Altered acetyl CoA acetyltransferase 1 (ACAT1) expression has been reported in diverse cancers. However, the expression of ACAT1 and its prognostic value in oral squamous cell carcinoma (OSCC) has remained unexplored.In this study, the expression of ACAT1 was analysed by immunohistochemistry (IHC) in 61 OSCC patients and compared between OSCC and adjacent pre-tumour tissue of 21 patients.The expression of ACAT1 in OSCC tumours is heterogeneous between patients. More specifically, 52.38% of the patients show low expression of ACAT1 in both tumour and adjacent pre-tumour tissues, 9.52% of the patients show high expression of ACAT1 in both tumour and adjacent pre-tumour, 19.05% of the patients have high expression of ACAT1 in tumour tissue and low expression of ACAT1 in adjacent pre-tumour tissue and another 19.05% of the patients have low expression of ACAT1 in tumour tissue and high expression of ACAT1 in adjacent pre-tumour tissue.Comparison of ACAT1 expression, one of the key enzymes in the ketone body metabolic pathway, divided OSCC patients into two groups: 1) similar expression and 2) different expression of ACAT1 in tumour and adjacent pre-tumour tissue. No significant association between ACAT1 levels and overall survival was observed. Oral squamous cell carcinoma (OSCC) is the most common malignancy of oral cancer with high mortality and 5-year survival is less than 50% . There iFormalin-fixed paraffin-embedded fresh human OSCC and adjacent pre-tumour biopsy specimens were selected to evaluate the expression of ACAT1 between OSCC and adjacent pre-tumour tissues. These included 21 OSCC samples with their adjacent pre-tumour tissues that were harvested at surgical removal from hospitals . All the OSCC patients were histopathologically diagnosed. Fresh tissues were immediately fixed in formalin for histological study. Adjacent pre-tumour tissues were harvested far from the margin of tumours. Additionally, 40 OSCC paraffin embedded tissues without adjacent pre-tumour tissues were obtained from the archives of the Oral and Maxillofacial Department, Tehran Medical University. Altogether, an overall of 61 OSCC patients with paraffin blocks were used to evaluate the association of ACAT1 expression with survival probabilities. This research project was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1399.528).The formalin-fixed tissues obtained at surgical removal were dehydrated and embedded in paraffin. Four-micrometer-thick paraffin sections were prepared from OSCC and adjacent pre-tumour tissues, followed by deparaffinization in xylene and rehydration in serial dilutions of ethanol. The tissues were incubated in PBS 3% hydrogen peroxide for 10 min. Next, the antigen retrieval was performed using citrate buffer (pH = 6) at 90 °C for 15 min. The tissues were then incubated with the ACAT1 primary antibody  for 1 h at RT, followed by three washes with PBS and incubation with primary antibody amplifier master for 20 min, after washing with PBS, slides were incubated with the secondary antibody for 30 min, followed by washing in PBS. The signal was developed with DAB substrate solution, then counterstained with hematoxylin and dehydrated with serial dilutions of alcohol and mounted.The stained slides were observed using a light microscope. For each slide, the intensity staining score was applied as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong)) . The scoThe statistical analysis was performed using Graphpad Prism software version 8. The ACAT1 percentage of positive cells and its total score were compared between OSCC and adjacent pre-tumour tissues using paired sample T test. Survival analysis was performed using the Kaplan–Meier method and log-rank test with Python software and the lifelines package. p-values < 0.05 were considered statistically significant.low/Normallow (11 out of 21 patients (52.38%)), ii/OSCClow/Normalhigh (4 of 21 patients (19.05%)), and iii/OSCChigh/Normallow (4 of 21 patients (19.05%)) and iv/OSCChigh/Normalhigh (2 of 21 patients (9.52%))  and low ACAT1 protein expression in 38 of 61 patients (62.3%). We then evaluated the ACAT1 expression in adjacent pre-tumour tissues and looked for possible differences with OSCC tumour tissue. To this end, we took advantage of 21 samples for which both OSCC tumour and adjacent pre-tumour tissues were available. Variable ACAT1 protein expression was observed in pre-tumour tissues, similar to OSCC tissues. Indeed, out of the 21 patients, high ACAT1 protein expression was found in the pre-tumour tissue of 6 patients (29%) and low ACAT1 protein expression in 15 patients (71%). We then wanted to evaluate whether the percentages of ACAT1 positively stained cells and ACAT1 total score staining were similar or different between tumour and their adjacent pre-tumour matched tissues in our 21 patients. We performed a paired sample T test and found a higher percentage of ACAT1 positive cells in tumour tissues compared to adjacent normal mucosa tissues  whereas no significant difference was observed between the total scores of tumour and adjacent pre-tumour tissues  . Additio(9.52%))  (Table S(9.52%)) .We then investigated the association of ACAT1 expression according to the percentage of positively ACAT1 stained cells, the score of percentage and ACAT1’s total staining score with the survival of OSCC patients. Kaplan–Meier analysis revealed no significant difference in overall survival between stratified groups according to high and low levels of ACAT1 .low/Normallow, OSCClow/Normalhigh, OSCChigh/Normallow and OSCChigh/Normalhigh. In 2016, Fan and colleagues reported an upregulated ACAT1 activity despite comparable gene and protein expression in human leukaemia cells, head and neck cancer and lung cancer cells, compared to their corresponding normal cells [low/Normallow and OSCChigh/Normalhigh).OSCC is the most common subtype of oral cancer. Overall, cancer is a disease with mitochondrial metabolism alterations which seems to benefit from dietary therapeutic approaches. The association of enzymes responsible for catalyzing ketone bodies with the progression of cancers, including prostate and kidney, has been recently reported ,8. AmongSince the expression levels of ACAT1, one of the key enzymes in the ketone body metabolic pathway, are variable between OSCC patients, a high-fat low-carbohydrate diet (ketogenic), which increases the amount of blood ketone bodies, would probably not be able to disrupt the metabolism and therefore would not increase the death of oral squamous cancer cells in all OSCC tumours. These results suggest that ACAT1 staining could help stratify patients into different groups with respect to their potential response to a ketogenic diet, although this statement would still need to be strengthened by analysing other ketolytic enzymes.In conclusion, our measurements of ACAT1 protein expression did not show any clear common trend regarding the relative expression levels of this enzyme between OSCC and adjacent pre-tumour tissues. Further studies would be required to elaborate on the reason behind these variations of ACAT1 expression among OSCC patients. The main limitation of this study was the evaluation of other ketolytic enzymes including 3-hydroxybutyrate dehydrogenase 1(BDH1), 3-hydroxybutyrate dehydrogenase 2 (BDH2) and succinyl CoA: 3-oxoacid CoA transferase 1 (OXCT1) in the tumour and pre-tumour tissues of the OSCC patients. To conclude on the clinical application of this study, OSCC patients with observed low ACAT1 expression in their tumour compared to the pre-tumour tissue may respond better to ketogenic diet therapy, although further investigations of BDH1, BDH2 and OXCT1 related to the ketolysis would be required.


With the increasingly serious energy and environmental problems, the R1234yf ejector refrigeration system (ERS) shows great development potential in the refrigeration industry due to its simplicity, low maintenance costs and environmentally friendly nature. However, poor ejector performance has always been the main bottleneck for system applications. In order to overcome this problem, this paper proposes a design method for R1234yf ejectors based on the gas dynamic method and optimizes the geometrical parameters including the area ratio (AR) and nozzle exit position (NXP) to improve its performance through the control variable optimization algorithms. Based on the validated simulation model, the results show that the entrainment ratio increases initially and then decreases with the increase in AR and NXP, respectively; the AR has a significant effect on the shock wave position in the mixing chamber and the NXP can directly influence the expansion state of motive fluid; the ejector performance increases by about 17% over the initial entrainment ratio by the control variable optimization algorithms. This work can guide the R1234yf ejector design and promote the development of the ERS with environmentally friendly working fluids. With the rapid development of the global population and economy, the number of refrigeration systems has increased rapidly. The improvement of people’s comfort inevitably brings energy and environmental problems. In this scenario, the efficient cooling cycles and environmentally friendly refrigerants have been significant research hotspots in the refrigeration industry ,2,3.2, which reduced the equipment investment. As a result, it can be concluded that the R1234yf is indeed an ideal alternative refrigerant for the refrigeration system.With the promulgation of Regulation No. 517/2014 (F-gas Regulation), the refrigerants with a high GWP will be phased out, such as R134A, R410A, R407C and so on  also gaiThe compressor is the main exergy-reduction component in conventional vapor compression refrigeration systems. The replacement of compressors with non-electricity-consuming components driven by waste heat or renewable energy is the main way to improve system overall performance. Currently, there are mainly three thermal-driven refrigeration systems: absorption, adsorption and ejector cooling systems. The adsorption cooling system has a difficult time achieving a higher refrigeration capacity and may cause system interruption when it works for a long time . FurtherRonanki et al.  investigAs for the optimization of ejector thermodynamic models with conventional working fluids in the ERS, Keenan  made twoFrom the above literature reviews, we can see that the research into improving the ejector performance with conventional working fluids has matured. However, there are few reports on the thorough design and performance optimization methods of the R1234yf ejector in the ERS, which will hinder the performance improvement of the ejector and the ERS with R1234yf because the design approach is the cornerstone of optimizing ejector performance. Consequently, this paper proposes the detailed design method for the R1234yf ejector based on the aerodynamic method and optimizes the geometrical parameters to improve its performance through the control variable optimization algorithms, which will promote the development of the ERS with R1234yf.The entrainment ratio The ejector is under steady conditions;(2)The ejector wall is adiabatic.