Escherichia coli (Ec) and Klebsiella pneumoniae (Kp). Bacteria acquire resistance to antibiotics via multiple routes, including by chromosomal mutation. In this study, spontaneous resistance to TBP among Enterobacterales, including those resistant to clinically relevant antibacterials, was assessedTebipenem (TBP), an oral carbapenem in development for treatment of complicated urinary tract infections and acute pyelonephritis, is active vs. uropathogens which produce extended-spectrum beta-lactamases (ESBLs) such as 8 CFU were plated on agar containing 4x or 8x the MIC and incubated for 24h. Resistance was confirmed by MIC testing (CLSI M07Ed11). Serial passages were performed with two Ec and two Kp isolates, each of which included a wild type and ESBL+ isolate, using TBP, MEM and ETP over a 14-day period. Whole genome sequencing was conducted and assembled mutant sequences were aligned vs. β-lactamase-encoding genes, porins OmpF/OmpK35, OmpC/OmpK36, and OmpK37 (Kp only) and penicillin binding proteins (PBPs) 1a, 1b, 2, and 3 and compared with parent strains.The frequency of resistance (FOR) to TBP, meropenem (MEM) and ertapenem (ETP) was assessed for Ec (n = 8), Kp (n = 9), and other Enterobacterales (n = 7). Approximately 10-9 to 5.54 × 10-5, with similar results obtained for all species and genotypes, including ESBL+ isolates and those with porin alterations. In the serial passage, MICs for each agent vs. four isolates over the 14-day period were ∼ five to seven 2x dilutions higher than baseline, resulting in similar MIC values to those observed in the FOR studies (0.12 to 2 µg/mL). Mutants isolated in the presence of TBP, MEM or ETP had similarly decreased susceptibility to each compound. In general, mutants selected during the single- and multi-step resistance studies showed alterations of OmpC in Ec and its homologue OmpK36 in Kp.FOR values for TBP, MEM and ETP at 4x to 8x the MIC ranged from < 6.87 × 10in vitro and was similar to MEM and ETP in both single-step and serial passage studies. Cross-resistance to TBP, MEM or ETP among mutants suggests common mechanisms contribute to elevated MICs, including through reduced permeability.These data suggest TBP has a low propensity for spontaneous resistance at 4x to 8x MIC Brian D. VanScoy, B.S., Adagio Therapeutics, Inc.: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics,: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc.: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp.: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc.: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Haley Conde, B.S., Adagio Therapeutics, Inc.: Grant/Research Support|Amplyx Pharmaceuticals, Inc: Grant/Research Support|AN2 Therapeutics,: Grant/Research Support|Antabio SAS,: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica,: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC,: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited,: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma,: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp.: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceutical: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc.: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Rodrigo E. Mendes, PhD, Spero Therapeutics: Grant/Research Support Nicole Cotroneo, B.S., Spero Therapeutics: Employee|Spero Therapeutics: Stocks/Bonds Ian A. Critchley, PhD, Spero Therapeutics: Employee|Spero Therapeutics: Stocks/Bonds Ian A. Critchley, PhD, Spero Therapeutics: Employee|Spero Therapeutics: Stocks/Bonds.


To the editor,In a recent trial including 650 COVID-19 patients with acute respiratory distress syndrome (ARDS) [The volume of fluid balance is calculated as fluid intake—fluid output. Although dozens of studies have reported that increased fluid balance volume is associated with poor prognosis in various diseases, it remains unclear which factor really matters with the prognosis: Fluid input? Fluid output or fluid balance? For instance, in the current study, the fluid intake volume within three days was quite close between the lower and higher tertiles, and the difference in fluid balance was mainly caused by the difference in urine output. However, is urine output or fluid balance the key factor in this relationship?In clinical practice, fluid intake, output and fluid balance have complex interactions. Based on our previous findings, we suggest that the fluid accumulation index, which is derived from both fluid balance and fluid intake , may play a vital role in the relationship with prognosis. For instance, studies have reported that patients with severe capillary leak may have a worse prognosis , 3. HoweFinally, Dr. Ahuja et al.’s study added important information to the current knowledge of COVID-19, and their findings are highly appreciated. We hope our hypothesis will be helpful for further research. In your journal, we recently showed that a higher cumulative fluid balance has an association with a longer duration of ventilation in patients with acute respiratory distress syndrome (ARDS) due to coronavirus disease 2019 (COVID-19), suggesting that restricted fluid management in these patients may be beneficial [Shen et al. correctly mention that fluid intake, fluid output and fluid balance have complex interactions, and that a ‘fluid accumulation index,’ an index derived from both the fluid balance and the fluid intake, may play a vital role in this association .To test this hypothesis, we reanalyzed the database of ‘Practice of Ventilation in COVID–19 patients’ (PRoVENT–COVID), the study we used to test our original hypothesis . Here, wP = 0.2853)  (Fig. The fluid accumulation index was not associated with successful liberation of ventilation, even after adjusting for potential confounders P = 0.853 (Fig.Based on these findings, we conclude that the fluid accumulation index has no added value in patients with ARDS due to COVID-19.


During the exploitation of deep and ultradeep oil and gas resources, the high-temperature problem of deep reservoirs has become a major challenge for water-based drilling fluids. In this study, a novel high-temperature-resistant filtrate reducer (LDMS) with low molecular weight was synthesized using N, N-dimethylacrylamide; sodium p-styrene sulfonate; and maleic anhydride, which can maintain the performance of a drilling fluid gel system under high temperature. Unlike the conventional high-temperature-resistant polymer filtrate reducer, LDMS does not significantly increase the viscosity and yield point of the drilling fluid gel systems. After aging at 210 °C, the filtrate volume of a drilling fluid with 2 wt% LDMS was only 8.0 mL. The mechanism of LDMS was studied by particle size distribution of a drilling fluid gel system, Zeta potential change, adsorption experiment, change of bentonite interlayer spacing, filter cake scanning electron microscope, and related theoretical analysis. The mechanism study revealed that LDMS could be adsorbed on the surface of bentonite particles in large quantities and intercalated into the interlayer of bentonite. Thus, it can improve the hydration degree of bentonite particles and the colloidal stability of the drilling fluid gel system, maintain the content of fine particles in the drilling fluid gel system, form a compact mud cake, and significantly reduce the filtrate volume of the drilling fluid gel system. Therefore, this work will promote the application of a low-molecular-weight polymer filtrate reducer in high-temperature-resistant water-based drilling fluid gel systems. Oil is an indispensable fossil energy source for human production and life. After a long period of continuous exploitation, shallow as well as conventional oil and gas resources have been gradually depleted. Shallow oil and gas resources have been unable to meet the energy demand of the rapidly developing society. Therefore, the current focus of oil and gas development has been extended from shallow reservoirs to deep and ultra-deep reservoirs. Taking China as an example, the large oil and gas reservoirs discovered in recent years are mainly distributed in deep and ultra-deep formations, where the depth of the formation generally exceeds 6000 m and the temperature at the bottom of the well is close to 200 °C ,2,3. WitDrilling fluid is a circulating fluid used in the oil and gas drilling process and meet the needs of drilling work with its multiple functions. A drilling fluid plays an important role ,6 in carResearchers have conducted massive research on improving the temperature resistance of filtrate reducers. At present, the filtrate reducers commonly used in the petroleum industry are mainly modified natural materials and synthetic polymers. Natural materials ,12,13,14Synthetic polymeric filtrate reducers are all water-soluble polymers. After the high-molecular-weight polymeric filtrate reducers were dissolved in the drilling fluid, the molecular structure was fully stretched. On the one hand, the filtrate reducers were adsorbed on the surface of bentonite particles, and retained in the pores of the filter cake by bridging, which reduced the permeability of the filter cake and reduced the filter loss of the drilling fluid. On the other hand, the filtrate reducers reduced the filter loss by significantly increasing the viscosity of the drilling fluid through the formation of a gel network structure in the drilling fluid with their irregular molecular structure. The main mechanism ,21,22 of−1 are –OH and crystal water in the product. The wavelength of 2934 cm−1 is the antisymmetric stretching vibration absorption peak of the C–H bond in the methyl group. The peaks at 1574 and 1406 cm−1 are the antisymmetric and symmetric stretching vibration absorption peaks of –COO− in the carboxyl group. The peaks at 1040 and 1010 cm−1 are the symmetric stretching vibration absorption peaks of –SO3− linked to the benzene ring. The out-of-plane bending vibration absorption peak of –CH on the benzene ring [−1. The FTIR characterization results showed that LDMS carried the characteristic functional groups of all the reacting monomers, and all the reacting monomers participated in the reaction successfully.The FTIR test results of LDMS are shown in ene ring  affected1H-NMR of LDMS dissolved in heavy water is shown in 2 in the polymer backbone. Peak b (2.48 ppm) should be attributed to–CH– attached to the amide group on the main chain. The proton peaks of the two methyl groups in DMAA appear at peak c (2.80 ppm). Peak d (2.89 ppm) corresponds to the proton peak of –CH– attached to the carboxyl group on the main chain. The two unsubstituted hydrogen proton peaks on the benzene ring [2O. The results of 1H-NMR also showed that the molecular structure of LDMS contained all the reacting monomers, and all the reacting monomers participated in the reaction successfully.The ene ring  in SSS cDrilling fluid additives are critical to maintaining the performance of drilling fluids under high temperature conditions, so a good thermal stability of additives is required. The thermogravimetric analysis curve (TG) and the derivative thermogravimetry curve (DTG) of LDMS are shown in w) of LDMS is 1325, and that of Driscal D Polymer is 626,763. The Mw and number-average molecular weight (Mn) of Driscal D Polymer are two orders of magnitude higher than those of LDMS. The Z-average molecular weight (Mz) of Driscal D Polymer is three orders of magnitude higher than that of LDMS. In the chemical category, low-molecular-weight polymers [3 to 6 × 103. LDMS satisfies the relative molecular weight requirements of low-molecular-weight polymers, indicating that the low-molecular-weight filtrate reducer LDMS was synthesized successfully. At the same time, Driscal D Polymer was selected as the representative of high-molecular-weight polymeric filtrate reducers to conduct comparative experiments in WBDFs.In order to compare and investigate the effects of different molecular weight filtrate reducers in WBDFs, the relative molecular weights of LDMS and the purchased filtrate reducer Driscal D Polymer were first determined by GPC. The distribution of the relative molecular weights and the measurement results are shown in polymers  are polyIn order to study the performance of LDMS in freshwater drilling fluids, different contents of LDMS and Driscal were added to the freshwater base fluid to prepare drilling fluids. The rheological parameters and API filtration of drilling fluids were measured at room temperature. The results of rheological parameters and filtration loss measurements are shown in The drilling fluids were prepared by adding 2.0 wt% LDMS and 2.0 wt% Driscal to the freshwater base fluid. After the drilling fluids were aged for 16 h at different temperatures, the rheological parameters and API filtration were measured at room temperature. The HTHP filtration of the drilling fluid was