Underground ant nests are typically made of soil and rubble mixed with dead plant bodies, ant secretions, and other organic substances. The clay content is high, and the natural clay materials show pozzolanic activity after calcination. In this study, the underground ant nest materials of Iridomyrmex anceps, which is a common ant in the Shanghai area, are calcined and ground, and the material properties of calcined ant nest clay powder (CANCP) are characterized from six aspects: chemical composition, particle morphology, specific gravity, specific surface area, particle size distribution and pozzolanic activity index. The pozzolanic activity of CANCP is evaluated by the strength contribution rate of pozzolanic activity, revealing that CANCP is beneficial to the strength of the mortar system from an early age. The influence of CANCP on the compressive strength of concrete is analyzed using three aspects, namely, content, curing age and calcination temperature, and it is found that the three aspects of CANCP have a strong influence on the compressive strength of concrete.  Ants are excellent architects in the animal world; their nests contain unique structures that feature excellent heat preservation, moisture retention and ventilation function. However, ants have strict demands on the building materials of nests, such as the soil particle size, weight, luster and color. Underground ant nests are typically made of soil and rubble mixed with dead plant bodies, ant secretions and other organic substances, and the content of clay is high. The clay of underground ant nests is composed of earth particles cemented together to form a hard brick-like material. Natural clays typically contain a mixture of clay minerals associated with non-clay minerals. Most natural pozzolans develop their activity from clay minerals such as kaolinite, montmorillonite, and illite. Each type of clay has an optimum calcination temperature range that causes the breakdown of the crystalline structure of the clay and the formation of amorphous silica and alumina . Some po+, Na+, Ca2+, and Mg2+ ions in anthills [2 production [Ants change the physical characteristics of the soil through nesting activities , which canthills  and alteanthills . Throughanthills . Such acanthills . Ant actoduction  and soiloduction . Throughoduction . Thus, rIridomyrmex anceps belongs to the Iridomyrmex category and is distributed across China. Ant nests are underground, and anthills consists of soil particles, the Pinus massoniana male flower, grass, and frass. Anthills are packed into a grave shape, spherical shape, or irregular shape. In this study, the underground nests of Iridomyrmex anceps were sampled from three locations. 2O3 at high temperature under aerobic conditions. The ant nest soil quality at 600 °C and 800 °C after calcination led to masses of 39.25 kg and 38.74 kg and a loss on ignition of 21.5% and 22.5%, respectively. The optimum calcination temperature and time of clay minerals depends on the types of clay minerals, and clay can exhibit potential pozzolanic properties when treated under appropriate calcination temperatures and times. Clay has the greatest activity when it is calcined at 600–800 °C . Thus, 62, Al2O3 and Fe2O3 in 800 °C CANCP and 600 °C CANCP reached 88.76% and 83.37%, respectively, both greater than the 70% specified in the ASTM standard for concrete mineral admixtures [An SM-500 ball crusher was used for milling of the ant nest soil for 60 min after calcination. The chemical composition of CANCP was analyzed using an X-ray fluorescence spectrometer. The total content of SiOmixtures . The resmixtures ,28,29,302/kg. After two tests, the margin of 45 μm square hole sieves was measured as 16.25% and 16.58%, close to the 12% requirement of first-grade fly ash . The speThe particle size distribution of CANCP was determined using a Winner laser particle size analyzer. The results are shown in The pozzolanic activity index of CANCP of 800 °C calcination was evaluated using two methods: the lime absorption method (direct) and the strength index method (indirect).2+ and OH− in solutions of different ages. Lower concentrations of Ca2+ and OH− indicate higher pozzolanic activity [2 at 40 °C). Thus, CANCP material has pozzolanic activity; see The lime absorption method involves mixing a pozzolanic material with a Portland cement slurry or a saturated lime solution, and the activity of pozzolanic material is quantitatively evaluated by measuring the concentrations of residual Caactivity . The actactivity , metakaoactivity , and claactivity  has prevactivity , and theThe strength index method compares the 28 days compressive strength of the contrast mortar of a certain mineral admixture with an equal mass of cement to that of a reference mortar, and the ratio of these two compressive strengths is defined as the activity index of the composite mineral admixture. Among all mineral admixtures, fly ash is the most commonly used and systematically characterized, and recycled powder can be used to grind waste sintered clay brick to a certain fineness, similar to CANCP; fly ash can also be treated through clay calcination and grinding processes. A comparison of the two types of materials would be meaningful; thus, according to GB/T 18736-2002 [PO 42.5 ordinary Portland cement conforming to GB175-2007  was used3, and the average particle size is 16.14 μm.Recycled powder in this study was prepared by smashing, grinding, drying and grading abandoned clay bricks and cement solids. The chemical composition and physical and mechanical properties are listed in The standard sand was ISO679 standard sand. Milled standard sand was obtained by ball milling of the ISO standard sand for 2 h with a SM-500 ball mill, and the sieving test was performed using a FSY-150E cement fineness negative-pressure sieve analyzer. The margin of a 45 μm square hole sieve was 17.55%, the fineness control requirements are close to that of CANCP, and the average size of the ground standard sand was 23.29 μm. The particle size distributions of CANCP, fly ash, recycled powder, and milled standard sand are plotted in Specimens for strength index tests were prepared following GB/T17671-1999 . The cemThe mass mix ratio of reference mortar CT was one part cement, three parts standard sand and half-part water. The reference mortar SS used standard sand instead of 30% quality cement, and the milled standard sand mortar GSS used milled standard sand instead of 30% quality cement. The replacement amount of other pozzolanic ash materials was 30%. The mix design was modified as shown in The CANCP mortar and contrast mortar of each group were cured for 3 days, 7 days, 28 days and 90 days to test their flexural strength and compressive strength following GB/T17671-1999 . FlexuraThe test results are shown in The reference mortar SS uses standard sand instead of cement of the same quality because the standard sand has no pozzolanic activity and because the filling action is weak; thus, the amount of cement decreases. This condition directly results in decreased compressive strength in the mortar, and thus the compressive strength is lower than that of the other mortar after 3 days. The mortar GSS uses milled standard sand instead of cement of the same quality. Milled standard sand also has no pozzolanic activity, but its compressive strength is higher than that of the reference mortar (SS); thus, the micro-filling effect of milled standard sand plays an active role. The compressive strength of CANCP mortar begins to exceed mortar GSS at 7 days, as shown in The activity index of CANCP, fly ash and recycled powder was 76.3, 71.9 and 71.3, respectively, and the activity index of CANCP was larger than that of fly ash and recycled powder and greater than the value of 65 specified in the GB/T2847-2005 standard . These rThe pozzolanic activity index obtained from the compressive strength ratio reflects only whether the admixture has pozzolanic activity; the index does not reflect the extent of pozzolanic activity of the admixture. Pu Xincheng  introducraR, i.e., the strength contribution of unit cement to concrete, is defined as follows:(1) The concrete compressive strength ratio of the mineral admixture raR is the specific strength of concrete containing an admixture (MPa), aR is the absolute value of the strength of concrete containing an admixture (MPa), and q is the percentage of cement content in the concrete cementitious material.In this expression, rrR is defined as follows:(2) The specific strength of reference concrete without mineral admixture rrR is the specific strength of concrete (MPa); aR is the absolute value of the strength of concrete.In the expression: rrR and raR is the specific strength of the pozzolanic effect contribution:(3) The difference between rpR reflects the contribution of the pozzolanic effect of mineral admixtures to the strength of the entire system. A negative value shows that mineral admixtures reduce the strength.hP is defined as follows:(4) The strength contribution rate of cement hydration reaction pP is defined as follows:The strength contribution rate of the pozzolanic effect The pozzolanic activity