(3)The velocity at the primary and secondary flow inlets and the ejector outlet is negligible compared with the supersonic velocity inside the ejector;(4)The fluid and friction loss that occurs in the ejector is considered by the isentropic coefficients;To simplify the calculation process, the following assumptions are presented:The entrainment ratio calculated is expressed as follows:The fluid converted mass velocity at the mixing chamber inlet is expressed as:The fluid converted mass velocity at the mixing chamber outlet is calculated as:The ejector works under the second limit state when the secondary fluid reaches the critical speed at a certain section in the mixing chamber; at this time, the entrainment ratio is maximum and is defined as:The maximum entrainment ratio is the optimization objective for ejector design with a satisfied pressure ratio, so it is considered a critical step for ejector design in computing the maximum entrainment ratio. The converging and diverging nozzle should be adopted to make the primary fluid fully expand when The diameter of the nozzle throat is:The nozzle outlet area is:The nozzle inlet area is expressed as:The mixing chamber outlet area is defined as:The mixing chamber outlet diameter is:The determination of the nozzle exit position mainly affects the expansion of motive fluid and then affects the mixing of two streams of fluid, which is considered to be an ideal value when the final section area of the primary fluid free flow beam coming out of the nozzle is equal to the mixing chamber inlet area. As a result, the free flow beam length of the motive fluid ((1)The free flow beam length of the motive fluid When the entrainment ratio is less than 0.5:(2)The fluid diameter at the end of the free flow beam  and nozzle exit position (NXP), which are two critical parameters related to fluid mixing and primary flow expansion, and finally obtains the optimal ejector performance. To ensure that the results are unaffected by other parameters, the simulation is carried out with fixed operation temperatures and geometry parameters, which are the same as the initial designed parameters. The control variable optimization algorithm is used to optimize the structural parameters in order to obtain optimum ejector performance, because these two geometrical parameters are coupled. The mass, momentum and energy conservation equations are the basis for solving the fluid flow characteristics inside the ejector, and they are expressed as follows based on above assumptions:The continuity equation:The momentum equation:The energy equation:The turbulent model is used to close the fluid flow equations in the Reynolds-Averaged Navier–Stokes (RANS) numerical simulation methods, and the two-equation model is more widely used in engineering computation due to it being more theoretical when dealing with complex flow processes. Moreover, the prediction accuracy about fluid characteristics is higher for the o et al.  reportedThe Software Fluent 19.0 is used to complete numerical simulations, and the ejector works under a steady state. The 2D axisymmetric model was adopted to simplify the computation process. The turbulent SST rom NIST . The motMichael et al.  confirmeThe mixing chamber is an important component for the ejector where the primary fluid and secondary fluid exchange energy and the fluid velocity changes dramatically. As a result, the fluid field characteristic is very sensitive to grid numbers. In other words, it can be considered that the errors at every position on the axis are within the acceptable range when the change in flow characteristics at this region satisfies the error requirements under different grid densities. The results of the numerical model are generally considered as reliable if the errors of the entrainment ratio from simulations and experiments are acceptable . The expFurthermore, the inappropriate AR will directly affect the flow area of the secondary flow, which in turn will cause the presentence of the backflow phenomenon. The better understanding of flow field characteristics is the basis of ejector performance optimization. As a result, this part analyzes the pressure field and velocity field characteristics inside the ejector under the optimal structural parameters to better understand the flow state and flow field characteristics of the fluid in the ejector.(1)Indicate the coupling laws between the area ratio and the ejector performance. When the area ratio increases, the entrainment ratio increases initially and then decreases; the recirculation area in the mixing chamber first decreases and then increases with the increase in the area ratio. There is no recirculation inside the ejector when the area ratio is 5.28, and the shock wave disappears exactly at the end of the mixing chamber. At this time, the entrained secondary fluid just passes through the mixing chamber without additional energy loss, and the maximum entrainment ratio is 0.602.(2)Reveal the relationships between NXP and ejector performance. The entrainment ratio first increases and then decreases with the increase in NXP, and the change in NXP directly affects the expansion state of the motive fluid. When ΔNXP is −2 mm, the expansion degree of the motive fluid is appropriate, and the final section area of the motive fluid beam is exactly equal to that of the mixing chamber inlet. There is no backflow phenomenon in the suction chamber, and the fluid streamline is clear, with the maximum entrainment ratio being 0.617.(3)Obtain the optimal entrainment ratio through the control variable optimization algorithms. The entrainment ratio increases by 17.34% compared with the initial value.This paper proposes a thorough ejector design method for R1234yf based on the gas dynamic methods and optimizes the ejector geometrical parameters to improve the R1234yf ejector refrigeration system performance through control variable optimization algorithms. Based on the verified numerical simulation model, the following results are obtained:In conclusion, this paper provides a design method for the R1234yf ejector based on the aerodynamic method and optimizes the ejector geometrical parameters through control variable optimization algorithms. In addition, the coupling laws between the area ratio, the nozzle exit position and the ejector performance are obtained, which can promote the development of the ERS with R1234yf.


Simulation is a useful education tool for high-stakes clinical skills and decision-making. Recommending whether to accept or reject an organ for transplantation based on infection risk is a critical core competency in Transplant Infectious Disease (ID), however there are no published data that learners have opportunities to practice this during training. We created a novel simulation to expose learners to this real-life clinical scenario and evaluated their clinical decision-making in these situations.We created 6 simulations with common ID consult questions about whether to accept or reject an organ for transplant based on infection risk (Table 1). During learners’ Transplant ID rotations, faculty periodically texted or paged them with the simulation cases as though they were the transplant coordinator. Learners had 15 minutes to ask follow up questions before deciding to accept or reject the organ and explain their decision-making process in a survey. Learners completed a survey 1 month after the simulation experience to evaluate its effectiveness.Between October 2021 and April 2022, 16 learners from 7 medical centers participated in the simulation (Table 2) and 94% (15/16) completed the follow up survey. Eighty-seven percent (13/15) of ID learners reported that the simulation was effective in teaching them when to accept or reject organs and 80% (12/15) felt more prepared to make these decisions in practice. Most learners correctly identified acceptable organs for transplant during the simulations . Of the 100 clinical reasoning decisions made during the activity, 19% were discordant, where the learner correctly decided to accept or decline the organ but with incorrect or incomplete reasoning for this decision .ID learners perceived our transplant ID simulation as an effective educational tool to learn when to accept or reject an organ for transplant. By evaluating the clinical reasoning behind these decisions our simulation provides ID educators with nuanced insight into their learners' thought process and allows for targeted coaching to correct deficits in reasoning.Rebecca Kumar, MD, Gilead: Grant/Research Support|Regeneron: Grant/Research Support Jonathan Hand, MD, GlaxoSmithKline: Grant/Research Support|Janssen: Grant/Research Support|Pfizer: Grant/Research Support Roderick Go, DO, Aptose Biosciences: Stocks/Bonds|Bristol Meyers Squibb: Stocks/Bonds|Cytodyn Inc.: Stocks/Bonds|Scynexis: Grant/Research Support Erica J. Stohs, MD, MPH, bioMerieux: Grant/Research Support.


Fluid pressure along faults plays a significant role in fault behaviors in seismic cycles in subduction zones. When a thermal pressurization event occurs, the fluid pressure rises; conversely, when a fault-valve behavior event occurs, the fluid pressure falls. The stress state changes with seismic cycles from a reverse fault regime to a normal fault regime, as observed in both geophysical observations and geological records. Fluid pressure has been estimated for both modern accretionary prisms and exhumed accretionary complexes. However, changes in fluid pressure on seismogenic faults have not been connected to seismic cycles. Here, we quantitatively show the dynamic change in fluid pressure in a seismogenic fault with geological evidence from an exhumed accretionary complex. We found extensional veins related to seismogenic fault records that exchanged stress states the during seismic cycles. We also constrained the fluid pressure quantitatively, both at an increasing stage during an event and at a decreasing stage after an event. In this procedure, we propose new methods to constrain the magnitude of vertical stress and rock tensile strength. The shear strength along a seismogenic fault changes with fluid pressure during seismic cycles, and the variations in the shear strength are time-dependent1. Recent experimental studies have also suggested that fluid pressure can affect the frictional stability of fault zones3.Variations in fluid pressure along seismogenic faults are important in understanding earthquake mechanisms6. The other is the fault-valve model, in which the fluid pressure decreases after an earthquake due to fluid escape along newly developed fractures or faults along a subducting plate interface, enhancing the shear strength along the fault. It has been shown that variations in shear strength due to changes in fluid pressure during an earthquake cycle, as well as stress changes, may be important in controlling the onset and recurrence of rupture on seismogenic faults along subduction plate interfaces7. Quantitative constraints on fluid pressure variations are therefore important because these two conflicting models can coexist at different timescales during an event, potentially leading to a better understanding of seismic mechanisms and recurrence timescales.There are two opposing models for the shift in fluid pressure caused by earthquakes. One is the thermal pressurization model, in which the fluid pressure rises due to frictional heating during a seismic slip. The increase in fluid pressure reduces the shear strength and promotes slip along a seismogenic fault9. The stress change from the reverse to normal fault stress regime occurred at least one day after the Tohoku earthquake8. A geophysical study has reported that the recovery of the stress state to almost the same stress state as that of the pre-earthquake period took six years following the Tohoku earthquake10. Similar changes in the stress state were also identified in on-land accretionary complexes and in the cores of the Taiwan Chelung-pu Fault Drilling Project (TCDP) using a micro-fault inverse method12. However, it is difficult to geologically estimate the time scale of the changes in the stress state.The change in the stress state from reverse to normal fault stress regimes was observed during the Tohoku earthquake13. However, because the study only focused on extensional veins formed during normal fault stress regime, it did not fully capture the change in fluid pressure during seismic cycles.In a previous study, a poro-elastic model was used to constrain the fluid pressure decrease during seismic cycles, as demonstrated by the extensional veins of the Nobeoka thrust in southwest Japan14. We examined the change in the stress state with seismic cycles using mixed Bingham statistics16 for extensional veins. Then, the rock failure theory17 and fluid pressure obtained from fluid inclusion microthermometry for the veins18 instead of the poro-elastic model13 were employed to constrain the change in fluid pressure during seismic slip and after a seismic event in seismogenic zones along the subduction plate interface.In this study, we focus on the underplating thrust zone in a mélange zone within an exhumed accretionary complex. The mélange adjacent to the thrust zone has extensional veins showing network texture, which is related to the activity of the underplating thrust zone because of their occurrences20 –245 °C and pressures ranging 107(+ 8/− 4.1)–149.4 MPa18. The