measured at the corresponding aging temperature. The measurement results of rheological parameters and filtration are shown in With the increase in the aging temperature, the AV, PV, and YP of the base fluid were very low and almost unchanged, and the API filtration and HTHP filtration increased sharply. The HTHP filtration of the base fluid at 210 °C was as high as 144 mL. It indicated that the gel network structure inside the base fluid had been destroyed. The viscosity and YP of the drilling fluid with LDMS decreased slightly with the increase in aging temperature. Compared with the base fluid, the viscosity and YP of the drilling fluid with LDMS were about twice that of the base fluid, which increased to a lesser extent, but the amount of filtration was significantly reduced. After aging at 210 °C, the API filtration of the drilling fluid with LDMS was only 8.0 mL, and the HTHP filtration was 41.0 mL. The viscosity and YP of the drilling fluid with Driscal changed dramatically with the increase in aging temperature. The viscosity and YP of the drilling fluid with Driscal decreased sharply with the increase in temperature before 180 °C, and the change was no longer significant after 180 °C, indicating that the temperature resistance turning point of Driscal is 180 °C. Compared with the base fluid, the viscosity and YP of the drilling fluid with Driscal increased greatly, and the filtrate volume also decreased. However, compared with the drilling fluid with LDMS, the drilling fluid with Driscal had a higher viscosity and yield point, and about twice as much the amount of filtration. After aging at 210 °C, the API filtration of the drilling fluid with Driscal was 18.4 mL, and the HTHP filtration was 91.0 mL.The comparison results showed that the performance of Driscal was greatly affected by temperature. With the increase in temperature, the macromolecular chain of Driscal was gradually broken and decomposed, resulting in a decrease in performance. Additionally, the performance decreased significantly after 180 °C, indicating that the temperature resistance turning point for Driscal is 180 °C in a WBDF environment. However, with the increase in temperature, the performance of LDMS was very stable, indicating that the temperature resistance of LDMS is better than that of Driscal. This is because the small molecular chain of LDMS contains a rigid benzene ring, and the main chain is a carbon–carbon bond with large bond energy, which is not easy to break at high temperature, so that LDMS is not easily degraded at high temperature. In addition, LDMS can be firmly adsorbed on the surface of bentonite particles under the action of hydrogen bonding . The theThe drilling fluids were prepared by adding different contents of LDMS into the freshwater base fluid. The drilling fluids were aged at 180 °C for 16 h. The Zeta potential of the drilling fluid was measured before and after aging, and the results are shown in 10 , D50 (median particle size), and D90 particle size value at 90% of the cumulative volume) of the bentonite particle size also decreased gradually  results of the dry bentonite treated with different concentrations of filtrate reducers are shown in The XRD patterns and interlayer spacing , and the results depicted in 10 of the bentonite particles in the base fluid was 2.19 μm, while the D90 was as high as 74.9 μm  are depicted in  74.9 μm . The loo 74.9 μm b had an  74.9 μm c. Many fThe premise of the effect of the filtrate reducer is that it can be firmly adsorbed on the bentonite particles in a large amount. In order to intuitively feel the adsorption of the filtrate reducer on the surface of bentonite particles, the changes of elements and element contents on the surface of the filter cake were measured by EDS. Since montmorillonite ,43 is co3− and –COO−, which have good hydration properties. LDMS adsorbs on the surface of bentonite particles under the action of hydrogen bonds and electrostatics, and binds more water molecules on the surface of bentonite particles. On the one hand, the negative charge of the bentonite particles is enhanced so that the repulsion between the bentonite particles increases, which is beneficial for the bentonite particles to maintain a small particle size. On the other hand, the thickness of the bentonite hydration layer is increased, and the colloidal stability of the drilling fluid is improved. At the same time, the small molecular chain of LDMS contains rigid groups, and compared with high-molecular-weight long-chain polymers, the small molecular chain is not easy to break, so it has better high temperature stability. Additionally, small molecular chains can intercalate into the interlayer of sodium bentonite. Under the action of hydrated ions of the LDMS molecular chain, the interlayer water content and hydration degree of bentonite are increased, and the resistance to high temperature dehydration is enhanced. In general, the unique chemical structure of LDMS, including rigid small molecular chain, strong adsorption groups, and highly negatively charged hydration groups, makes it have excellent temperature resistance and filtration reduction performance.Based on the comprehensive analysis of the above experimental results, the mechanism of the low-molecular-weight filtrate reducer LDMS was obtained. In order to better describe the mechanism of LDMS, w is 1325 Dalton). As a result, when 2 wt% LDMS was added to the base fluid, the apparent viscosity of the drilling fluid gel system was 8.5 mPa·s and the API filtration loss was only 8.0 mL after high temperature aging at 210 °C. In addition, the mechanism of LDMS was further studied. It was confirmed that due to the unique chemical structure of LDMS, including a rigid small molecular chain, strong adsorption groups and highly negatively charged hydration groups enabled it to be adsorbed on the surface of bentonite particles in large quantities and intercalated into the interlayer of bentonite. Thus, the hydration degree of the bentonite particles and the colloidal stability of the drilling fluid gel system were improved, the aggregation of the bentonite particles was inhibited, the content of fine particles in the drilling fluid gel system was maintained, and a compact filter cake was formed, which significantly reduced the filtration of the drilling fluid. Overall, this work will facilitate the application of a low-molecular-weight polymer filtrate reducer in high-temperature-resistant water-based drilling fluid gel systems.In this work, a novel high-temperature-resistant filtrate reducer  with low molecular weight was synthesized using DMAA, MAH, and SSS as reaction monomers. TGA experiment showed that the polymer LDMS has good thermal stability, the initial decomposition temperature is as high as 238 °C, and it has a good application prospect in a high-temperature-resistant drilling fluid gel system. At the same time, unlike the commonly used macromolecular polymer filtrate reducers, LDMS does not significantly increase the viscosity and yield point of a drilling fluid gel system due to its low molecular weight , maleic anhydride , and sodium p-styrene sulfonate  were purchased from Aladdin Reagent Co., Ltd. . Ammonium persulfate (AR), anhydrous ethanol, and sodium hydroxide (AR) were purchased from Sinopharm Chemical Readditive Co., Ltd. . Driscal D Polymer  was purchased from Chevron Phillips Chemical Company . The sodium bentonite used for water-based drilling fluids (WBDFs) was purchased from Huaian Tengfei Bentonite Development Co., Ltd., China (Huaian).In total, 9.9 g DMAA, 9.8 g MAH, and 10.3 g SSS were mixed evenly in a beaker containing 75 g deionized water. The beaker was heated in a water bath at 40 °C. The reaction monomer was completely dissolved by constant stirring, and the pH of the solution was adjusted to 7 with a dilute NaOH solution. Next, the solution was transferred from the beaker to the flask. When the water bath was heated to 70 °C, 0.15 g ammonium persulfate was added to the flask. The reactants were reacted under nitrogen atmosphere for 6 h to obtain a viscous liquid product. The product was purified with acetone and ethanol, dried under vacuum at 50 °C, and pulverized to obtain a low-molecular-weight filtrate reducer, namely, LDMS. The synthesis process and possible molecular structure of LDMS are shown in (1)Fourier-transform infrared (FT-IR)−1, the resolution ratio was 4 cm−1, and the sample scanning times were 32.The synthetic product was characterized by the potassium bromide (KBr) tabletting method using an IRTRacer-100 infrared spectrometer . A small amount of sample was mixed well with KBr in a mortar and ground thoroughly, and the ground powder was pressed into a complete and transparent sheet using a tablet press. The scanning wavelength range was from 500 to 4000 cm(2)1H-NMR)Nuclear magnetic resonance hydrogen spectrum . The signal was collected by dissolving 3 mg of sample in heavy water and cycling 16 times.(3)Thermogravimetric analysis (TGA)2 environment. The experimental temperature range was 50.0 to 800.0 °C, and the heating rate was 10.0 °C/min.The thermal stability of LDMS was measured using a thermal analyzer  in a N(4)Gel permeation chromatography (GPC)The relative molecular mass distribution of the filtrate reducer samples LDMS and Driscal was determined by gel chromatography using a Waters 1515 GPC type gel chromatograph . The mobile phase of the test was 0.1 mol/L aqueous sodium nitrate solution, and the standard was polyethylene glycol (PEG).Sixteen grams of bentonite, 0.56 g of sodium carbonate, and 400 mL of distilled water were added into a high-speed mixing cup and stirred at 10,000 rpm for 20 min, and then maintained for hydration at room temperature for 24 h to derive the base fluid. The filtrate reducer sample was added to the base fluid and stirred at 4000 rpm for 20 min to obtain the drilling fluid sample to be tested.600 and θ300 are the values of the rotary viscometer at the corresponding speed.The rheology of the drilling fluids was tested by a six-speed rotary viscosimeter . The following equations calculate the apparent viscosity (AV), plastic viscosity (PV), and yield point (YP):According to American Petroleum Institute (API) standards, the API filtration of drilling fluids was measured by a medium pressure filtration meter  at a fixed pressure of 100 psi for 30 min. The drilling fluids were aged in the aging kettle for 16 h. The rheological parameters and API filtration were measured before and after the aging experiments, using a high-temperature and high-pressure filtration apparatus , maintaining a fixed pressure difference of 3.5 Mpa, and measuring the high-temperature and high-pressure (HTHP) filtration of the drilling fluids for 30 min at the corresponding aging temperatures.The Zeta potential value of the drilling fluid samples was measured by a nanoparticle potentiometer . The particle size distribution of the drilling fluids was determined using a laser particle size analyzer .The drilling fluid samples were prepared by adding different contents of the filtrate reducer to the base fluid. The drilling fluids were cooled and centrifuged after aging at 180 °C for 16 h. The bentonite obtained by centrifugation was rinsed three times with deionized water, dried at 105 °C for 4 h, and ground through a 200 mesh sieve to obtain the bentonite treated with filtrate reducers. The lattice spacing of bentonite was measured by X-ray diffraction (XRD) using an X-ray diffractometer . The experimental test used a Cu target, the test voltage was 40 kV, and the test current was 40 mA. Thermogravimetric analysis of the bentonite was performed using a thermogravimetric analyzer  with a nitrogen atmosphere. The heating rate was 10.0 °C/min.0 is the mass of bentonite in the drilling fluid (g); m1 is the mass of the filtrate reducer in the drilling fluid (g); m2 is the mass of the filtrate (g); L is the mass percentage of elemental carbon in the molecule of the filtrate reducer (%); and P is the amount of carbon element in the filtrate (mg/L), which is the measured value of the TOC experiment.The filtrate was collected when measuring the HTHP filtration volume of drilling fluids containing different contents of the filtrate reducer at 180 °C. The total organic carbon content (TOC) in the filtrate was measured by a total organic carbon tester . The adsorption amount of the filtrate reducer on the drilling fluid bentonite was calculated by the following equation :(4)K=m1−The filtrate volume of the drilling fluid was measured according to API standards, and the filter cake was obtained after the measurement. After the filter cake was dried, its microscopic morphology was observed with scanning electron microscopy , and the distribution of elements on the surface of the filter cake was studied by an energy dispersive spectrometer .