of CANCP, standard sand, milled standard sand, fly ash, and recycled powder was evaluated using this method. The results are shown in The strength contribution rate of the pozzolanic effect of mortar SS was negative for all ages, and the standard sand had no pozzolanic activity. Thus, standard sand instead of equal-quality cement reduces the strength of cement mortar. The 3 days strength contribution rate of the pozzolanic effect of mortar GSS is 11.73 because it has no pozzolanic activity. For intuitive comparison, the pozzolanic effect strength contribution rate is used to describe its strength in cement paste, and the strength of the pozzolanic effect at this time is contributed by the filling effect of the milled standard sand. The strength contribution rate of the pozzolanic effect of mortar GSS is zero or negative with increasing age, indicating that when milled standard sand is replaced by equal-quality cement, the strength of the cement mortar can decrease, and the contribution of the filling effect of milled standard sand to the strength of the cement mortar appears primarily at early ages. The strength contribution rate of the pozzolanic effect of mortar FA increases with age, and the 3 days strength contribution rate of the pozzolanic effect of mortar FA is negative. This result shows that fly ash has an adverse effect on the early strength of the complete mortar system. The strength contribution rate of the pozzolanic effect is positive starting from the age of 7 days, and the fly ash begins to have a beneficial effect on the strength of the entire mortar system; the 90 days strength contribution rate of the pozzolanic effect of mortar FA is 18.96. The strength contribution rate of the pozzolanic effect of the recycled powder RP is positive at all ages, and the 3 days strength contribution rate of pozzolanic effect is as high as 11.27. Thus, the beneficial influence of recycled powder on the entire mortar system is reflected primarily in the early strength, and the 90 days strength contribution rate of pozzolanic effect is 11.93, with a long-term pozzolanic effect that is less than that of fly ash. The strength contribution rate of the pozzolanic effect of mortar CAP increases with age and is positive. CANCP begins to show a favorable effect on the strength of the mortar system from an early age, and the 90 days strength contribution rate of the pozzolanic effect of mortar CAP is 13.91. The long-term pozzolanic effect of CANCP is larger than that of recycled powder but smaller than that of fly ash.PO 42.5 ordinary Portland cement conforming to GB175-2007  was usedA cubic specimen measuring 100 mm × 100 mm × 100 mm was used to study the compressive strength of concrete following GB/T 50081-2002 . The mol3, the ratio of water to binder was 0.35, and the sand content was 42%. The CANCP with 800 °C calcination was used to replace cement in proportions of 5%, 10%, 15%, 20% and 30%, and the CANCP with 600 °C calcination was used to replace cement in proportions of 10% to study the effect on concrete properties. By adjusting the dosage of water-reducing agent, concrete slump was controlled near 200 mm. The mix proportion and concrete slump are shown in In this experiment, CT was the reference concrete, i.e., ordinary concrete, and a standard mix proportion of design strength of C50 was selected. The calculated mix proportions for the ordinary concrete CT were (cement: water: sand: stone = 486:170:741:1023). For the other six sets of concrete, the total amount of cementitious material was 486 kg/mThe compressive strength of concrete was tested following GB/T 50081-2002 . The tesFor a more intuitive observation and analysis of test results, the ratio of the compressive strength of concrete with varying CANCP content to the compressive strength of reference concrete was used as the relative compressive strength of CANCP concrete. The relationship between the compressive strength of concrete and the CANCP content is shown in The content of CANCP has a strong influence on the compressive strength of concrete. With increasing CANCP content, the cubic compressive strength of concrete first increases and then decreases. Each curve has a peak value, and the compressive strength of the concrete containing 10% CANCP is the largest. The compressive strength for curing times of 3 days, 7 days, 14 days, and 28 days increases by 20.8%, 15.5%, 18.9%, and 18%, respectively, and the early strength increases quickly. When the CANCP content is 20%, the compressive strength of concrete is essentially identical to that of ordinary concrete CT