network veins formed after the mélange formation because they cut the mélange fabrics. Each vein in the network crosscuts with others without any specific orientation of veins, suggesting that multiple stress states were repeatedly exchanged . As a result of rotation, the dip azimuth and dip angle of the maximum, intermediate, and minimum principal stresses were , , and  for Stress 1, , , and  for Stress 2, and , , and  for Stress 3, respectively , a, a\documely Fig.  are diviram Fig.  indicateram Fig. , includi18. As the previous study did not classify the extensional veins based on stress regime, the range of fluid pressures from the fluid inclusion was expected to be included in different stress regimes because the extensional veins were collected randomly in the previous study. When the differential stress is the maximum value of 34, and using Eqs. (3 and 2 in the conversion). The cross-cutting relationship in the network veins suggests that the extensional veins formed at similar depths and rock tensile strengths in the normal and reverse fault stress regimes. Therefore, the cross point of the two slopes in the relationships between 19. The constrained values for tensile strength, depth and geothermal gradient are reasonable, which supports our expectation that extensional veins include both reverse and normal fault stress regimes.In order to constrain these parameters, we used previous estimates of fluid pressure based on fluid inclusion analysis from the network veins, which estimated that the fluid pressure was between 107+ 8/− 4.1)–149.4 MPasee the “” sectioning Eqs. , the rel.1–149.4 The difference between the lower and upper limits for fluid pressure in the een Eqs.  for the een Eqs.  and 15)\document26. In our study, the dynamic fluid pressure reaching an over-lithostatic pressure indicated thermal pressurization during a seismic event. Our quantitative constraints indicate that fluid overpressures of about 6.7–11.3 MPa above the minimum requirement for the formation of extensional veins in a reverse fault stress regime were achieved at the time of the seismic event. The unstable condition with 37.The absolute value of the fluid pressure ratio was also estimated using the constrained t A Fig. , which e in Eqs.  and 15)\document37. Our results also quantified the fluid pressure drop from the maximum to minimum fluid pressure after a seismic event, which was approximately 42 MPa from point A to point B in Fig. However, our findings show that the fluid pressures in the reverse and normal fault stress regimes differ. The fluid pressure in the reverse fault stress regime was higher than that in the normal fault stress regime Fig. . The sma31 when the differential stress becomes larger than 13. Shear veins were observed close to the shear zone within the mélange in this study area, although the relationship between the shear veins in the mélange and the shear zone below the mélange needs to be examined carefully.In our study, we constrained fluid pressure variations only in the extensional veins along a seismic fault, but we cannot rule out the possibility that the fluid pressure can decrease further after an earthquake to a level where extensional veins cannot form but instead promote the formation of shear veins32.In summary we proposed new methods to constrain the depth, rock tensile strength, and dynamic fluid pressure variations from geological data. Our study successfully constrained the dynamic change in fluid pressure quantitatively in a seismic cycle, both during the increase stage caused by thermal pressurization and during the decrease stage caused by fault-valve behavior in a seismogenic fault zone. To fully evaluate fluid pressure fluctuations in seismogenic fault zones, investigations on other modes of failure, such as shear veins and hybrid extensional shear veins, must be expanded16. The software GArcmB16 (http://bs.kueps.kyoto-u.ac.jp/tsg/software/GArcmB/) was used for the mixed Bingham statistics. The software fits the dataset with Bingham distributions and statistically identifies multiple Bingham distributions when multiple stress states are mixed in the network veins. Each Bingham distribution provides the principal stress orientations, and concentration parameters, 16 , for the driving pressure ratio  and 42. Hence, extensional veins were formed whenBrittle-rock failure is controlled by coefficients of internal friction, differential stress, and rock tensile strength (In the case of the normal fault stress regime, the maximum principal stress is equal to the vertical stress. Therefore, the minimum fluid pressure and vertical stress during the extensional vein formation under a normal fault stress regime can be approximated as follows:Because the minimum principal stress in a reverse fault stress regime is equal to the vertical stress, the fluid pressure during extensional vein formation can be expressed as13 is defined as:The fluid overpressure (from Eq. . The flu13 as follows:The driving fluid pressure ratio (d in Eq. , and it We can thus express ging Eq.  as folloIn the case of a normal fault stress regime, the minimum principal stress can be substituted with mentclasspt{minimaIn a reverse fault stress regime, because the minimum principal stress is equal to the vertical stress, the fluid pressure during extensional vein formation can be expressed using Eq.  as15\docSupplementary Information.


Testicular tumours are the most common cancer found in young men, and germ cell tumours account for the majority. This article describes the use of conventional ultrasound in germ cell tumours and how to perform the ultrasound, including how to improve patient position and scanning technique. The classification of germ cell tumours is also provided, and the salient features of different types of tumour are described. Some pitfalls and difficulties of ultrasound are discussed, as well as other uses of ultrasound in this group of patients.Testicular tumours are the most common tumours found in young males and germ cell tumours account for 95% of testicular tumours. Ultrasound is the first-line radiological investigation for imaging of the testis. This article outlines how to undertake an ultrasound examination, including optimal patient position, scanning technique and imaging parameters. Classification of germ cell tumours is provided, and salient imaging features of different tumours are described. Difficulties and pitfalls of ultrasound are described, including tumours found after a trauma presentation, orchitis causing diagnostic difficulties and imaging of small testicular lesions. Other uses of ultrasound are outlined, including looking for a primary testicular tumour following the discovery of retroperitoneal lymph nodes, imaging when tumour-makers increase, local recurrence in the scrotum, and for solid organ biopsy in metastatic disease. Conclusion: Ultrasound remains the first-line of investigation for imaging of the testis, and conventional ultrasound still plays a large role in imaging. On ultrasound alone, accurate morphological characterisation of tumours remains a challenge, despite the imaging features that can be seen in different tumour types. Therefore, histology following orchidectomy of a germ cell tumour remains the gold standard for accurate tumour characterisation. Testicular tumours are the most common tumours in young males, and there is a peak incidence between 25 and 34 years . Germ ceSeveral risk factors are known, including previous undescended testis, orchidopexy, family history, previous contralateral testicular tumour, and syndromes, including Klinefelter syndrome and dysplastic nevus syndrome. Previous studies suggest that 5–20% of patients with a previous history of an undescended testis will develop a tumour in the contralateral testis , and havTesticular microlithiasis was previously thought to be a risk factor, but newer evidence has shown that it is only a risk factor for a testicular tumour in the presence of another independent risk factor. Ultrasound is useful for surveillance in this group of patients and is recommended annually until the age of 55 years, following which regular self-examination is advised . If testUltrasound (US) is the first-line radiological examination for imaging of the testes due to its high sensitivity, patient acceptance and relatively low cost . UltrasoUltrasound is very useful in distinguishing intratesticular from extratesticular lesions. Ultrasound is able to confirm the presence of an intratesticular mass and is mainly used for identification and initial characterisation. Extratesticular lesions are much less commonly malignant in nature than an intratesticular lesion , with apThe differentiation of different types of germ cell tumour is challenging using ultrasound, and recent research suggests that tumour morphological features can be suggestive of a particular type of germ cell tumour . AdditioRecent advances in ultrasound include the use of elastography and contrast-enhanced ultrasound.This narrative review focuses on the use of conventional ultrasound in germ cell tumours, and features as a part of a special edition on testicular tumour imaging for this journal.This is an important topic, as conventional ultrasound is used routinely as the first-line investigation for suspected testicular malignancy; therefore, a good understanding of how to perform and interpret ultrasound is needed.The database PubMed was used to search for previously published articles using relevant key words such as ‘testicular germ cell tumours’, ‘seminoma’, ‘teratoma’ ‘ultrasound’, with the applicable papers reviewed for the article. This paper is written as a narrative review in order to include both the relevant literature as well as knowledge and experience from the authors in their routine clinical practice.Before performing the ultrasound, it is recommended that the operator questions the patient about where they have noticed symptoms, what they have felt on palpation and to find out the exact location of the abnormality. If a lesion cannot be identified during the ultrasound examination, it is useful to ask the patient to demonstrate exactly where they can feel the abnormality, and then perform the ultrasound directly in that location.Testicular ultrasound should be performed with the patient in the supine position, with the penis placed onto the anterior abdominal wall so that the scrotum is clearly visible, and the patient is as comfortable as possible. A pad or cushion can be placed under the scrotum in order to elevate the contents and make the ultrasound assessment of all the scrotal structures easier to perform .A high-frequency linear transducer (7–14 Hz) should be used, and a systematic approach is required. Ideally the gel should be warmed prior to the investigation, and enough gel should be used to ensure no gas is trapped in the skin folds of the scrotum. If gel is left on the scrotal skin and the examination is too timely, the gel can dry and become trapped in between scrotal folds and hair, causing some artefacts that can make the images difficult to interpret .Both testes should be evaluated in longitudinal and transverse planes with B-mode ultrasound . Colour The asymptomatic side should be scanned first in order to allow the appropriate setting of the gain parameters when performing a direct comparison between the two testes. The careful examination of the asymptomatic side is particularly important if a testicular tumour is seen, to exclude a synchronous tumour, although the risk of simultaneous bilateral tumours is low, between 1–5% . It is iAny mass that is seen within the testis should be clearly examined using 2D ultrasound, and colour Doppler should be applied to assess for the vascularity. If a lesion is seen, further images should be taken and the lesion carefully evaluated to look for evidence of cystic change, haemorrhage, calcification, and to assess the overall echogenicity and margins of the lesion.If there is a large hydrocele and the testes cannot be clearly identified, changing to a curvilinear probe is recommended as this will often help identify the testes, although the resolution is not as clear.Images should be optimised by using the whole screen on the ultrasound machine and ensuring that the image is in the centre of the