The last line of the Financial support section currently states “The community survey was supported by the Wellcome Trust via a strategic award to AHRI (grant number 201433/Z/16/Z)”. The sentence should state “The community survey was supported by the Wellcome Trust via a strategic award to AHRI (grant number 201433/Z/16/A)”.An error appeared in the corrected proof publication of this article [Govender I, Karat AS, Olivier S, et al. Prevalence of Mycobacterium tuberculosis in Sputum and Reported Symptoms Among Clinic Attendees Compared With a Community Survey in Rural South Africa. Clin Infect Dis The author regrets this error.


The following case study demonstrates that the effectiveness of Deep Tissue Massage (DTM) can be monitored in real time with bioimpedance. DTM techniques are used as a medical treatment to help reduce swelling of the calves of congestive heart failure patients. Bioimpedance monitoring shows immediately how fluid is redistributed within the intravascular, interstitial and intracellular fluid compartments, and how long the redistribution lasts. Bioimpedance spectroscopy, as used in this study, is a non-invasive procedure that can be used to monitor compartment fluid volumes and changes during many fluid management procedures. There are numerous types of physical massage modalities. Most massage therapies are done to relieve stress, reduce inflammation, ease sore muscles and/or restore the body's energy. Only Deep Tissue Massage (DTM) and Deep Lymphatic Massage (DLM) are done to reduce the fluid accumulation that may take place in different segments of the body.DTM differs from DLM in its purpose, focus and mode of application . DTM empLittle information is available regarding the depth or effectiveness of either DTM or DLM. The objective of this pilot level study was to assess how well this information can be obtained using the bioimpedance device described below. In particular, how DTM affects the three fluid compartments that may accumulate fluids in congestive heart failure patients. Congestive heart failure not only leads to fluid retention but also triggers a change in fluid distribution (intra- and extra-cellular and vascular fluid shifts).An electrical impedance spectrograph (EIS)  was used to monitor segmental intracellular and extracellular compartment volumes. A custom analytical program was then used to divide the extracellular compartment volume into its intravascular and interstitial components. Detailed descriptions of the EIS and analytical procedures used in this study can be found in Sasser and Gerth , Gerth aThe subject that took part in this pilot level study is a male who has congestive heart failure. He is 83 years old, weighs approximately 130 pounds and is 5 ft. 9 in. tall. He is on a limited fluid intake regime of 2 liters/day and closely monitors his food (sodium) intake to be under 600 – 800 mg/day. He is taking a diuretic in the form of 100 mg oral Torsemide (DEMADEX) administered orally once a day in the morning. This dosage is adjusted based upon his current weight, swelling and blood pressure. The goal for blood pressure is to maintain it at approximately 95/65 throughout the day.To date, this program has been rather successful in preventing severe edema in the thigh, torso and chest. However, by the end of the day his calves exhibit mild pitting edema of approximately 1 mm in depth .The objective of DTM is for the pressure applied by the therapist to be more fully transmitted to the underlying muscles where it can mobilize and move the accumulated fluids back toward the heart .The DTM in this study was administered as described by The Wellness Council  and HealThe leg is placed in a horizontal position and allowed to rest prior initiation of the massage,Aveeno (dimethicone skin protectant – Johnson & Johnson Co.) daily moisturizing lotion was then used to lubricate the calf before and during the deep tissue massage.The therapist then applied circumferential force to the calf through long slow strokes from the ankle toward the knee using the hands and thumbs as shown in DTM was continued for five minutes after which the leg remained horizontal for a recovery period.After the recovery period the subject sat upright with the leg perpendicular to the floorto allow the fluid in the calf to return to pretest levels.The timing sequence of the various phases of the DTM was:Elapsed Time (minutes):The EIS was applied to the calf by using four ECG electrodes attached to the subject’s dominant lower leg as shown in Informed consent has been obtained from all individuals included in this study.The research related to human use has been complied with all relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration.To our knowledge, this is the first study to use bioimpedance to simultaneously quantify the intravascular, interstitial and intracellular fluid compartment volumes during DTM. DTM was found to affect each fluid compartment volume in two different ways: the amount of the fluid change during the five-minute application of DTM  and the The salient findings of the current study are as follows:1The Ri, Re and Cm values are used to calculate the separate intravascular, interstitial and intracellular fluid compartment volumes of the calf, as given in During the time period Elapsed Time 60 to 120 minutes, when the subject sat upright, Vcell increased by 146 mL and Vinstl gained 31 mL. During this period Vblood decreased an additional 49 mL.There is a time delay between the time of starting DTM and the time at which the external massage force affects a given compartment volume. Vblood loses fluid immediately upon the start of DTM. The effect of DTM is delayed approximately 1 min for Vinstl and 2 min for Vcell, respectively.2Between Elapsed Times 20 min. and 60 min., the leg remained horizontal. All three compartments lost fluid for 10 min. At Elapsed Time 30 min. both Vinstl and Vcell increased in volume. Vblood continued to lose volume until the leg was repositioned on the floor.The prolonged recovery period found in this study is similar to that reported by Roffay et al. . followiSeveral authors ,16,17 haDowney  describeHunt et.al.  suggestsRoffay et. al.  reports This pilot study has demonstrated that DTM does affect all three fluid compartments of the calf which can be quantified using bioimpedance spectroscopy.It quantifies the amount of fluids present in and transferred between each compartment before, during and after DTM.Possible mechanisms of how the cell structure is changed by DTM and how it may alter the intracellular, interstitial and intravascular compartment volumes are summarized from the literature.This information may be useful in the study of massage and other fluid management techniques. Additional research should be done using the same EIS systems to confirm these results and their applications to other clinical and environmental conditions.


