns. The tumour cells had uniformly round to oval nuclei and often had perinuclear halos but lacked significant irregularities. Immunohistochemically, the tumour cells showed a diffuse and strong positivity for CK7 and were negative for CD117. The tumour cells were also positive for GATA3, E-cadherin, Pax-8, Succinate dehydrogenase B (SDHB) and Fumarate hydratase (FH), and negative for vimentin, Carbonic anhydrase 9 (CA9), CD10, P504s, CK20, TFE3, TFEB, HMB45, ALK and Forkhead box protein I1 (FOXI1). Next-generation sequencing identified genetic variations in these tumours, including MTOR gene mutations (4/5) and PIK3CA gene mutation (1/5). All patients were alive without disease progression at a median follow-up of 32 months (range 10–57 months).Conclusion: LOT is an emerging renal entity of indolent behaviour that has morphologic overlap with some renal tumours with eosinophilic cytoplasm, primarily with oncocytoma and eosinophilic variant of chromophobe renal cell carcinoma. Familiarity with the distinctive morphological features, immunophenotype and molecular genetics of LOT helps avoid misdiagnosis. Low-grade oncocytic tumour (LOT) of the kidney is an emerging renal entity that was first described by Trpkov K and Hes O in 2019 , 13 cases of renal oncocytoma, 16 cases of papillary renal cell carcinoma (PRCC) with eosinophilic features, 8 cases of clear cell renal cell carcinoma (CCRCC) with eosinophilic features, and 6 cases of unclassified oncocytic renal tumour. We only identified 5 tumours as LOT characterized by CK7 positivity and CD117 negativity. Of the five cases, four were originally diagnosed as E-ChRCC, and one as renal oncocytoma. The clinical and pathological findings were obtained from the medical records and pathology reports. Follow-up information was obtained by direct telephone communication with the patients and/or their relatives.Immunohistochemical staining was performed on 4-µm-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections using a Roche Benchmark XT Automated Staining System. The antibodies used in this study  includedNext-generation sequencing (NGS) was performed by Illumina MiSeq (Illumina). The tissue DNA was extracted from FFPE tumour tissues using QIAamp DNA FFPE tissue kit . Fragments between 200 and 400 bp from the sheared tissue DNA were purified , hybridized with capture probes baits, selected with magnetic beads, and amplified. Target capture was performed using a commercial panel consisting of 520 genes  chosen bThe clinical features of the five patients are summarized in Histologically, all tumours were well circumscribed but lacked a fibrous capsule. The growth patterns included solid, nested , trabecuMTOR gene exon 53 p.L2427Q mutation [c.7280T>A (p.Leu2427Gln)], and one tumour had a PIK3CA gene exon 10 p.E542K mutation [c.1624G>A (p.Glu542Lys)].Immunohistochemically, all tumours showed diffuse and strong positivity for CK7 and were negative for CD117. GATA binding protein 3 (GATA3) , E-cadheLow-grade oncocytic tumour (LOT) of the kidney has emerged as a new diagnostic entity in renal tumour pathology in recent years –13. LOT The clinicopathological features of LOT in this study are highly consistent with those of previously published cases. The tumours are found accidentally during physical examination in older patients as a single tumour, have a slight female predilection and have an indolent behaviour. Several LOTs have been reported in patients with tuberous sclerosis complex (TSC) , 6, 8 orAll the five tumours in this study had a typical immunophenotype characterized by diffuse positivity for CK7 and negativity for CD117, and were also positive for PAX-8, E-cadherin, FH and SDHB, but negative for vimentin, CD10, P504s, CA9, CK20, TFE3, TFEB, HMB45, and ALK. Rare neoplasms can show weak focal CD117 staining . We alsoCCND1 rearrangements were not found in LOT by using fluorescence in situ hybridization (FISH)  and mTOR complex 2 (mTORC2). mTORC1 is regulated by the tuberous sclerosis complex (TSC)  . An incrH)  . 4EBP1 aex (TSC) . All of TSC/MTOR mutations . They observed a specific trend in TSC/MTOR mutation in different tumours. ESC RCC and TSC-mt RCC-NOS displayed consistent TSC1/TSC2 mutations, EVT demonstrated equal mutation distributions in TSC and MTOR genes, and all LOT cases but one bearing TSC1 mutation displayed MTOR gene mutations.Pivovarcikova et al.  discusseutations . Recentlutations  further FLCN germline mutations. Hybrid oncocytic/chromophobe tumour (HOCT) was initially described in patients with BHD syndrome harbouring FLCN gene mutations   expression of CD117 and FOXI1 may help to avoid misdiagnosis. In challenging cases, the molecular genetic features of