screen. Labelling of the images should be performed prior to saving any image, and it is imperative to ensure that the correct side marker is in place.The ultrasound report should clearly state the side of the lesion, along with specific imaging features, including size, location within the testis, vascularity, presence of calcification, solid and cystic components. The appearance of the contralateral testis should also be documented, including the presence of microlithiasis.) [Germ cell tumours originate from spermatogenic cells and account for 90–95% of primary testicular tumours ,19, wher) .Non-seminomatous lesions include pure embryonal carcinoma, teratoma, yolk sac tumour and choriocarcinoma. Tumours are often a combination of multiple cell types, termed mixed germ cell tumours.In 2016, the WHO updated its classification system of germ cell tumours. It introduced a new title for the precursor lesion: germ cell neoplasm in situ (GCNIS) [The accurate diagnosis of the tumour type has significant implications both prognostically and therapeutically but can be difficult to ascertain using ultrasound. Studies have been conducted to predict the type and size of testicular tumours, and there are some features that are more suggestive of seminomatous tumours compared with NSGCT .Seminomas are the most common type of germ cell tumour, comprising 40–50% of GCTs, with a peak incidence between 35–45 years . At presOn ultrasound, seminomas are usually homogeneous and of low echogenicity . They maThis comprises all germ cell tumours that are not seminomas, and these may be found as isolated tumours, or in combination as a mixed-germ cell tumour. The peak incidence is earlier than seminomas, between 20–35 years, where they are more likely to present with advanced disease . On ultrIn comparison to seminomas, embryonal cell carcinomas are more aggressive . These tPure teratomas in post pubertal males are rare and are more commonly seen in children . They arIn the pure form, yolk sac tumours predominately affect children under the age of 2 and are rarely seen in post-pubertal males ,25. TheyChoriocarcinomas are the most aggressive of all the NSGCTs . It is cPatients may present following trauma, and a previously unknown testicular tumour may be found incidentally during the ultrasound examination. The appearance may mimic a haematoma on grey-scale ultrasound ,27. VascRupture of the testis due to an unknown testicular tumour has also been described ; therefoThe testes can be hypervascular in the presence of orchitis, which can make interpretation difficult. Cases have been described in the literature where patients presented with an enlarged, heterogeneous, hypervascular testis on ultrasound and it was difficult to distinguish a tumour from orchitis on the initial ultrasound  Figure . TubercuSmall (<5mm) testicular lesions can be challenging as they can be seen as an incidental finding on ultrasound due to the improved resolution of ultrasound transducers . PreviouIf a patient is found to have a retroperitoneal nodal mass on cross-sectional imaging, or a primary retroperitoneal tumour, ultrasound of both testes is advised to ensure there is no testicular tumour, including a previous burned-out tumour . Burned-Ultrasound should be performed prior to a retroperitoneal nodal mass being biopsied, but in clinical practice the biopsy often takes place before a testicular ultrasound has been performed.Ultrasound can be performed in patients with a previous germ cell tumour whose tumour markers begin to increase to ensure that there is no tumour in the residual contralateral testis. The 15-year risk of developing a germ cell tumour in the contralateral testis is reported as approximately 1.2% . UltrasoPatients may develop local recurrence in the scrotum, which may be felt as a palpable lump, and ultrasound can be used to assess any new lesion. This may be due to direct disease recurrence or nodal disease . This isIn a patient presenting with metastatic disease who needs to start a chemotherapy regime prior to primary orchidectomy, ultrasound can be useful to acquire a specimen for histology, particularly in the presence of a liver metastasis . PrimaryUltrasound remains the first-line investigation for a patient presenting with a palpable scrotal lump and can often provide immediate reassurance to the patients when a benign lesion, such as an epididymal cyst, is confirmed. Many departments can provide access to ultrasound without a long delay, ensuring that patients with a germ cell tumour can have the lesion identified early, hopefully before nodal or metastatic disease has developed.B-mode and colour Doppler ultrasound remain the conventional ultrasound imaging techniques and identify the majority of intratesticular and extratesticular lesions. The majority of intratesticular lesions that demonstrate increased vascularity are malignant. There are some mimics of malignant tumours, including haematomas, infarcts, congenital adrenals rests, abscesses and epidermoid cysts, that may be mistaken for a malignant tumour on ultrasound. If there is a history of trauma or recent infection, repeat ultrasound should be recommended for further evaluation of the visualised abnormality to ensure that there is no underlying testicular tumour.Characterising testicular lesions with ultrasound remains a challenge, particularly in NSGCT which are often of mixed cell types. There are features that can be suggestive of one type of tumour, but histology remains the gold standard in giving a formal tumour composition report. Higher resolution ultrasound machines may enable the further characterisation of intratesticular lesions and further studies can be performed to correlate ultrasound and histological findings. Newer ultrasound techniques, including elastography , contrasWith advancing ultrasound techniques and constantly improving ultrasound equipment, morphological characterisation may be more possible in the future. However, patients with suspected testicular tumours on ultrasound are likely to undergo orchidectomy, and many departments can provide a histological report with accurate morphological information about the tumours in a timely manner, allowing suitable treatment to commence without patient harm. Characterising a tumour as mixed cell is unlikely to be possible with ultrasound alone; therefore, histological examination is likely to remain the gold standard investigation for tumour characterisation.Ultrasound remains the first-line investigation for patients presenting with a suspected tumour. Identification of a tumour allows the clinicians to undertake further management, including the measurement of tumour markers, orchidectomy and staging examinations. However, morphological characterisation remains a challenge on ultrasound alone despite imaging features that can be seen in different tumour types. With advancing ultrasound techniques and improved images and equipment, morphological characterisation may continue to improve in the future and is an area where further research can be performed.


Understanding the pathophysiology of fluid distribution in acute high-risk abdominal (AHA) surgery is essential in optimizing fluid management. There is currently no data on the time course and haemodynamic implications of fluid distribution in the perioperative period and the differences between the surgical pathologies.Seventy-three patients undergoing surgery for intestinal obstruction, perforated viscus, and anastomotic leakage within a well-defined perioperative regime, including intraoperative goal-directed therapy, were included in this prospective, observational study. From 0 to 120 h, we measured body fluid volumes and hydration status by bioimpedance spectroscopy (BIA), fluid balance (input vs. output), preload dependency defined as a > 10% increase in stroke volume after preoperative fluid challenge, and post-operatively evaluated by passive leg raise.We observed a progressive increase in fluid balance and extracellular volume throughout the study, irrespective of surgical diagnosis. BIA measured variables indicated post-operative overhydration in 36% of the patients, increasing to 50% on the 5th post-operative day, coinciding with a progressive increase of preload dependency, from 12% immediately post-operatively to 58% on the 5th post-operative day and irrespective of surgical diagnosis. Patients with overhydration were less haemodynamically stable than those with normo- or dehydration.Despite increased fluid balance and extracellular volumes, preload dependency increased progressively during the post-operative period. Our observations indicate a post-operative physiological incoherence between changes in the extracellular volume compartment and inadequate physiological preload control in patients undergoing AHA surgery. Considering the increasing overhydration during the observational period, our findings show that an indiscriminate correction of preload dependency with intravenous fluid bolus could lead to overhydration.Trial registration clinicaltrials.gov. (NCT03997721), Registered 23 May 2019, first participant enrolled 01 June 2019.The online version contains supplementary material available at 10.1186/s13054-023-04309-9. Inadequate fluid management after initial haemodynamic resuscitation can have detrimental consequences . SeveralThere is some evidence  that theThe term acute high-risk abdominal (AHA) surgery , 13 encoAs such, the assessment of hydration status in AHA surgery and consequent fluid treatment are still complex and require an in-depth knowledge of body fluid homeostasis to establish a strategy that optimizes tissue perfusion and identifies the transition from necessary fluid resuscitation to harmful fluid volume accumulation , 19. AddThe present study aimed to assess the perioperative fluid changes and haemodynamics in intestinal obstruction, perforated viscus, and anastomotic leakage following elective surgery within a goal-directed therapy approach.This was a single-centre, prospective observational cohort study from 01–06-2019 to 25–02-2021 at the department of Anaesthesiology and Intensive Care and the department of Gastrointestinal Surgery at Hvidovre University Hospital. The ethics committee approved the study (H-19010653), The Danish Data Protection Agency (VD-2019-121) and registered at clinicaltrials.gov. (NCT03997721). We followed Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.After verbal and written consent, we included all adults (18 y/o or over) undergoing AHA surgery for primary intestinal obstruction , perforated viscus , and anastomotic leakage following elective surgery.Intestinal ischemia, abdominal bleeding, reoperations not including anastomotic leakage following elective surgery, and subacute surgeries  were excluded. Elective surgeries converted intraoperatively to acute were also excluded. Patients with a negative find  missing data on fluid administration, vasopressor and inotropes administration, plasma electrolyte levels, and patients transferred to another hospital immediately following surgery were also excluded Fig. .Fig. 1FlThe primary outcome was to describe the hydration status and fluid distribution (including the association with registered volume administration) measured by bioimpedance spectroscopy analysis (BIA) during the early perioperative period in patients with intestinal obstruction vs. perforated viscus vs. anastomotic leakage following elective surgery. Secondarily, we wanted to explore the association between perioperative haemodynamics, and hydration status measured by BIA.At this surgical centre, a well-established multimodal standardized protocol is applied to patients undergoing AHA surgery , followiAll study-related measurements were performed by the primary study investigator, and attending physicians were unaware of the results. After initial patient triage and diagnostic workup leading to a decision to operate, the study protocol was initiated. Figure BIA assesses body composition and estimates total body water (TBW) and extracellular water (ECW) volumes based on the tissue’s capacity to conduct electrical impulses .The BIA device displays absolute fluid overload/overhydration (AFO), the difference between normal, expected ECW and the measured ECW, expressed in litres, as well as