Pdyn was mainly expressed by Drd1-expressing cells in the dense cell layer (DCL) of the OT, whereas Penk was expressed primarily by Drd2-expressing cells in the DCL. We also confirmed the presence of a larger population of Pdyn-Penk-Drd1 co-expressing cells in the DCL of the anteromedial OT compared with the anterolateral OT. These observations will help understand whether and how dynorphins and enkephalins in the OT are involved in diverse odor-induced motivated behaviors.The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence  The olfactory tubercle (OT) plays an essential role in acquiring odor-induced motivated behavior , dwarf cells, and granule cells  or D2 (Drd2), dwarf cells express Drd1, and granule cells express D3 (Drd3) and weakly Drd1  : Pdyn, 5'-AGGAAAAGTTCAGGGGTCTCTC−3' – 5'-TCTCACAGTTCCCATGCAATAC−3'; Penk, 5'-TTCCTGAGGCTTTGCACC-3' – 5'-TCACTGCTGGAAAAGGGC-3'; andPomc,5'-CGACGGAAGAGAAAAGAGGTTA-3' – 5'-CTTGGAATGAGAAGACCCCTG-3'. We used a commercial mouse brain cDNA library  as amplification templates. The PCR products were subcloned into pGEM-T Easy plasmids (Promega) for in vitro transcription of RNA probes. Plasmid templates for Drd1 and Drd2 probes were provided by Dr. Kazuto Kobayashi  according to the manufacturer's protocol. We prepared both antisense and sense probes for opioid precursors and dopamine receptors, and confirmed that ISH using sense probes yielded no detectable signals (data not shown). All data presented in this report were obtained by ISH using antisense probes.Mouse Pdyn, Penk, and proopiomelanocortin (Pomc) cDNAs were subcloned by conventional PCR using the following primers in accordance with the Allen Brain Atlas . The sections were treated overnight with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) color development substrate mixture (Promega) at room temperature in a dark room for color development. They were then rinsed in PBS and mounted in Mount-Quick Aqueous (Daido Sangyo).ISH using a single probe for Pdyn, Penk, Pomc, Drd1, and Drd2  was perfTriple fluorescence ISH using DIG-, FLU-, and BIO-labeled probes , 3 was pIn The BCIP/NBT color-developed samples were examined using a bright-field virtual slide system  . A confopost-hoc Tukey's test using GraphPad Prism 7  with We first confirmed that Pdyn and Penk mRNAs were expressed in the anterior OT, dorsal striatum, and NAc using single-probe ISH in mice . Penk mRThe cytoarchitecture of the OT can be defined by the expression of the Drd1 and Drd2 mRNAs. We compared the Drd1 and Drd2 ISH signals with the Pdyn and Penk signals in the neighboring OT coronal sections . The OT Curran and Watson reported Pdyn-, Penk-, and Drd1-coexpressing cell clusters in the rat ventral striatum  . We alsoThe ventral pallidum anteriorly protrudes into the OT deep layer (multiform layer), where pallidal cells are distributed  and anteromedial DCL . None of the DARPP-32 immunonegative cells in the multiform layer of the anteromedial OT showed Penk or Drd1 mRNA signal . SF was supported by JSPS KAKENHI (Grant Numbers 21H03086 and 21K19559); the Japan Agency for Medical Research and Development ; and a Health Labor Sciences Research Grant [R3-Nantitou (nan)-Ippan-21FC1013]. YF was supported by JSPS KAKENHI (Grant Numbers 19H03323 and 20H05058).KM received research funding from the LOTTE Foundation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The correlations of Treg cells with TILs, invasion depth, and tumour morphological risk were analysed. We identified a high mean proportion of Treg cells within the tumour  despite a relatively low median of TILs , supporting the classification of BCC as a cold tumour. A significant positive correlation was observed between the proportion of Treg cells and sTILs , suggesting a predominant role of TILs in the infiltration of Treg cells. An inverse correlation discovered between Treg cells and tumour invasion depth  might indicate Treg cells’ anti-tumour capacity in BCC.Basal cell carcinoma (BCC) is the most common skin malignancy worldwide. Current evidence suggests tumour-infiltrating lymphocytes (TILs) may influence the clinical outcomes of patients with BCC. The present study aimed to profile the infiltrative characteristics of stromal TILs and regulatory T cells (Treg cells) in the tumour centre (TC), tumour periphery (TP), and normal adjacent tissue (NAT) of BCC. A total of 111 samples from 43 cutaneous BCC cases were examined for TIL (CD3 Basal cell carcinoma (BCC) is the most diagnosed kind of skin cancer worldwide. The mainstay treatment for BCC is surgical resection with tumour-free excision margins . The dis+ TILs—was reported to be high in BCC [+ TILs, subsequently decreasing the host’s immune response to tumourigenesis and invasive processes [+ CTLs by Treg cells might explain the correlation between the high number of Treg cells with lower recurrence rates in patients with estrogen receptor-negative (ER-) breast cancer [In their case study, Sabbatino et al. proposed that the poor response of BCC to immunotherapy correlates to a low number of tumour-infiltrating lymphocytes (TILs) found in the tumour microenvironment. In contrast to a low number of TILs, the proportion of regulatory T cells (Treg cells)—a subset of CD3rocesses . The prorocesses  and heparocesses . Howeverrocesses ,8. The at cancer ,8, cleart cancer , and blat cancer . The con+ sTILs were evaluated in the tumour centre, tumour periphery, and the normal adjacent tissue beyond the tumour borders. Similar to sTILs, CD3+/FOXP3+ Treg cells were also evaluated in the three different tumour areas. We also analysed the correlations of Treg cell proportions with tumour morphological risk and tumour invasion depth in our samples. The evaluation of sTIL and Treg cell infiltration patterns, and also their correlations with tumour characteristics hopefully gives better insights into the role of sTILs and Treg cells in the development and progression of BCC. The present study aimed to evaluate the proportion of stromal TILs (sTILs) and Treg cells to profile their infiltration characteristics among different tumour zones within each patient with BCC. As recommended by the International Immuno-Oncology Biomarker Working Group , CD3+ sTDuring the years 2016–2021, 132 BCC cases were identified in the Anatomical Pathology Installation of Dr. Sardjito Hospital, Yogyakarta, Indonesia. Among 132 cases, 69 cases were classified as cutaneous BCC. Of 69 cases, 26 were excluded because of the absence of both normal adjacent tissue and tumour periphery in their respective paraffin blocks; hence, they were unable to be used for comparative analyses, leaving 43 cases to be included in our study. In total, 111 slides from 3 different tumour zones were acquired for the subsequent immunohistochemical assessment: 43 from the tumour centre (TC), 34 from the tumour periphery (TP), and 34 from the normal adjacent tissue (NAT). TP is defined as the zone centred on the tumour border separating the normal adjacent tissue from the tumour nests with an extent of 2 mm. TC is the zone next to TP toward the centre of the tumour, while NAT is the dermal zone beyond the TP. Immunostaining procedures were conducted in the Anatomical Pathology Installation of Dr. Sardjito Hospital, Yogyakarta, Indonesia. Ultratek HRP Anti-polyvalent (DAB) staining system kit  was utilised to perform deparaffinisation, rehydration, blocking of endogenous peroxidase, antigen retrieval, and counterstaining. AEC substrate chromogen was purchased separately from the staining system kit. The antibodies used for double staining were FOXP3 mouse monoclonal antibody  diluted to a 1:400 ratio and CD3 mouse monoclonal antibody  diluted to a 1:100 ratio. Hematoxylin and Bluing Reagent were used to counterstain the slides. Normal spleen tissue was used as a positive control.+ cells over the area of the stroma. Tumour invasion depth was assessed at the thickest area at the centre of the tumour. Tumour morphological risk was classified as low-risk and high-risk based on the latest version of the World Health Organization (WHO) classification of skin tumours [Immunostained IHC slides were examined for FOXP3 and CD3 expressions by two independent observers. Each slide was viewed at 400× magnification and 5 fields were selected to be digitally captured using the OptiLab Viewer . The captured images were manually counted for regulatory T cells (Treg cells) defined as FOXP3/CD3 double-positive stained cells (brown nucleus and red cell membrane), and sTILs defined as CD3 single-positive cells (red cell membrane) with the aid of an on-screen count tracker in the software ImageJ . Treg cell proportion was defined as the fraction of Treg cell count (double-positive cells) over the sum of Treg cells and non-Treg cell lymphocytes (the sum of double-positive and single-positive cells). Meanwhile, the sTIL proportion was defined as the area occupied by all CD3 tumours . t-test, respectively. A p value of <0.05 was considered statistically significant.Statistical analyses were performed using SPSS 26.0 . The difference in sTIL and Treg cell counts amongst three different tumour zones  were analysed using the Kruskal–Wallis test. Meanwhile, the difference in Treg cell proportions amongst the three different tumour zones was measured using one-way ANOVA. The correlation of sTILs with tumour invasion depth was analysed using Spearman’s rank correlation; meanwhile, the correlation of Treg cell proportion with tumour invasion depth was investigated using Pearson’s correlation coefficient. The difference in sTIL and Treg cell proportion between low and high-morphological-risk groups were calculated using Mann–Whitney U test and independent p = 0.002). A post hoc analysis confirmed a significant difference in sTILs between the TP and NAT (p = 0.012), as well as between the TC and NAT (p = 0.004). No significant difference in sTILs between the TC and TP was found.A total of 111 samples from 43 cutaneous BCC cases were evaluated for sTILs, Treg cell counts, and Treg cell proportions. Samples were divided into three different tumour zones: tumour centre (TC), tumour periphery (TP), and normal adjacent tissue (NAT) . Minimalp = 0.014), with post hoc analysis affirming the disparity between the counts in the TP and NAT to be most prominent (p = 0.01). However, the difference in Treg cell proportion among the three tumour zones was found to be statistically insignificant (p = 0.195), with a mean of 46.9% in the TC, 56.1% in the TP, and 51.8% in the NAT (p < 0.001)  .p = 0.017), but not in the TP    . Inconsi7 in TP) .+ T cells, and T helper 1 (TH1) cells in the TME [+ T cells, and TH1 cells are found in high numbers among phenotypically hot tumours [+ to CD4+ T cell proportion, with a mean of 45%, concordant with our finding of 51.3% [In the present study, the tumour microenvironment (TME) of our BCC samples demonstrated a high proportion of tumour-associated Treg cells, despite a relatively low proportion of sTILs. These results suggest BCC cases in our study belong to the category of phenotypically “cold” tumours. Cold tumours are malignancies characterised by the dominance of the Treg cell proportion over the amount of all of the other sTILs, such as natural killer (NK) cells, CD8 the TME . In cont tumours . The claof 51.3% . Other mof 51.3% . Meanwhiof 51.3% . In the of 51.3% .+, CD4+ (TH1 and TH2 cells), CD8+, and FoxP3+ Treg cells. Even though the presence of Treg cells in tumours has been suggested to affect clinical outcomes, controversy regarding whether it is the accretion [Even though the quantification of TILs has been included as part of a routine diagnostic procedure in pathology , the indccretion ,21 or deccretion  that corccretion ,8.Aside from tumour subtypes, the tumour zones in which Treg cells were examined seem to contribute to inconsistent results among different studies. In the present study, we found significantly different Treg cell counts between the TC, TP, and NAT. Interestingly, in alignment with our findings, the number of Treg cells was found to be significantly higher within the tumour sites of BCC ,23, breaWithin the tumour, we found higher sTIL and Treg cell proportions in the TP than in the TC. This phenomenon might be explained by the different main activities between these tumour zones. The pathways activated in the TC are mainly related to altered cellular metabolisms, which include tumour cell adaptation to hypoxia and necrosis . On the H1 and TH2 cells [H1 and TH2 cells, resulting in a continuous cycle of TIL–TAM upregulation, which enhances the density of Treg cells within the TME [In our study, we discovered a significant positive correlation between the proportion of Treg cells and sTILs in all three observed zones. We also did not find the increase in Treg cell proportion in the TC and/or TP compared to the NAT to be significant. It can be inferred from these results that the infiltration of Treg cells into the TME seems to be largely regulated by sTILs, rather than independently by the tumour cells. Previous studies have shown that the recruitment, expansion, and conversion processes of peripheral Treg cells into active cells involve various chemokine–chemokine receptor interactions regulated predominantly by cytokines released by tumour-associated macrophages (TAM) and partly by NK cells ,30. The H2 cells . In resp the TME . With respect to tumour characteristics, we found a significant correlation of Treg cell proportion with the tumour invasion depth. Tumour invasion depth is largely accepted as an independent prognostic factor in skin melanoma . Albeit We found no significant difference in sTIL and Treg cell proportion between different tumour morphological risk groups. The morphological risk group stratifies BCC into two categories (low and high-risk) based on the risk of recurrence . The lowIn summary, the present research findings imply that the diagnostic approach through the quantification of specific components of TILs rather than TILs by themselves seems to pose a greater benefit for the determination of patient management and prognosis. The selection strategy for tumour zone sampling needs to be appraised considering the heterogenous distribution of TILs and their components surrounding the tumour. The correlation between tumour invasion depth, Treg cells, and potentially patient outcomes in BCC further enhances the benefits of Treg cell quantification in clinical practice. The strength of our study lies in the use of multiple tumour zones to analyse the variations of sTIL and Treg cell distribution within different tumour zones. However, this multiple-tumour-zone sampling method limits our sample selection to only BCC cases with paraffin blocks containing tissue from the tumour periphery and adjacent tumour-free tissue. From the 69 cutaneous BCC cases found in our hospital, 23 cases were excluded because tissues containing the tumour periphery and tumour-free area were absent in their respective paraffin blocks. Due to the sampling method, our cases are more representative of incompletely resected BCC cases with a relatively higher invasion depth (mean = 6.4 mm) than the general BCC cases.