relative fluid overload/overhydration (RFO), absolute fluid overload/extracellular water ratio (AFO/ECW), expressed in percentages. A negative AFO indicates underhydration, while a positive one indicates overhydration. Based on RFO values, overhydration was defined as RFO > 15% , 23. BIAPreload dependencyPreoperative preload dependency was assessed by the initial fluid challenge, performed routinely during the resuscitation phase before general anaesthesia and neuraxial analgesia, with 250 mL human albumin solution as a bolus over 5 min. Preload dependency was defined as a > 10% increase in stroke volume [e volume .post-operative preload dependency was assessed by the passive leg raise manoeuvre , and theStroke volume-guided fluid management or passive leg raise measurements were not a part of standard post-operative routine and were regarded purely as a research parameter. No treating physician or nurse were at any time aware of the results, thereby not influencing the post-operative fluid management. It is important to underline that no fluid was administered as a direct consequence of the result of the PLR manoeuvre. Post-operative fluid administration was done at the discretion of the treating physician with no interference from the primary investigator.Daily fluid balance was defined as the difference between total input  and total output , not including insensible losses. Cumulated fluid balance from 06.00 to 06.00 the following morning was calculated as the algebraic sum of daily fluid balance during the first five post-operative days. Fluid balance was determined from six o’clock in the morning from the previous day.Haemodynamic and bioimpedance variables were collected at; baseline (before surgery) and 6 h after surgery. The same procedure was applied on the 1st, 3rd, and 5th post-operative day.The following variables were recorded upon inclusion: demographic, clinical history (comorbidities), American Society of Anaesthesiology classification, ECOG performance score , CarlsonBased on a recent study , we estiData are presented by descriptive statistics (nonparametric distribution: medians with 25th–75th inter-quartile ranges (IQR) and range, normal distribution means with 95% CI and range). Normal distribution was assessed from Q–Q plots and histograms and Kolmogorov–Smirnoff's test. Categorical data were analysed using the chi-square test. Continuous data were analysed with Kruskal–Wallis test. The unpaired t test or Mann–Whitney test was used to compare data between the groups. Univariate analysis for association with outcome was applied.p value at a 0.05 level of significance. All analyses were performed using R statistical software. (www.r-project.org).All statistical assessments were done by a two-sided test using a From 01-06-2019 to 25-02-2021, 275 patients were assessed for eligibility, and 202 patients did not meet the inclusion criteria, where 34 were not included due to logistic reasons   Fig. A, and thp < 0.001) than before surgery. This was consistent for all 3 groups . Absolute overhydration increased significantly by 1.77 ± 1.4 L (p < 0.0001), from 0.68 ± 2.5 L preoperatively to 2.4 L ± 2.7 post-operatively, equivalent to a significant 10% rise in ECW. Total body water and intracellular water increased without reaching statistical significance .Table p = 0.001. Inflammatory response peaked at 1st POD in both intestinal obstruction and perforated viscus IO and PV, with ΔCRP increase 80 ± 104 mmol/L and ΔCRP 119 ± 119 mmol/L (p < 0.001), respectively. The C-reactive protein remained unchanged (ΔCRP 1 ± 104 mmol/L) in patients with anastomotic leakage, beginning a downward trajectory on 3rd POD , patients with perforated viscus had a significantly higher need for ultrasound-guided pleural drainage (39%) than intestinal obstruction (4%) and anastomotic leakage (10%), p = 0.048. Also, 35% of the patients with anastomotic leakage were admitted to the ICU due to respiratory failure, compared to intestinal obstruction (4%) and perforated viscus (15%), without reaching statistical significance (p = 0.105).The incidence of post-operative major complications including death was significantly higher in patients with perforated viscus (65%), than in patients with intestinal obstruction (26%) and anastomotic leakage 35%), 5%, p = 0During the first five days after AHA surgery, we found progressive overhydration, measured by bioimpedance spectroscopy, irrespective of surgical diagnosis. Simultaneously, we observed a progressive increase in preload dependency, as evaluated by passive leg raise, again irrespective of surgical diagnosis.Overhydration was most prevalent in patients with perforated viscus compared to intestinal obstruction and anastomotic leakage, with a persistent increase in extracellular volume coinciding with fluid administration. The increase in extracellular volume was present irrespective of diagnosis, although most pronounced in patients with perforated viscus. Interestingly, intracellular volume decreased for patients with intestinal obstruction but remained unchanged or slightly increased for perforated viscus and anastomotic leakage.There is extensive literature on the perioperative fluid status and the impact of overhydration on mortality in critically ill patients –3. HowevSince patients undergoing AHA surgery may have prolonged derangement of cardiovascular, pulmonary, and gastrointestinal function for days to weeks after primary surgery, judicious fluid replacement is needed to prevent multi-organ failure. In this study, we found that patients with overhydration were less haemodynamically stable, with significantly higher cardiac biomarker proBNP as well as incidence of septic shock, with no difference in preoperative cardiac pathology  anaesthesia suppress the sympathetic tone, reducing preload and afterload, potentially inducing or amplifying preload dependency. The effect of an epidural is greatest in the initial post-operative period, where we found no or very low incidence of preload dependency Fig. .Still, post-operative vasoplegia due to continuous neuraxial blockage and opioid therapy, combined with post-operative inflammatory response, should be considered when assessing preload dependency in a surgical ward. Considering the increasing overhydration during the observational period, our findings show that if we were to apply correction of preload dependency with a fluid bolus as a primary basis for post-operative fluid therapy, as practiced in GDT protocols, we might create unnecessary overhydration.The inflammatory response was on the downward trajectory by 5th POD Fig. , but theDuring the post-operative period, preload dependency was assessed by passive leg raise, which is a way of challenging preload without administering fluid and thereby avoiding unnecessary fluid administration, provided cardiac output monitoring . SeveralOur study has several strengths. This was the first study to assess the fluid status and fluid shifts in patients undergoing AHA surgery, a group equated to the international term emergency laparotomy, beyond the immediate intra- and post-operative period while considering the fundamental pathophysiological differences of diagnosis AHA surgery. This study suggests that specific fluid resuscitation strategies should depend on the diagnosis and highlight the discussion about the place of vasoconstriction therapy in the context of GDT protocols , 42.Limitations of the study include a single-centre study and thus prone to inclusion bias. However, it was a prospective study, and the patient enrolment was unselected. Our results indicate a significant variation in fluid administration, compared to elective surgery, and a considerable amount of fluid is administered. However, there were no data on the indications for post-operative fluid management, which would have been important.Bioimpedance spectroscopy fluid analysis has been validated in several studies evaluating different patient populations, both elective and emergent , 43, 44.In conclusion, despite progressive overhydration throughout the perioperative period, post-operative preload dependency assessed by PLR increased steadily in patients undergoing AHA surgery, indicating a physiological incoherence between fluid status and preload dependence, where patients appeared to be volume deficient but still overhydrated. Considering the increasing overhydration during the observational period, our findings show that an indiscriminate correction of preload dependency with intravenous fluid bolus could lead to overhydration.Additional file 1. Appendix 1: Preload dependency and hydration status in acute high-risk abdominal surgery. RFO: relative fluid overload (measured by Bioimpedance spectroscopy)—the absolute fluid overload/extracellular water ratio (AFO/ECW), expressed in percentages; normo- or dehydration: RFO < 15%; overhydration: RFO > 15%; Preload dependency defined as stroke volume increase during fluid challenge or passive leg raise manoeuvre.Additional file 2. Appendix 2: Changes in haemodynamic variables during the observational period stratified according to hydration status. RFO: relative fluid overload (measured by Bioimpedance spectroscopy)—the absolute fluid overload / extracellular water ratio (AFO/ECW), expressed in percentages; normo- or dehydration: RFO < 15%; overhydration: RFO > 15%; POD: post-operative day; *p<0.05.Additional file 3. Appendix 3: Postoperative preload dependency stratified according to the days with epidural analgesia.


The purpose of this research was to see how the physicochemical properties and porosity of matrix tablets containing various types of hydroxypropyl methylcellulose (HPMC) K series affected the release of propranolol hydrochloride (PNL). PNL is a class I drug (high solubility and permeability) according to the Biopharmaceutics Classification System (BCS), making it an excellent model drug used for studying extended-release drug products. The direct compression method was used to prepare the HPMC-based matrix tablets. PNL and the excipients were found to be compatible using Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), and differential scanning calorimetry (DSC). The surfaces of all the compressed HPMC-based matrix tablets were rough, with accumulated particles and small holes. The compressed HPMC-based matrix tablet porosity was also determined by using mercury porosimetry. The compressed HPMC-based matrix tablets made of low viscosity HPMC had tiny pores (diameter < 0.01 μm). The shorter polymeric chains are more prone to deformation, resulting in a small pore proportion. The compressed HPMC-based matrix tablets sustained the release of PNL for over 12 h. The release exponent values (n), which reflect the release mechanism of the drug from the tablets, ranged from 0.476 to 0.497. These values indicated that the release was governed by anomalous transport. The compressed HPMC-based matrix tablets have the potential for a sustained release of PNL. Hydrophilic matrices are monolithic systems of a matrix containing one or more hydrophilic excipients and an active pharmaceutical ingredient . From a Hydroxypropyl methylcellulose (HPMC), also called hypromellose, has a cellulosic backbone that is partly substituted with methoxy and hydroxypropoxy groups. It is an enzyme-resistant, water-soluble hydrophilic polymer that is stable over a pH range of 3.0–11.0 . HPMC isThis study used different HPMC viscosity grades to prepare the compressed HPMC-based matrix tablets at the constant compaction force, and the manufactured tablets were characterized. Propranolol HCl (PNL), a beta-blocker, was used as a model drug. PNL is a class I drug (high solubility and permeability) according to the Biopharmaceutics Classification System (BCS), making it an excellent model drug used for the study of extended-release drug products ,22. The ®) such as K4M (H-K4M), K15M (H-K15M), and K100M (H-K100M) were manufactured by Dow Chemical Company  and kindly supported by Rama Production Co., Ltd. . The viscosity of K4M, K15M, and K100M is 4000, 15,000, and 100,000 mPa·s, respectively [Various grades of HPMC (Methocelectively . PNL wasca. 28 °C), and humidity was around 35 %.Preparing compressed HPMC-based matrix tablets via direct compression was adapted from a previous study in Kraisit P. . PharmacThe top view of the compressed HPMC-based matrix tablets and the broken-tablets side view were analyzed morphologically using SEM  at a 20 kV acceleration voltage. Prior to obtaining the micrographs, the tablets were adhered to a metal stub with double-sided adhesive tape and covered with a fine gold layer under a vacuum.−1 at a resolution of 4 cm−1.The FTIR spectra of samples were determined using FTIR spectrophotometer . The sample was ground, blended with KBr powder, and compressed at a pressure of 5 tons using a hydraulic press. The disc was placed in a sample holder and scanned from 4000 to 400 cmThe samples’ X-ray diffraction patterns were determined at 30 kV and 15 mA using a MiniFlex II . The relative intensities were determined over a 5°–45° 2θ range using a Cu Kα radiation wavelength of 1.5406 Å.The thermal characteristics of the samples were determined using the DSC 8000 . Two to five mg of sample were accurately weighed and sealed in a solid aluminum pan. The measurement temperature ranged from 30 to 300 °C, with a heating rate of 10 °C/min. The nitrogen purge rate was set to 20 mL/min for the analysis.The total pore surface area, mean pore diameter, and pore diameter were determined using a Mercury intrusion porosimeter  operating at pressures ranging from 0.5 to 60,000 psi. Each compressed HPMC-based matrix tablet was removed from a vacuum desiccator and precisely weighed with an analytical balance. The tablet was placed into a penetrometer and filled with mercury. tot) was calculated by Equation (1) as follows [−1), Vtot is the total intrusion volume of mercury (mL g−1), γ the surface tension of mercury (485 dynes cm−1), and θ the contact angle of mercury (130°). Total pore surface area (A follows :(1)Atot=m) was calculated by Equation (2) as follows [Mean pore diameter (D follows :(2)Dm=4·Pore diameter (D) was calculated by Equation (3) as follows :(3)D=−4γThe 50 percent value derived from cumulative pore size distribution data were used to calculate the median pore diameter .n = 6).The basket method at 50 rpm was used to examine the release of PNL from the compressed HPMC-based matrix tablets. The dissolution medium in each vessel used in the release testing was 900 mL of 0.05 M, pH 4.5 of phosphate buffer solution, and kept at 37 °C. The release medium (5 mL) was removed and replaced with an equal amount of fresh dissolution medium at the specified times . PNL was analyzed by spectrophotometry at 320 nm , and PNL was calculated as a percentage of total PNL released , kH denotes the Higuchi constant k denotes the Korsmeyer–Peppas constant, and n denotes the drug release mechanism’s release exponent. The drug release mechanism was predicted using the value of n, and the equations with the highest R2 were chosen as the best mathematical release model.The compressed HPMC-based matrix tablets‘ drug release kinetics were compared to Higuchi and Korsmeyer–Peppas equations (Equations (4) and (5), respectively).p-values less than 0.05 considered significant.All determinations were performed in at least triplicate. The data are presented as mean values ± SD. The deviation between groups was determined using one-way analysis of variance (ANOVA), with −1 represented the signals from C–O–C stretches, secondary alcohols, and methoxy groups in HPMC [−1 and the characteristic peak at 2934 cm−1 representing the presence of O–H stretching and the vibration of the C–H group, respectively [−1, the vibration of the secondary amine group at 2964 cm−1, and the C–O–C stretching in aryl alkyl ether at 1107 cm−1 [−1 can be assigned to the O–H stretching modes of the crystallization water in the dibasic calcium phosphate dihydrate molecule [FT-IR spectroscopy was used to look for possible interactions between PNL and the excipients in tablet formulations. The FT-IR spectra shown in  in HPMC . The FT-107 cm−1 . For allmolecule . The wavA DSC investigation was conducted on the PNL sample, powder of various HPMC grades, and each tablet formulation. According to DSC thermograms , all usePXRD results of pure PNL, powder of various HPMC grades, and each tablet formulation were given in The scanning electron microscopy (SEM) imaging technique can provide data on the surface morphology of the tablet’s outer surface or cross-sections ,38. The Mercury porosimetry is based on mercury penetration into the sample . The meaThe pore size distribution curves can be represented in the following two formats: pore diameter vs. log differential intrusion for a broad pore distribution range and pore diameter vs. differential intrusion for capillary pore distribution .tot values of Tab-K4M, Tab-K15M, Tab-K100M, and Tab-Mixed were 5.840, 6.917, 1.136, and 1.156 m2/g, respectively. The number of pores with a minimal diameter significantly impacts the total pore area determined by mercury porosimetry [m of Tab-K4M, Tab-K15M, Tab-K100M, and Tab-Mixed were 0.06521, 0.06433, 0.30179, and 0.32479 μm, respectively. The smaller pores substantially influence the mean pore size [The Aore size . The meaore size .2 value was observed for the Korsmeyer-Peppas model, indicating that the release data of all HPMC matrices were best fitted with the Korsmeyer–Peppas model. The Korsmeyer–Peppas model provides insight into the drug release mechanism in the swellable polymeric matrices [The release profiles of PNL and the total amount of PNL released at each time point are shown in matrices . Althougmatrices . The promatrices . Therefo25), 50% (T50), and 75% (T75) of PNL from various matrix tablet formulations. The differences in the release of PNL from the distinct compressed HPMC-based matrix tablet formulations were relatively small. However, according to release parameters T50 and T75, the release of PNL from Tab-K100M was significantly faster than the others (p < 0.05). The difference in T25 values of Tab-K100M and Tab-Mixed was not statistically significant (p > 0.05), while the T25 values of Tab-K4M and Tab-K15M were significantly higher than that of Tab-K100M (p < 0.05). Several research works reported that the pore size influenced the drug release, and the matrix with the greatest pore diameter had the fastest drug release [ release . When a  release . The dru release . However50 and T75, PNL release from Tab-K100M was significantly faster than that of the other formulations. An anomalous controlled-release mechanism governed the sustained PNL release from the compressed HPMC-based matrix tablets. As a result, the various types of HPMC could be beneficial for the extended release of PNL from compressed HPMC-based matrix tablets. The preparation of HPMC-based matrix tablets is a potential and economically favorable way of developing oral extended-release matrices in the future. Compressed HPMC-based matrix tablets containing PNL were manufactured by direct compression. According to a compatibility study, HPMC and chosen excipients were compatible. Mercury porosimetry was used to determine the porosity of the tablets. The tablets made up of low viscosity HPMC contain tiny pores. Shorter polymeric chains are more likely to deform during compression, resulting in a small pore proportion. PNL was released continuously for more than 12 h. The differences in PNL release between the various formulations of compressed HPMC-based matrix tablets were relatively minor. Nevertheless, based on the release parameters T


In the published article, there was an error in the Funding statement.This study was supported by the NSCF (grant number 31870520) and China Agriculture Research System of MOF and MARA (grant number CARS-37).The correct Funding statement appears below.This study was supported by the NSCF (grant number 31872520) and China Agriculture Research System of MOF and MARA (grant number CARS-37).The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


The second paragraph of Acknowledgments should read as follows: “We gratefully acknowledge funding from the following sources: Czech Science Foundation grants 20-23513S to H.H., 18-17529S to A.Z., and 20-04150Y to O.G.; Czech Ministry of Education grant OPVVV16_019/0000759; and Czech BioImaging grant LM2015062.”Volume 6, no. 3, e00327-21, 2021,


Dr. Kirsten Harvey should be included in the author byline. She should be listed as the fourteenth author, and her affiliation is: Department of Pharmacology, UCL School of Pharmacy, University College London, London, UK. Contributions of this author are as follows: Funding Acquisition and Supervision.The Funding Disclosure should contain Dr. Kirsten Harvey’s funding details. Please see the correct Funding Disclosure below:This work was supported in part by the National Institutes of Health, NIA, Intramural Research Program (MRC), by NIH extramural grant NS110188 (MJL), the Wellcome Trust grant 106805/Z/15/Z , the Medical Research Council grant MR/M00676X/1 (KH) and the NIA IRP Postdoctoral grant scheme (AM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


The movement of fluid into, through, and out of the brain plays an important role in clearing metabolic waste. However, there is controversy regarding the mechanisms driving fluid movement in the fluid-filled paravascular spaces (PVS), and whether the movement of metabolic waste in the brain extracellular space (ECS) is primarily driven by diffusion or convection. The dilation of penetrating arterioles in the brain in response to increases in neural activity (neurovascular coupling) is an attractive candidate for driving fluid circulation, as it drives deformation of the brain tissue and of the PVS around arteries, resulting in fluid movement. We simulated the effects of vasodilation on fluid movement into and out of the brain ECS using a novel poroelastic model of brain tissue. We found that arteriolar dilations could drive convective flow through the ECS radially outward from the arteriole, and that this flow is sensitive to the dynamics of the dilation. Simulations of sleep-like conditions, with larger vasodilations and increased extracellular volume in the brain showed enhanced movement of fluid from the PVS into the ECS. Our simulations suggest that both sensory-evoked and sleep-related arteriolar dilations can drive convective flow of cerebrospinal fluid not just in the PVS, but also into the ECS through the PVS around arterioles.The online version contains supplementary material available at 10.1186/s12987-022-00326-y. The circulation of cerebrospinal fluid (CSF) is thought to play the important role of clearing harmful solutes like amyloid-β from the brain , 119. ThSeveral mathematical models have attempted to understand the nature and drivers of fluid and solute transport through the PVS , 116. Ho., . [. [In this study, we improve on our previous modeling of transport through the PVS and brain by using 3D poroelastic models. Poroelastic models based on mixture theory , 20, 13 ., . , is the ., . [.  