Transcriptomic profiling of several drugs in cancer cell lines has been utilised to obtain drug-specific signatures and guided combination therapy to combat drug resistance and toxicity. Global metabolomics reflects changes due to altered activity of enzymes, environmental factors, etc. Integrating transcriptomics and metabolomics can provide genotype-phenotype correlation, providing meaningful insights into alterations in gene expression and its outcome to understand differential metabolism and guide therapy. This study uses a multi-omics approach to understand the global gene expression and metabolite changes induced by Disarib, a novel Bcl2-specific inhibitor in the Ehrlich adenocarcinoma (EAC) breast cancer mouse model. RNAseq analysis was performed on EAC mouse tumours treated with Disarib and compared to the controls. The expression of 6 oncogenes and 101 tumour suppressor genes interacting with Bcl2 and Bak were modulated upon Disarib treatment. Cancer hallmark pathways like DNA repair, Cell cycle, angiogenesis, and mitochondrial metabolism were downregulated, and programmed cell death platelet-related pathways were upregulated. Global metabolomic profiling using LC-MS revealed that Oncometabolites like carnitine, oleic acid, glycine, and arginine were elevated in tumour mice compared to normal and were downregulated upon Disarib treatment. Integrated transcriptomic and metabolomic profiles identified arginine metabolism, histidine, and purine metabolism to be altered upon Disarib treatment. Pro-angiogenic metabolites, arginine, palmitic acid, oleic acid, and myristoleic acid were downregulated in Disarib-treated mice. We further validated the effect of Disarib on angiogenesis by qRT-PCR analysis of genes in the VEGF pathway. Disarib treatment led to the downregulation of pro-angiogenic markers. Furthermore, the chorioallantoic membrane assay displayed a reduction in the formation of the number of secondary blood vessels upon Disarib treatment. Disarib reduces tumours by reducing oncometabolite and activating apoptosis and downregulating angiogenesis. Breast cancer is a heterogeneous disease with a high diversity between and within subtypes. Recent advances in cancer development have led to selective targeting of deregulated molecules and pathways to achieve a rational cancer therapy. Such targets include mutant kinases, cancer stem cells, and tumour microenvironment, etc. . The BclMany small-molecule inhibitors, such as Gossypol, AT101, Obatoclax, ABT-737, ABT-263, Tw37, YC137, and HA-14 break, and the interaction between Bcl2 and its partner Bak/Bax (pro-apoptotic) has been developed ,6,7,8,9.One of the strategies explored to get insight into the drug-induced changes at the transcriptional level is RNA-Seq. RNA-seq allows deciphering the biological effects of the drug, in vitro, and in vivo . One of Bcl2 is an antiapoptotic protein which regulates cancer cell survival by altering the pro-apoptotic/antiapoptotic balance in the cell and promotes angiogenesis ,18. AngiThis study employs integrated transcriptomic and metabolomic approaches to study the effect of Disarib in vivo in an EAC-induced breast cancer model.All the chemicals used for the experiments were purchased from Biorad , Thermo Fisher Scientific , MP biomedicals , Life Technologies , KAPA , Sigma Chemical Co. , and BioLegend . Primers were ordered from Bioserve Biotechnologies India Pvt Ltd. . NGS reagents were purchased from Illumina  and New England Biolabs . Analytical quality water used for all the studies was obtained from the MilliQ water purification system from Thermo Fisher Scientific, USA. All other chemicals used were of analytical grade. Disarib, the BCL2-specific inhibitor was synthesized in-house .Swiss albino mice experiments were conducted per the Institutional Animal Ethical Committee guidelines of IBAB and as per Indian national animal care and use law (Ref. IAEC/IBAB/07/10-7-2019). Female Swiss albino mice of weight 19–22g 4–6 weeks old were purchased from Liveon Biolabs Pvt. Ltd., Bangalore, India. Animals were maintained in a room with a 12 h dark/12 h light cycle and controlled humidity and temperature (23 + 3 °C). Ventilated polypropylene cages were used for the animals, and they were supplied with a standard pellet diet  and water ad libitum. Components of the standard pellet diet is 21% protein, 5% lipids, 4% crude fibre, 8% ash, 2% vitamin, 3.4% glucose, 1% calcium, 0.6% phosphorus, and 55% nitrogen-free extract (carbohydrates).6 cells/animal) into the left thigh was given to 10 animals for developing a solid tumour. The no-tumour control animals were divided into two groups; Group I served as tumour control and received no treatment, and Group II received 50 mg/kg Disarib orally. EAC injected animals were divided into 2 groups containing 5 animals each (Group III and IV). Group III animals were EAC tumour controls that did not receive any treatment, and group IV received 50 mg/kg of Disarib orally. The treatment was given after 7 days of tumour development and continued until the 22nd day (12 doses). The tumour growth was monitored by measuring the diameter of the tumour using vernier callipers every alternate day until a 70–80% reduction of the tumour was observed. The tumour volume was calculated using the formula V = 0.5 × a × b2, where ‘a’ and ‘b’ indicate major and minor diameter, respectively. Blood was collected from the tail vein of the animals belonging to groups I, II, III and IV at three different time points; (a) after tumour development, (b) midway through Disarib treatment, and (c) once the tumour was reduced by around 70–80%, plasma was isolated from the blood samples and subjected to metabolomics analysis [A total of 20 Swiss albino mice were used for the study. Among these, 10 served as no tumour control. Intramuscular injection of EAC cells  was applied to obtain a significant DEG list. A list of oncogenes and tumour suppressor genes specifically for breast cancer was procured from https://oncovar.org/ and https://bioinfo.uth.edu/TSGene/, accessed on 25 June 2021, respectively. Downregulated oncogenes and upregulated tumour suppressor genes were obtained from the significant gene list of Disarib-treated EAC tumours. Heatmap was plotted to analyse oncogenes and tumour suppressor genes using pheatmap, an R package. String database was used to generate a network of the oncogenes, tumour suppressor genes and BCL2. Significantly up and downregulated genes were separately given as an input to the Reactome database [From the EAC tumour treated with Disarib samples, a cutoff of database  to obtaidatabase .Complementary DNA was synthesised using NEB reagents from the intact RNA ,38. 4 µg® Green chemistry. Primers for angiogenesis marker genes were used with GAPDH primer as an internal control  made with 50% methanol and incubated in ice for 30 min. Samples were sonicated after the incubation for 15 min. The supernatant was then filtered using an amicon 3kDa cutoff filter. A 2 µL of the supernatant was then analysed in Agilent 6490 iFunnel triple quadrupole LC/MS system.Waters X-Bridge amide 3.5 µm, 4.6 ×100 mm column was utilised for the study (positive mode). 100% acetonitrile with 0.1% formic acid was solvent-B (organic), and water with 0.1% formic acid was solvent-A (aqueous). The solvents were subjected to sonication for 15 min before use ,47. The This study used a triple-quadrupole mass analyser for the targeted metabolomics. Quadrupole analysers are simple, robust and with good low mass resolution and accuracy . The ionhttps://www.metaboanalyst.ca/) accessed on 25 June 2021.  Edmonton, Alberta, Canada. Both univariate and multivariate regression analysis was performed on the metabolites. Univariate analysis (t-test) generated metabolites with t-stat and False discovery rate (FDR) value. Significant metabolites (FDR ≤ 0.25) were subjected to pathway analysis, and a heatmap was plotted using the same software for all the time points [p-value adjusted less than 0.05, and log2 fold change (<−1 and >+1)) and metabolites with VIP scores ≥ 1 were used for generating interaction networks using Metaboanalyst software Edmonton, Alberta, Canada.Agilent MassHunter Qualitative Analysis B.06.00 and Agilent MassHunter Quantitative analysis B.06.00 software  was used for analysis. The list of metabolites was analysed based on Retention time, area under the peak and signal-to-noise ratio. The selected metabolites were then normalised w.r.t internal standard and subjected to binary logarithmic transformation (base 2). Statistical analysis was performed using Metaboananalyst (Version 5.0) e points . Multivae points . SignifiCAM is an extra-embryonic membrane comprised of a high density of blood and lymphatic vessels . CAM hast-test was applied to calculate the significance. p values less than 0.05 were considered significant, and the bar graphs are represented as Mean + SEM. p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = **** were used for representation.Statistical analyses and graphing were done using GraphPad Prism 7.0 software  and R packages, Vienna, Austria. Deseq2 uses the Wald test statistic with a probability to generate a significant gene list. The Benjamini–Hochberg False Discovery Rate (FDR) method was used for choosing significant pathways from the Reactome database. For comparative qRT-PCR analysis, a two-tailed EAC tumour control and Disarib treated tumour RNA samples were sequenced (2 replicates each). Around 50–70 million reads were obtained for all the samples after sequencing . The quap-value-adjusted less than 0.05 and log2fold change greater than 1 and less than −1 to filter out the significant genes. A total of 1215 DEGs were significant, out of which 184 were downregulated, and 1031 were significantly upregulated. There was equal up and downregulation of genes in the total DEG list. However, most of the significant DEGs showed upregulation. The number of DEGs for each sample is summarised in The quantile normalised read counts were subjected to differential gene expression analysis using deseq2 (R package). The analysis revealed 17,623 differentially expressed genes between the EAC control and Disarib-treated tumour samples . 