consider., . [. , 62. Our., . [. . This coUsing 3D poroelastic models, we considered two modes of solute transport through the PVS: dispersion and convection. Dispersion could improve solute transport over diffusion by oscillatory fluid exchange between the PVS and the SAS or the PVS and the ECS, while convection can drive directional fluid and solute transport from the SAS to the ECS, via the PVS. Several published models of transport through the PVS suggest that dispersion is the main mechanism of solute transport through the PVS , 61, 73.ransport . TherefoIn this study, we demonstrate the possibility of convective transport through the PVS driven by a combination of the non-linear flow response of the fluid spaces in the brain and asymmetry in the waveform of arterial wall motions. The possibility of directional fluid flow through the PVS has been previously explored through mathematical models and numerical simulations , 92, 116The role of arterial dilations and subsequent return to baseline during functional hyperemia in driving fluid and solute transport through the PVS will be the major focus of this study. Functional hyperemia  is the dThe geometry of the model was created to represent the anatomy of a single penetrating arteriole in the mouse cortex, while keeping the shape relatively simple. The dimensions of the model geometry are shown in Fig. entclass1pt{minimaentclass1pt{minimaillaries , 38, 51.entclass1pt{minimae cortex , 97. Theent}50μm , 18. Theent}20μm , 96, witing data , 78, 91.ing data  to avoid1 to represent the parameters in the fluid-filled spaces, and 2 to represent the parameters in the brain tissue. An incompressible Darcy-Brinkman model was used for fluid flow through porous spaces, with a mass density , \documurements , 103. Thterioles , 81 and fixation , as wellfixation . For theent}2kPa , 117 wasactivity , 61, 111The boundary conditions for the model were chosen to represent a segment of the cerebral cortex in an active region of the brain, i.e., a section of the cortex containing a dilating arteriole surrounded by other arterioles dilating with similar dynamics. This choice of boundary conditions  into the “large” CSF chambers in the cranial space (cisterna magna or the ventricles) and observe their movement , 70, 77.We started the particle tracking simulations with 243 equally spaced fluid particles (27 rings of 9 particles) in the PVS  the fluid particles moving from the PVS to the ECS compared to vasodilation with a symmetric waveform (9%) Fig. c over thThe PVS fluid penetration into the ECS is not an artifact of the directional fluid flow imposed through the pressure difference across the two ends of the SAS Fig. a. To vermentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimAn attractive hypothesis for the physiological purpose of sleep is to remove waste from the brain , 66, 122diameter , once evOur models suggest that both the increased extracellular volume (and permeability) and the larger CBV oscillations could contribute to larger PVS fluid movement into the ECS. Figure mentclass2pt{minimOur models also showed that the lower frequency arteriolar diameter changes that occur during sleep  could alHere, we simulated fluid flow in a poroelastic model of the brain during the dilations of penetrating arterioles. We found that temporally asymmetric vasodilation drove directed fluid flow, with fluid flow from the PVS into the ECS during dilation, and fluid flows from SAS into the PVS during the return to baseline diameter. This could explain the importance of the speedup of vasodilation by noradrenergic stimulation , and howThe Reynold’s number and the spatial average flow velocities through the PVS in Additional file urements , 78, 86. studies , 50, 89,Several studies have suggested that heartbeat-driven pulsations of arteries and arterioles can cause directional fluid flow through the PVS of arterioles , 56, 78.There are several ways that the model could be improved. The problem with the computational cost of the model could be addressed by implementing a stabilization techniques , 76, 85 Our results could shed a new light on some aspects of the glymphatic hypothesis of solute transport in the brain. There has been controversy whether diffusion or convection dominates in the brain , 82. OurIn contrast to the fluid pathway from the arteriolar PVS to the venular PVS proposed by the glymphatic hypothesis, our model suggests a pathway of fluid circulation, into the ECS through the PVS of arterioles and out through the surface of the brain. Although the model does not simulate the PVS of venules, the high flow resistance of the ECS and the small size of the PVS of venules compared to arteriolar PVS  suggest https://github.com/kraviteja89/poroelastic3DPVS).The geometry was created using Autodesk Inventor 2020 . The two domains \documentWithin each domain, we have five equations, Eqs. , 6), , , and 1313, that At the interface of the mostly fluid-filled spaces i.e., the SAS and the PVS with the brain parenchyma, there is a sharp change  in the volume fraction of the fluid (porosity) and the composition of the solid skeleton. Therefore, there is a sharp change in the fluid permeability and elastic modulus which could result in a sharp change in the fluid velocity and the distribution of traction between the solid and fluid components at the interface. To deal with the sharp change, the mass and traction continuity at the interface were implemented through special boundary conditions called jump conditions , 98, 99.The solid phases in both the domains are in contact with each other at the interface and therefore the solid displacement and the velocity are continuous across the interface, as indicated in Eq. . For an u et al.  showed td in Eq. :16\documtotal traction force across the interface be continuous. In Eq. total traEquations –20) are are20) an in Eq. . Using En in Eq. , 15), a, a\documn in Eq. , the tran in Eq.  and 23)\documentFor the segment of the arteriole in the SAS \documentAt the bottom face of the fluid-filled domain  has feas of Eq. , here wea in Eq. . The valy number  is concerea Eqs. , 57. A cAs shown in Additional file As mentioned earlier in the paper, we chose Peclet numbers to present some of our results even though Peclet numbers do not arise from a formal dimensional analysis of our field equations. As such, we do not view Peclet numbers as flow indices. Rather, we view them as a way, admittedly unconventional, to bring into the discussion of our result considerations on transport driven by a process of Fickian diffusion as opposed to a process of Darcy-like diffusion, promoted by the pressure gradients engendered by arterial wall motion.Two Peclet numbers were defined to compare the convective and diffusive transport driven by vasodilation. We used non-traditional definitions for the Peclet numbers to represent the flow of PVS fluid through the ECS. A characteristic length of entclass1pt{minima-V=150μm  was usedWe considered two possible pathways of fluid transport from arteriolar to venous PVS. The axial Peclet number  20% asymmetric dilation with default parameters, (c) 20% asymmetric dilation with a small (0.001 mmHg) pressure difference across the SAS, (d) 20% symmetric dilation with default parameters and (e) 40% dilation with simulated sleep state (increased ECS permeability and porosity). Negative values indicate flow in the negative-z direction and into the PVS, in the direction of blood flow. (a) shows the cross sections where the average velocity was calculated.


Salmonella (NTS) often focus on serological measures of immunity, ignoring potentially relevant oral mucosal responses. In this study we sought to assess the levels and detectability of both oral fluid and serum IgG and IgA to NTS antigens, in endemic and non-endemic populations.Immuno-epidemiological studies of orally acquired, enteric pathogens such as nontyphoidal Salmonella Typhimurium and Salmonella Enteritidis O antigen and phase 1 flagellin were assessed using Enzyme Linked Immunosorbent Assay (ELISA). Paired oral fluid and serum samples were collected from groups of 50 UK adults, Kenyan adults and Kenyan infants. Additionally, oral fluid alone was collected from 304 Kenyan individuals across a range of ages.IgG and IgA antibodies specific for Antigen-specific IgG and IgA was detectable in the oral fluid of both adults and infants. Oral fluid antibody increased with age, peaking in adulthood for both IgG and IgA but a separate peak was also observed for IgA in infants. Oral fluid and serum responses correlated for IgG but not IgA. Despite standardised collection the relationship between oral fluid volume and antibody levels varied with age and country of origin.Measurement of NTS-specific oral fluid antibody can be used to complement measurement of serum antibody. For IgA in particular, oral fluid may offer insights into how protective immunity to NTS changes as individuals transition with age, from maternal to acquired systemic and mucosal immunity. This may prove useful in helping to guide future vaccine design. •Salmonella-specific IgG and IgA was detected in the oral fluid of adults and infants.Nontyphoidal •Oral fluid antibody increases with age, peaking in adulthood.•O-antigen- and flagellin-specific oral fluid and serum IgG but not IgA responses correlate. Nontyphoidal; Oral fluid; IgG; IgA; O-antigen; Flagellin. InvasivImmuno-epidemiological studies from Africa support the concept that serum antibody acquisition with age may provide protection against invasive NTS disease , 4, whilSalmonella Typhi disease [Despite these observations, there are currently no confirmed correlates of protection against invasive NTS disease in humans. Whilst a number of studies have investigated the role of the mucosal innate immune response in protection against invasive NTS serovars , 9, few  disease , relevanOral fluid is composed of a number of fluids of different origins, each contributing differently to its antibody composition. Gingival crevicular fluid is derived from periodontal tissue located between the lower gum and cheek. It is composed of serum and locally generated materials such as tissue breakdown products, inflammatory mediators, and antibodies originating from the serum. Saliva, in contrast, originates from the parotid, submandibular and sublingual salivary glands , 17, 18.E. coli, antibody responses in the salivary glands were shown to correlate better with gut mucosal immune responses than serum responses [Specific oral fluid antibodies, in particular IgG and IgA have been identified against numerous bacterial , 22, 23 esponses , howeveresponses . Oral flesponses . In oldeesponses . Oral flIn young children, obtaining serum through venepuncture can be stressful to both child and parent. As a result, patient retention or recruitment of healthy subjects can be difficult, as parental consent is often withdrawn once their child has recovered from an illness, or is not given in the first place. The method of oral fluid collection is non-invasive, making it safer for use in individuals with acute disease such as with Ebola virus  or co-inIn this study we sought to assess the value of oral fluid for measuring NTS-specific antibody. Using IgG and IgA standardised ELISAs, we compared antibody response in both oral fluid and serum across different populations and age groups, with a focus on individuals from a representative Low- and Middle-Income Country where invasive and diarrhoeal NTS disease are endemic.22.1In the UK, paired blood and oral fluid samples were collected from 50 heathy adults at the Centre for Clinical Vaccinology and Tropical Medicine (CCVTM) in Oxford.In Kenya, sample collection was split into two phases. In phase one, paired blood and oral fluid samples were collected from 50 healthy adults and 50 healthy infants at local health facilities in the coast town of Kilifi, located north of Mombasa. In phase two oral fluid samples alone were collected from 304 individuals across five different age groups  at participant's homes. Across both Kenyan cohorts, individuals within the age-groups specified were selected randomly from four local districts  using the Kilifi Health and Demographic Surveillance System (KHDSS) .2.210 mL of blood from adults and 5 mL from infants were collected by qualified health workers into a plain Vacutainer tube and allowed to clot naturally and stored at room temperature prior to processing. In Kenya, samples were stored and transported from the field sites to the laboratory in temperature controlled cool boxes. At both sites, samples were collected in the morning or early afternoon to ensure processing the same working day in the laboratory. Blood samples were centrifuged at 600g for 10 min before serum was carefully removed and divided equally between a minimum of two 2 mL cryovials. Samples were stored at -80°C until required.https://www.malmed-oracol.co.uk/oracol-plus/). In a review of existing literature we found little standardisation in the collection and storage of oral fluid even using the same collection devices [A single oral fluid sample was collected using an Oracol+ oral swab  and phase 1 flagellin (CVD1943 FliC) from S. Enteritidis, were generated as described [2CO3 & 4.2g NaHCO3 per litre dH20, pH 9.6) and 50μL added per well to an ELISA plate. Plates were incubated overnight at 5 °C then blocked for 1 h using preformulated 1% Casein in PBS (Thermo Scientific). Human test sera were diluted in the same Casein buffer to a starting concentration of 1 in 50 and further diluted in sequential 10-fold dilutions where antibody levels were above the linear portion of the ELISA standard curve. The standard curve and internal controls were created from an in-house human control serum containing antibodies to all four antigens. The positive control was pooled serum from Malawian donors containing antibodies to all four antigens. Plates were washed using PBS containing 0.1% Tween then sera added to triplicate wells and incubated at room temperature for 2 h. Plates were again washed before addition of 50μL per well HRP secondary antibody diluted 1 in 1000  or 1 in 2000  for 1 h then washed 6 times with PBS/0.05%Tween. Plates were developed with 100μl of highest sensitivity TMB solution (AbCam) and stopped with 100μl of Stop Solution (AbCam) once the internal control reached an O.D of 1. Plates were read at 450nm and data analysed using GenV ELISA software. Internal and positive control readings were assessed to ensure they were within 20% of expected values. For plates where control readings fell outside this range, the ELISA was repeated. CVs were determined from individual serum triplicates and repeated where >20%.O-antigen, O:4,5 (CVD1925) and phase 1 flagellin (CVD19255FliC) from escribed , 32, 33.Oral fluid ELISAs were performed as for serum ELISAs but using 1% BSA in PBS as dilution buffer. Oral fluid was tested at a final dilution of 1 in 50 for IgA ELISA and 1 in 10 for IgG ELISA.2.4Data were analysed using GraphPad Prism version 9 for Windows . Groups analyses were performed using Kruskal-Wallace with Dunns multiple comparison tests. Correlations were assessed using Spearman’s Rank test.2.5For the UK, ethics approval was provided by the Medical Sciences Interdivisional Research Ethics Committee (IDREC). For Kenya, ethics approval was first provided by Oxford Tropical Research Ethics Committee (OxTREC) and by the Kenyan Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU). Informed consent was obtained from all participants or their parents or guardians.33.1Study participants for specific groups were recruited from either the UK, or Kenya in one of two phases . For UK 3.2Assay standardisation was performed using samples from the UK adult population. Oral fluid collection volumes varied significantly between individuals ranging from 60μL to 1500μL  giving final volumes of 120–3000μL after a 1 in 2 dilution of transport medium. In the Kenyan phase 1 population, smaller volumes were obtained, with initial volumes of 6μL to 800μL in adults  and 40μL to 720μL in infants , which also doubled with addition of transport medium.S. Typhimurium and S. Enteritidis) at a 1 in 2 dilution requires a minimum volume of 1200μL (150 μL per ELISA). During preliminary laboratory work, it was established that a starting assay dilution of 1 in 10 for IgG ELISAs (15μL per ELISA) and 1 in 50 dilution for IgA ELISAs (3μL per ELISA), ensured that all assays could be conducted with most available volumes, good initial measurement of detectable antibody, and excess volume for repeats. In total there were 5 Kenyan adults and 2 Kenyan children with missing data for one or more antigen due to insufficient volume. The difference between IgG and IgA was in large part due to the ease of detection of IgA in oral fluid compared with IgG.Performing eight oral fluid ELISAs  from IgA to all antigens was detected in oral fluid from all UK adults, with the exception of one adult with undetectable IgA to O:4,5. For many UK adults, even using a 1 in 10 dilution, IgG to one or more antigen was not detectable. To gauge the limits of assay sensitivity, samples with undetectable IgG at the starting assay dilution were retested at reduced dilutions of 1 in 2, where sufficient sample was available. Only for IgG, and in particular O:4,5 IgG, did we find a notable increase in detection of antibody when repeating ELISA using a dilution of 1 in 2 . It was 3.3Salmonella antibody levels using sera from UK adults, and Kenyan adults and children collected in phase 1  and S. Enteritidis . However, Kenyan adult oral fluid IgG levels to both forms of flagellin (0.21 & 0.16 A.U.) were not significantly different from levels in either UK adults or Kenyan infants. There were less Kenyan infants with detectable oral fluid flagellin IgG compared with both UK and Kenyan adults, for both S. Typhimurium flagellin (81.3% vs 92% & 100%) and S. Enteritidis flagellin (68.8% vs 88% & 89.1%).Differences in age and location had a greater impact on O-antigen antibody levels compared with flagellin antibody levels A. In conl groups  C, D. Ke3.3.2S. Typhimurium (12.42 A.U.) and S. Enteritidis flagellin IgG (17.09 A.U.) compared with both UK adults  and Kenyan Adults    G, H. Ou3.3.3S. Typhimurium flagellin IgA (19.73 A.U.) compared with both Kenyan adults  and Kenyan infants  (S. Enteritidis flagellin IgA (15.07 A.U.) compared with Kenyan infants  but not Kenyan adults (12.23 A.U.)  compared with Kenyan adults  but not Kenyan infants (6.27 A.U.) I. Despit 0.0001) K. Simila23 A.U.) L. There 3.3.4S. Typhimurium flagellin IgA (3.66 A.U.) compared with both UK adults  and Kenyan Adults    compared with both UK adults  and Kenyan infants  M. Levels<0.0001) O. A simiarisons) P. Almost3.4S. Typhimurium antigens; O:4,5  and S. Enteritidis flagellin (p= <0.0001). Oral fluid O:9 IgG was significantly higher in adults compared with infants (p < 0.0001). No significant differences were observed between older children, adult and elderly groups across all antigens. Fewer infants had detectable oral fluid IgG for NTS O-antigens  compared with NTS flagellin . Kenyan adults had similar proportions of detectable oral fluid IgG for O:4,5 (74.2%) and O:9 (72.6%). The proportion of individuals with detectable oral fluid IgG against flagellin antigens were notably higher than for O antigens but similarly comparable to one another .Oral fluid IgG and the percentage of individuals with detectable IgG to all NTS antigens gradually increases with age and peaks in adulthood D. IgG le3.6.2S. Typhimurium 88.1%; S. Enteritidis 88.1%) had detectable and on average higher IgA levels (S. Typhimurium and S. Enteritidis flagellin IgA levels were significantly higher than those observed in young children (p = 0.0057 & <0.0001 respectively) but not significantly higher than those in older children or adults. O:4,5 and O:9 IgA levels in adults were significantly higher than both younger children (p = 0.0001 for both comparisons) and older children (p < 0.0001 for both comparisons), whilst S. Typhimurium and S. Enteritidis flagellin IgA levels in adults were only significantly higher compared to younger children (p=<0.0001 for both comparisons). The proportion of Kenyan adults with detectable IgA at the peak of the response were comparably high for all antigens  and higher than those observed for IgG. A drop in IgA levels, but not the percentage of individuals with detectable IgA were observed for all antigens in the elderly group.Oral fluid IgA and the percentage of individuals with detectable IgA to all NTS antigens gradually increases with age and peaks in adulthood, but with the major exception that a large percentage of infants , and its acquisition with age has been well established [To date the potential role of serum antibody in protection against non-typhoidal ablished . In thisS. Typhimuriumserovar compared O:9 S. Enteritidis [Direct comparisons between NTS antibody levels in endemic and non-endemic populations are currently under-represented in the literature, but robust functional antibody responses have been previously observed in both populations , 37. In eritidis , 39 whiceritidis , 40, 41 Our second aim was to establish whether there was a clear relationship between oral fluid and serum NTS-specific IgG and IgA. Oral fluid and serum IgG correlated for different NTS antigen and in different subject groups across the study. This suggests IgG in the two media originates from a common source. In contrast, oral fluid and serum NTS-specific IgA did not correlate. Many infants had high levels of oral fluid IgA but absent serum IgA for a specific antigen, or high levels of serum IgA and absent oral fluid IgA. This suggests that IgA in the two media may have different age-associated origins.At least one previous study has described protective immunity associated with maternal placental NTS specific IgG . DecliniInfants and young children in endemic countries are the main targets for future NTS vaccines. The lack of sensitivity for detecting oral fluid IgG in these groups, particularly for the leading vaccine target, O-antigen, makes oral fluid NTS-specific IgG less viable as a replacement for serum NTS-specific IgG, despite good correlation between the two. If future vaccination drives high levels of oral fluid IgG or results in a change in this relationship, it could be worth revisiting oral IgG as a measurement of immunity to NTS.For other pathogens, correlations between antigen-specific IgA in oral fluid and serum have been described , 23 and Salmonella. Oral IgA derived from both serum and secretory IgA [Our results suggest that NTS-specific oral fluid IgA is best viewed as an independent measure of immunity to that in serum. It is best used to supplement the measurement of systemic IgA, but may yet prove useful as a surrogate for mucosal IgA that is more difficult to measure . As an otory IgA , may platory IgA , 51, 52,tory IgA , 20. Thitory IgA , 53, 54.tory IgA , 56. It tory IgA . Breast tory IgA , 58 and tory IgA . Despitetory IgA  or matertory IgA .There are several drawbacks to the approaches used in this study. Addressing these could provide insights and clarifications for some of the ideas presented above. Firstly, there was variability in the sensitivity of some oral fluid NTS ELISA compared to equivalent serum ELISA. For serum ELISA, use of high titre endemic pooled serum or serum from vaccinated individuals as standards are commonly used to improve sensitivity . Oral flAlternatively, we could address this issue during the sample collection step. Collection of oral fluid as its constituent salivary and crevicular components would firstly help negate the effect of salivary dilution of crevicular antibody and secondly help assess their relative contributions to total NTS-specific antibody . Whilst In this study antibody cross reactivity was also not accounted for. Cross-priming of common flagellin epitopes from different serovars expressing phase 1 and 2 antigens has been well described . In thisSalmonella- positive and negative populations or those with recent infection. There is a possibility that individuals included in this study were NTS carriers. Whilst there is a lack of data supporting a relationship between carriage and serum antibody, we cannot at this stage discount a potential impact on oral fluid antibody.Our results also highlighted limitations in our study design. Firstly we did not directly control for breast feeding before sampling infants . Recent In conclusion, we suggest a potential future role for oral fluid in the study of immunity to NTS. This should not be in isolation, but as part of a concerted effort to better study localised immunity associated with NTS sites of entry, colonisation, translocation and carriage. This study in particular highlighted that mucosal IgA and in particular secretory IgA may potentially offer valuable additional insights that complement previous work focusing on the role of systemic IgG. There are clear next steps. Collection methods need to be improved to delineate individual components, sensitivity of existing assays need to be improved and new methods developed to investigate functionality. Once established, measuring oral fluid specifically, and mucosal immunity more generally, alongside systemic immunity may help provide opportunities to measure novel correlates of protection associated with both natural- and vaccine-induced immunity that to date we have struggled to find.Sean C Elias, DPhil: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Esther Muthumbi; Alfred Mwanzu; Perpetual Wanjiku; Agnes Mutiso: Performed the experiments.Raphael Simon: Contributed reagents, materials, analysis tools or data.Calman A. MacLennan: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.GCRF Networks in Vaccines Research and Development which was co-funded by the MRC and BBSRC. BactiVac Grant Ref: BVNCP-12.This work was supported by the Bacterial Vaccines (BactiVac) Network funded by theData included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.