8979  was given as input to the Reactome database for EAC samples. ECM proteoglycans, MHC class II antigens, programmed cell death, chemokine signalling, and platelet response pathways were upregulated a. ABT-73EAC tumour was injected in swiss albino mice. Once the tumour developed, Disarib was administered 50 mg/kg body weight, 12 doses, daily. Blood was collected from the tail vein at three different time points during the experiment: (a) before the treatment, (b) at the midpoint during the treatment, and (c) at the end of the experiment. Blood was collected from four different groups of animals: EAC tumour control, EAC tumour group receiving Disarib treatment, Normal animals, and Normal mice receiving Disarib treatment. Each group had 5 animals. Plasma was isolated from the blood of all the animals at three different time points and was injected into LC-MS. The peak intensities obtained after targeted metabolomic profiling of the mice samples were normalised based on the internal standards and log transformed. Normalised values of the metabolites were subjected to principal component analysis. An apparent clustering of normal vs. tumour control samples was observed . Differep-value and FDR values are provided in It was evident from the previous analysis that significant metabolites in tumour animals are representative of the oncogenic progression. It was followed up by Disarib treatment in tumour-bearing mice, with the aim of understanding the impact of Disarib on oncometabolites in EAC tumour mice. To begin with the metabolite analysis, the normalised peak intensities of metabolites were subjected to principal component analysis. Distinct segregation of tumour control and Disarib-treated samples was observed . Additioβ-alanine metabolism was observed. β-alanine is known to control tumorigenesis by altering energy metabolism in cancer cells [To understand if changes in the metabolites correlate with changes in the transcriptome, the significant gene list from Disarib-treated EAC tumour samples and the significant metabolite list with VIP scores greater than one at the endpoint was given input to metaboanalyst software. The joint pathway analysis option was chosen in metaboanalyst with the hypergeometric test as the enrichment factor and degree of centrality as the topological measure. The output was a list of pathways having both metabolite and corresponding genes. After data integration, amino acid metabolism (histidine and arginine), β-alanine metabolism, and purine metabolism were picked up . Histidier cells . Citruller cells . Adenosier cells . Adenyl The pathway analysis of Disarib-treated EAC tumours showed that genes participating in angiogenesis were significantly downregulated. We wanted to investigate further whether Disarib possesses anti-angiogenic property. VEGF and its partners are significant players in angiogenesis and are upregulated in breast cancer . TherefoThis study explored the global gene expression and correlated it with changes in metabolites post-Disarib treatment in an EAC Breast cancer mouse model. A significant reduction of oncometabolite and upregulation of tumour suppressor metabolites led to tumour regression in vivo. Integrated transcriptome and metabolome led to the identification of the amino acid metabolism and angiogenesis, among other pathways to be altered upon Disarib treatment.Breast cancer is a heterogeneous disease and involves the loss of tumour suppressors and amplification of oncogenes leading to activation of oncogenic signalling and tumorigenesis . The DisOne of the major issues of previous Bcl2 inhibitors has been thrombocytopenia. ABT-737 and ABT-263 were withdrawn from the trials because they severely decreased platelet counts ,60. InteCancer cells are known to modulate the energy metabolism pathways for their survival. The ultimate measure of the alterations in the phenotype can be attributed to the changes in the biochemical action in a cell due to a drug. The assay to detect the action of the metabolic pathways is to measure metabolites. We performed metabolomic profiling using targeted LC-MS on plasma samples of normal EAC tumour controls and Disarib-treated EAC tumour animals at different time points. For the positive mode, data were normalized using internal standard L-Zeatine, and L-Tryptophan was for the negative mode of acquisition. To take care of the batch effects, sample replicates from previous batches were injected and verified. Pooled plasma was employed as quality controls at the start, middle, and end of the batch run. EAC tumour control showed high levels of oncometabolite that remained constant throughout the tumour development. Kynurenine, Sarcosine, Myristoleic acid, Glycine, Dimethylarginine, and tryptophan are oncometabolite known to play a role in tumour progression ,75,76,77ABT-199, an FDA-approved Bcl2 inhibitor, is known to cause resistance in leukemic cells by elevating levels of a metabolite—phosphoinositides—in cells exhibiting a high glycolytic rate . InteresThrough transcriptomic analysis of Disarib-treated EAC mouse tumours, we obtained overall changes caused by Disarib. We identified potential pathways that cause resistance and thrombocytopenia to be downregulated. We confirmed that Bcl2 inhibition could potentially block angiogenesis. By integrating transcriptomics and metabolomics, we could infer that Disarib reduces tumours by activating apoptosis, regulating angiogenesis, and altering the levels of key oncometabolite.


PTIP-associated protein 1 (PA1) is a unique component of MLL3/4 complexes, which are important mammalian histone 3 lysine 4 (H3K4) methyltransferases. PA1 has generated research interest due to its involvement in many essential biological processes such as adipogenesis, B cell class switch recombination, spermatogenesis, and embryonic development. In addition to the classical role of PA1 in H3K4 methylation, non-classical functions have also been discovered in recent studies. In this review, we systematically summarize the expression pattern of PA1 protein in humans and sort the specific molecular mechanism of PA1 in various biological processes. Meanwhile, we provide some new perspectives on the role of PA1 for future studies. A comprehensive understanding of the biological functions and molecular mechanisms of PA1 will facilitate the investigation of its complicated roles in transcriptional regulation. Methylation modification on histone lysine residuals is linked to a wide variety of essential cellular processes such as transcription and DNA repair . One of PA1 is highly expressed in the cerebellum while the protein of PA1 was abundant in the B lymphocyte, fetal gut, testis, and CD8+ T cells, indicating the multiple potential functions of PA1 in different organs  domain within PTIP which is required for the interaction of PA1 and PTIP with MLL3/4 was deleted, the CSR process was not affected  , which ichanisms . In addichanisms .Pa1 in the precursor cell which could develop into BAT and skeletal muscle cells in the back would cause the death of newborn pups due to defective breath muscles. Further investigation showed that in the primary preadipocytes, PA1 could interact with phosphorated CREB and ligand-activated GR to induce the expression of C/EBPβ and C/EBPδ, which are pivotal transcription factors during the early phase of adipose differentiation. Also, the induction of PA1 on C/EBPβ was found independent of the MLL3/4 complex, suggesting the non-canonical function of PA1 protein in this process  and muscle development . The spe process . Given tntiation .Pa1 knockdown or Ptip knockdown in U-2 OS cells both could cause defective expression of TGF-β responsive genes instead of BMP responsive genes  which is an important factor for extraembryonic development in Pa1−/− embryos was significantly downregulated  of breast cancer patients with BRCA1 mutation accompanied with PA1 deficiency were significantly lower than those without PA1 deficiency   . The pres (CNVs) . Some evs (CNVs) . CNVs ats (CNVs) . Given tet al. found a homozygous missense variant of the PA1 gene  in three cases of Ashkenazi Jews ancestry from two completely unrelated families, exhibiting severe neurodevelopmental disorders including prenatal clinical features of microcephaly, polyhydramnios, severe developmental delay, dysmorphism, neurological deficits, and infancy death. This was the first time that the PA1 gene was reported to be associated with Mendelian genetic diseases (PA1 is a potential autosomal recessive causative gene whose homozygous mutation can lead to severe syndromic neurodevelopmental disorders.Recent studies have shed light on the role of PA1 in the development of the human nervous system . Hagit Ddiseases . Based oThis review summarizes the research into PA1 protein and its biological functions in various processes including its classical H3K4 modification and non-classical function via interacting with transcription factors, as well as those still unknown mechanisms. However, owing to the relatively limited data of the research findings on PA1 functions, a review in this regard needs to be constantly refined. Research could shed more light on the unclarified molecular mechanisms of PA1 in various biological processes and explored its new roles such as in neurodevelopment and tumor development.


Recently, the emergence of immunotherapy has revolutionized traditional tumour treatment. However, effective treatments for patients exhibiting αPD-1 resistance are still lacking. In our study, a combination of cytosine–phosphate–guanine oligodeoxynucleotides (CpG-ODNs), anti-OX40 and cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) injection in situ systematically generated a robust antitumour immune response in TC1 and B16 cells, which are αPD-1-resistant malignancies. More precisely, this method activates both adaptive and innate immunity. Additionally, in situ vaccination with CpG/αOX40/cGAMP fully activates the production of cytokines. However, the combination of αPD-1 does not improve the efficacy of triple therapy, prompting further questions. Collectively, the combination of CpG/αOX40/cGAMP causes the regression of various αPD-1-resistant tumours through the full mobilization of innate and adaptive immunity. In addition, we explored the therapeutic effect of triple therapy on the αPD-1-sensitive cell line CT26. The results showed that triple therapy could significantly enhance the therapeutic effect of αPD-1, and some mice even achieved complete tumour regression after the combined application of αPD-1 and triple treatment.The online version contains supplementary material available at 10.1007/s00262-021-03095-z. The incidence and mortality rates of cancer are increasing gradually and are primary factors threatening human health. According to global cancer statistics, in 2018, approximately 18.1 million new cases of cancer were diagnosed worldwide, and approximately 9.6 million deaths occurred [In recent years, the advent of immunotherapy has opened a novel chapter in the diversified treatment of various tumours, providing new hope for patients with cancer, particularly for some patients with advanced cancers. Currently, tumour immunotherapy is mainly divided into four categories according to the method: non-specific immune modulators, adoptive cell therapy, immune checkpoint inhibitors and tumour vaccines. Among them, the most common immunotherapy is PD-1/PD-L1 immune checkpoint inhibitors, which have been administered to many patients with cancer and have achieved a high response rate and lasting remission, significantly improving the survival rate of patients with recurrent or refractory Hodgkin's lymphoma –4, non-s+ T cells, and the combination of CpG with agonistic anti-OX40 antibody further enhances the antitumour effect of immune cells, systematically shrinking the tumours in mice, particularly lymphoma [In addition, in situ vaccination is a promising antitumour treatment method in which immune-enhancing agents are directly injected into the tumour site to stimulate the antitumour immune response throughout the body by inducing antigen release, promoting antigen presentation, activating immune effector cells, etc. . A growilymphoma . Temizozlymphoma . Accordilymphoma . Based olymphoma , 16. In lymphoma . HoweverAn extensive preliminary study and screening were performed to identify an in situ vaccine with good efficacy against αPD-1-resistant tumours. Notably, the combination of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), a STING agonist and CpG/αOX40 exerted a powerful vaccine effect; specifically, systemic tumour growth was significantly slowed, and surprising results were observed. In addition, in situ vaccination with CpG/αOX40/cGAMP increased the infiltration of immune cells in distant tumours and throughout the body, fully mobilized the production of cytokines and simultaneously activated adaptive and innate immunity  were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. All mice were housed under specific pathogen-free conditions and maintained at a constant temperature (22 ± 1 °C) and humidity (60–70%). In addition, all animals were provided ultrapure water and clean food and housed on 12 h light/dark cycles. All animal experimental procedures were conducted in accordance with guidelines for the care and use of laboratory animals from the National Institutes of Health and were approved by the Animal Experimental Ethics Committee of Wenzhou Medical University.2.The TC1, B16 and CT26 cell lines were purchased from the Cell Bank of Chinese Academy of Science . They were cultured in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) (Sigma), 100 μg/mL penicillin and 100 μg/mL streptomycin in a humidified incubator at 37 °C with 5% COCpG-ODN 2395 was provided by Synbio Technologies . The agonistic anti-OX40 antibody was provided by BioXcell . 2′3’-cGAMP was purchased from InvivoGen (Cat.# tlrl-nacga23m). CD4-, CD8- and NK-depleting antibodies were purchased from BioXcell (USA).5 TC1/B16 cells (50 μL of PBS) on day 0. When the tumour volume reached 10–20 mm3 on day 6 or 7, the mice were randomly divided into 2 groups: the PBS and αPD-1 groups. PBS or αPD-1 (100 μg/mouse) was administered by intraperitoneal injection every 2 days for 3 injections.For the construction of a mouse model for the αPD-1 treatment experiment, the left flank of each C57BL/6 mouse was subcutaneously injected with 2 × 105 TC1/B16 cells (50 μL of PBS) (primary tumour) on day 0, and the same number of TC1/B16 cells was injected subcutaneously on the other side on day 2. On day 7, when the TC1 tumour volume reached 10–20 mm3, the mice were randomly divided into the following 5 groups: PBS, CpG, cGAMP, CpG/αOX40 and CpG/αOX40/cGAMP. CpG (50 μg/mouse), αOX40 (30 μg/mouse) and cGAMP (10 μg/mouse) were administered by in situ injection only into the primary tumour every 2 days for 3 injections. When the B16 tumour volume reached 5–10 mm3, the treatment began.For the mouse model of systemic tumour, the left flank of each C57BL/6 mouse was subcutaneously injected with 2 × 10+ T cells, CD8+ T cells or NK cells were successfully knocked out. The tumour dimensions were measured with Vernier callipers every 2 or 3 days, and the tumour volume was calculated using the following equation: length × width2/2 (mm3). Mice were killed when the tumour volume reached 1500 mm3.For the cell depletion experiment, anti-CD4 , anti-CD8  or anti-NK  mAbs were intraperitoneally injected at a dose of 200 μg/mouse one day before therapy. When the treatment began, each mouse was injected with 100 μg of the anti-CD4, anti-CD8 or anti-NK mAbs every 2 days for 5 injections. Finally, a flow cytometry analysis of blood samples was performed to validate whether CD45 CT26 cells into the left flank of Balb/C mice (primary tumour) and 2 days later injected the same number of CT26 cells into the right side (distant tumour). Mice were randomly divided into 4 groups—the PBS group, αPD-1 group, CpG/αOX40/cGAMP triple group and CpG/αOX40/cGAMP/αPD-1 quadruple group—and treated on day 5 with the same drug dose as previously described. The tumour size and body weight of the mice were observed every 2–3 days. The mice were killed when the tumour volume reached 1500 mm3.To explore the efficacy of the triple drug in the PD-1-sensitive cell line CT26, we subcutaneously injected 4 × 106 cells) was stained with CD45-APC-A750, CD3e-FITC, CD4-APC, CD8-PC7, CD3-APC, CD11c-FITC and MHC II-PE antibodies and incubated at 4 °C in the dark for 30 min. After washing with staining buffer, a CyFLEX flow cytometer (Beckman Coulter) was used to conduct flow cytometry. The voltage was regulated by a negative control and single dye tube. The final data were analysed using CytExpert software.Mouse spleen tissues were completely ground in the filter and digested with a tumour dissociation kit (Miltenyi Biotec Inc.) at 37 °C for 1 h, and the suspension was filtered through 40 μm sieves to obtain a single-cell suspension. Next, the single-cell suspension was placed in 0.02% saline for approximately 20 s and then neutralized with an equal volume of 0.16% saline to lyse the erythrocytes. One hundred microlitres of the single-cell suspension  after incubation at room temperature for 2 h, and the supernatant was collected to analyse the changes in cytokine levels in the treated mice. The serum levels of Th1 , Th2  and Th17 (IL-17A) cytokines were detected using a CBA kit according to the manufacturer’s instructions.P < 0.05 was considered significant.All statistical analyses were performed using GraphPad Prism 7.0 software and SPSS 16.0. All data are presented as means ± SEM. Two-way ANOVA was used to examine the differences between groups, and Mice bearing TC1/B16 tumours were treated with αPD-1 (100 μg/mouse) 3 times, but no difference in tumour volume was observed between the treatment group and the PBS group Fig. a–b. TherA mouse model of αPD-1-resistant tumours was established, and all the treatments were only administered to the primary tumour to study the systemic efficacy of an in situ vaccine in αPD-1-resistant tumours Fig. a. For miP < 0.01. The survival time of mice in the other treatment groups was not significantly different from that of the PBS group . Regardless of what cell type was knocked out, triple therapy still exerted a strong antitumour effect on the treated side of the tumours, and no difference in the tumour volume was observed compared with the triple therapy group without antibody knockout , Th2  and Th17 (IL-17A) cytokines were noticeably increased, particularly in the triple therapy group, including both immunostimulatory cytokines and immunosuppressive cytokines  to maintain and strengthen the antitumour response of CTLs. On the other hand, even in the absence of CD8+ T cells, CD4+ T cells also directly kill tumour cells through an IFN-γ-dependent mechanism [+ T cells, CD8+ T cells and NK cells were all knocked out, indicating that both adaptive immunity and innate immunity played an essential role. Previous studies have found that an intratumoural injection of CpG/cGAMP also induces adaptive immunity and innate immunity, which further confirmed our findings [In situ injection of antitumour drugs is a practical strategy for cancer immunotherapy , 22; in bination . However” tumour , 24. Altechanism –28. In tfindings .+ T cells and inhibits the progression of B16-F10 melanoma with lung metastasis [., CpG/αOX40 therapy increases the expression of the cytokines IL-12, TNF and IFN [In addition, triple therapy fully mobilized various cytokines, including Th1, Th2 and Th17 cytokines, which play important roles in regulating innate immunity and adaptive immunity. Th1 cytokines mainly include IL-2, IFN-γ, TNF and IL-12, which promote the activation and proliferation of cytotoxic T cells and dominate cellular immunity , 30. Meatastasis . Howevertastasis –43. AccoFinally, our study found that a new combination therapy also substantially suppressed the progression of tumours that failed to respond to PD-1 immune checkpoint inhibitors. Currently, two types of tumour immunotherapy are usually used: one is to relieve the inhibitory effect of cancer cells on immune cells by administering drugs, such as αPD-1/αPD-L1 and αCTLA-4 antibodies, an approach often compared to "releasing the brakes", 45; theCompared with other tumour immunotherapies, such as adaptive cell therapy, immune checkpoint inhibitors and tumour vaccines, the in situ injection of CpG/αOX40/cGAMP triggered an effective antitumour immune response while limiting the risk of systemic exposure and associated toxicity, which not only alters the tumour microenvironment at the injection site but also produces a systemic immune response and suppresses the development of distant tumours. Of course, our research also has some limitations. First, in situ injections are only achieved when the tumour is sufficiently large and accessible, which leads to the practical limitations of tumour treatment. Second, our analysis of the antitumour mechanism of triple therapy was not performed at a sufficient depth, and the reasons why αPD-1 does not enhance the curative effect of CpG/αOX40/cGAMP are not clear; therefore, the escape mechanism requires further exploration in future studies.In summary, we designed an effective and safe drug combination. In situ CpG/αOX40/cGAMP injection generates a systemic antitumour immune response and simultaneously activates adaptive and innate immune responses, which is expected to overcome the bottleneck of ineffective treatment with PD-1/PD-L1 immune checkpoint inhibitors and provide benefits to certain cancer patients.In general, our study found that in situ vaccination with CpG/αOX40/cGAMP can be an effective treatment in a murine model of anti-PD1-resistant tumours. This method can not only activate both adaptive and innate immunity but also fully activate the production of cytokines, including Th1, Th2 and Th17 cytokines, exerting a non-negligible "heating" effect on the tumour immune microenvironment Supplementary file2 (PDF 283 KB)Supplementary file2 (DOCX 13 KB)Below is the link to the electronic supplementary material.


Gnathostoma spinigerum infective larvae (GsL3), crude somatic antigen (CSAg), excretory‐secretory antigen (ESAg) and partially purified antigens  to differentiate IgE, IgG, IgG1–4 and IgM for human gnathostomiasis diagnosis; and (2) application of the selected ELISA for following up stored sera of patients treated with ivermectin (IVM) and albendazole (ABZ).The aims of the study were two‐fold: (1) antigen (Ag) preparation and evaluation of three antigens of Different antigens were analysed by antibodies of gnathostomiasis cases, other parasite infections and healthy controls using indirect ELISA to differentiate IgE, IgG, IgG1–4 and IgM. Then, prominent antigen and immunoglobulin were used in antibody predictions of gnathostomiasis cases treated with albendazole or ivermectin.Gnathostoma worm was extracted from each two treated patients.Sensitivity of all evaluated ELISAs: IgM‐, IgG‐, IgG1‐ and IgG4‐ELISA, was 100%. IgM‐ELISA with CSAg and P3Ag exhibited the highest specificity of 99%. IgG‐ELISA with P2Ag resulted in the highest specificity of 92.3%. IgG1‐ELISA with P2Ag and P3Ag showed excellent results with 100% specificity. Finally, P2Ag evaluated IgG1 of the followed‐up cases with ABZ and IVM. Decreasing antibody IgG1 levels were mostly found in both treatments at Month 9 and long follow‐up was over 12 months. A Using IgG1‐ELISA against P2Ag and P3Ag gave excellent results with 100% sensitivity and specificity. These tests can be an alternative to immunoblotting for gnathostomiasis. IgG1 decreased at least 9 months in most cases, so long‐term treatment should be performed over 1 year. Gnathostoma larva migrates through the human body, causing cutaneous gnathostomiasis or visceral larva migrans. Since the larvae cannot develop into adults in humans [Gnathostoma worm infection is routinely applied. IgG and IgG1‐4 are preferred in studies for detecting gnathostomiasis and, based on ELISA, crude somatic antigen (CSAg) and purified antigens of G. spinigerum larvae (GsL3) are employed, although conflicting results have been obtained. As known, the IgG to purified antigens showed excellent results, but CSAg always caused low sensitivity and specificity. For example, IgG‐ELISA using CSAg of GsL3 showed 87% sensitivity and 96.7% specificity [G. binucleatum larvae cross‐reacted with 7 of 8 sera of 5 parasitoses using IgG‐ELISA, but IgG‐immunoelectrotransfer showed no cross‐reaction. Both methods provided 100% sensitivity with 11 sera of gnathostomiasis binucleatum [Human gnathostomiasis is mostly found in the tropical and subtropical regions. The n humans , 2, the arvae GsL are emplcificity . In contAll stored sera were permitted to be used by the Immunodiagnostic Unit for Helminthic Infections, the Department of Helminthology, Faculty of Tropical Medicine, and approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand No. TMEC 14‐067). Thirty gnathostomiasis (homologous) sera were positive by worm recovery or immunoblotting test . Among o4‐067. ThThirty‐five gnathostomiasis patients with symptoms and signs of migratory swelling, local swelling, history of fish consumption, positive 24 kDa immunoblot and GsL3 recovery after treatments with ABZ and IVM were selected from the records. Occasionally, worms migrating near skin surfaces were found in ABZ‐ or IVM‐treated patients. Experienced physicians would extract those worms by needle or biopsy. Stored sera were divided as follows: group 1, 23 patients treated with 400 mg of albendazole twice daily for 21 days; and group 2, 12 patients treated with ivermectin at 0.2 mg/kg as a single dose. Before treatments, each patient's serum was collected as Month 0 (M 0), and the treatments with ABZ or IVM sera were collected every 3 months of post‐treatment (mpt) called M3, M6, M9, M12 and others. The antibody patern of post‐treatments was followed up until regularly decreased to a normal level . Such decreasing OD value to a normal level indicated successful ABZ or IVM treatment.GsL3 was obtained from the livers of naturally infected swamp eels purchased from a market in Bangkok. Larvae were used in preparations of three kinds of antigens: crude somatic antigen (CSAg), partially purified antigens  and excretory‐secretory antigen (ESAg). CSAg was prepared by grinding larvae with alumina powder in distilled water, then sonicating and finally obtaining the antigen by centrifugation . The par2 for 6 days. The medium was collected, changed to a new medium every 24 h, pooled, dialysed against ddH2O and subsequently concentrated by Amicon at the cut‐off range of 5 kDa (PM‐5 membrane). The protein concentration of ES was determined as mentioned earlier.Excretory‐secretory antigen was prepared as previously reported with some modifications . BrieflyIndirect ELISA was employed to evaluate Ig isotypes and IgG subclasses against various antigens as well as follow‐up analysis after anthelminthic treatments. The ELISA was performed as described previously with some modifications . After cThe best sensitivity and specificity of the ELISA were used in determining the antibody pattern of post‐treated patients every 3 months of the follow‐up period.Thirty gnathostomiasis sera and 209 heterologous sera  were used for receiver operating characteristic (ROC) curve analysis. The ELISA cut‐off point was chosen using ROC curve analysis as sensitivity versus 1‐specificity or false positive. SPSS version 15 was used as a tool for ROC curve analysis . The accGsL3 antigens; CSAg, ESAg, P1Ag, P2Ag and P3Ag, were analysed for their protein qualities and patterns by SDS‐PAGE before analysis by ELISA Figure . For stuThe appropriate conditions were obtained from checkerboard titration: IgM‐ELISA, (1) 0.125 µg/ml CSAg, 1:1600 human serum and 1:8000 HRP‐conjugated mouse anti‐human IgM; and (2) 0.5 µg/ml of each of ESAg, P2Ag and P3Ag, 1:1600 serum, and 1:4000 of such conjugate. For IgG‐, IgG1‐ and IgG4‐ELISAs, their conditions were as follows: (1) 1 µg/ml CSAg, 1:800 serum and 1:8000 HRP‐conjugated mouse anti‐human IgG, ‐IgG1 and ‐IgG4; and (2) for ESAg, P2Ag and P3Ag, 0.5, 4 and 4 µg/ml, respectively, 1:800 serum, and 1:4000 of such conjugates. The optimal conditions of IgE‐, IgG2‐ and IgG3‐ELISAs using all antigens, CSAg, ESAg, P1Ag, P2Ag and P3Ag could not discriminate between pooled positive and pooled negative sera. Therefore, these tests were not further evaluated. In addition, all Igs with P1Ag showed the same evidence of reactions.Human gnathostomiasis cases, other parasitoses and healthy control sera were employed to determine the sensitivity, specificity and predictive values of ELISA.All antigens to IgM‐ELISAs presented high sensitivity of 100% and high specificity in the range of 91%–99%, with CSAg and P3Ag presenting 99% specificity following cut‐off values of 0.0835 and 0.130, respectively. The OD values of gnathostomiasis cases to CSAg were not far from each other and in the range of 0.086–0.200, while those values of this group to P3Ag were spread across a wider range of 0.131–0.354, with OD values of three cases being outliers. For angiostrongyliasis and paragonimiasis cases, two false positives were using CSAg, giving OD values slightly over the cut‐off value, while P3Ag showed two false positives for strongyloidiasis Figure .IgG‐ELISA against P2Ag demonstrated the highest sensitivity and specificity of 100% and 92.3% respectively /ABZ, six cases demonstrated decreased antibodies from the first 3‐month observation to at least 9 months of follow‐up. These cases appeared to have been cured of gnathostomiasis Figure . Seven cGnathostoma worm was extracted from each of the patients treated with ABZ or IVM.In the history of treatments, one G. spinigerum infection, use of various types of Ag and infections with a large number of different parasites [Immunodiagnoses of gnathostomiasis have been consecutively developed to improve their efficiency. Several strategies to improve the reliability of diagnosis suggested promising results based on the evaluation of immunoglobulins specific to arasites , 17, 18 In our study, the checkerboard titrations of IgE‐, IgG2‐ and IgG3‐ELISAs using CSAg, ESAg, P1Ag, P2Ag and P3Ag, could not discriminate between pooled positive and pooled negative sera. Those Ags might be non‐immunogenic components to the positive controls; therefore, the detection of IgE, IgG2 and IgG3 was not continued. This study attempted to conclude 25 parasitic infections with many samples (n = 179) to achieve the best specificity of tests. For IgE‐ELISA, the low specificity presented in this study was consistent with a previous study showing low sensitivity and specificity against CSAg and ES of GsL3 . MoreoveIgM‐ELISAs demonstrated a sensitivity of 100% for all Ags, along with a remarkable specificity of 91.9%–99%, while CSAg and P3Ag showed 99% specificity. A study on IgM for gnathostomiasis is uncommon, but our work demonstrated the excellent specificity for CSAg and P3Ag. Regarding the timeline of the responsive antibody, IgM rapidly responds and can be detected at an early stage during the first 2 weeks, followed by IgG antibody , 23. IgGIgG‐ELISA against CSAg and ESAg in our study presented low specificity, which was consistent with a previous study that showed high cross‐reaction of heterologous infections using CSAg . HoweverFor IgG subclass‐ELISA, IgG1 was an excellent immunoglobulin to bind on these four Ags at 100% sensitivity and 95%–100% specificity. Using P2Ag and P3Ag, IgG1‐ELISA achieved perfect results of 100% sensitivity and 100% specificity. However, P2Ag can well separate positive gnathostomiasis controls from heterologous sera compared with P3Ag at their cut‐off values Figure . GeneralAnthelminthic treatment is a standard method for curing gnathostomiasis, with albendazole (ABZ) or ivermectin (IVM) being the drugs of choice . It is kRegarding the data above , 27, 29,IgM and IgG1 were shown to be optimal for human gnathostomiasis diagnosis using CSAg, P2Ag and P3Ag (100% sensitivity and >99% specificity). The use of IgG1‐ELISA with P2Ag and P3Ag achieved perfect results of 100% sensitivity and 100% specificity. These tests can be an alternative method for the immunoblotting test of human gnathostomiasis. Screening and early detection tests require CSAg and P3Ag to IgM (100% sensitivity and 99% specificity). Therefore, the follow‐up duration should be at least 12 months.The authors declare that they have no competing interests.Figure S1Click here for additional data file.


Funding statement. We failed inadvertently to acknowledge one of our funding bodies in the “Funding” section. The Funding statement read as “This work was supported by a grant from Stavros Niarchos Foundation (ST and MZ), by RSF (Grant No. 16-14-00215) (VT and II), by RFBR (Grant No. 18-04-01366) (IK) and by BioStruct-X (Contract No. 283570/3521).”In the published article, there was an error in the The correct statement is: “This work was supported by a grant from Stavros Niarchos Foundation (ST and MZ), by RSF (Grant No. 16-14-00215) (VT and II), by RFBR (Grant No. 18-04-01366) (IK), by the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (PG),  and by BioStruct-X (Contract No. 283570/3521).”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.