without CANCP. When the CANCP content is more than 20%, the compressive strength of concrete begins to decrease, and the compressive strength for 3 days, 7 days, 14 days, and 28 days curing is reduced by 3.2%, 1.6%, 15.8%, and 15.4%, respectively. The early strength decreases less. Adding CANCP to concrete can improve the strength of concrete, but the content should be less than 20%. The optimum content in this experiment is 10% of the cementitious materials.The development curves for the compressive strength of concrete containing varying CANCP content with age are shown in The compressive strength of concrete containing various CANCP content increases with age. The development of early strength is faster, and the 3 days compressive strength reaches the 28 days strength of 60%, the 14 days compressive strength reaches the 28 days strength of 85%, and the compressive strength between 14 days and 28 days of curing is smaller; the 90 days compressive strength is more than 1.05 times the strength at 28 days. To characterize the changes in the compressive strength of CANCP concrete with age, the relative compressive strength of CANCP concrete at different ages was determined by the ratio of the strength at each age to the strength at 28 days, as shown in cu(t)/fcu(28)) was defined as the age coefficient of concrete (Y). Using the mathematical model Y = A ln (T) + B, where A and B are constants, regression analysis was performed on the relative compressive strength and age of concrete with CANCP content of 5%, 10%, 15%, 20%, and 30%, as shown in The ratio of the compressive strength of CANCP concrete at a certain age to the 28 days compressive strength 2 crystallizes as plates with a large size, small specific surface area, low surface energy, weak interfacial bonding, and a simple orientation arrangement on the surface of the aggregate. Ca(OH)2 acts as an isolation effect between the C-S-H gel and the aggregate and plays a negative role in interfacial adhesion. To increase the strength of concrete, the amount of C-S-H gel should be increased, and the amount of Ca(OH)2 should be reduced [The action mechanism of CANCP in concrete can be explained by the three microstructural phases of concrete, which are hydrated cement paste, aggregate, and the interfacial transition zone between cement paste and aggregate. The influence of the interfacial transition zone on the mechanical properties of concrete is much greater than that of other factors . Interfa reduced . Figure 2O3 and SiO2 formed by the removal of hydroxyl groups can undergo a second hydration reaction with the cement hydration product Ca(OH)2, and this reaction, called the pozzolanic reaction, can generate C-S-H gel. During the pozzolanic reaction, Ca(OH)2 in the hydrated cement paste is consumed, and the amount of C-S-H gel increases. CANCP can also react with the hydration product C-S-H gel in the hydrated cement paste to create additional C-S-H called the pozzolanic C-S-H gel. The composition and properties of pozzolanic C-S-H gel and traditional C-S-H gel are different; the latter is relatively stable after polymerization with OH- and Al2+ and can improve the performance of the traditional C-S-H gel to improve the performance of cement mortar, therefore improving the compressive strength of concrete. In CANCP has strong pozzolanic activity. The amorphous Al2 exists in the interface area of concrete, which makes the interface a weak area. CANCP has a large specific surface area and a large surface energy and can rapidly react with Ca(OH)2 for secondary hydration reactions, reducing the content of Ca(OH)2 in the interface region, also decreasing the crystal size of Ca(OH)2 [2 is found in the hydrated cement paste of the ordinary concrete, but Ca(OH)2 is not found in the cement hydration paste of concrete containing 10% CANCP. CANCP can react with Ca(OH)2 for secondary hydration reactions and can consume the content of Ca(OH)2 in the interface region. The product of reactions between CANCP and concrete can improve the interface structure between cement paste and aggregate. A large amount of Ca(OH) Ca(OH)2 . TherefoThe average particle size of CANCP is 23.35 μm, making it a relatively fine powder particle. Concrete is a granular accumulation system with continuous gradation, and the voids of each aggregate are filled with graded particles smaller than the voids. CANCP particles fill a wide range and can fill the fine pores in hardened cement paste, reducing the porosity of the paste. However, hydration reactions can produce hydrated calcium silicate and calcium sulphoaluminate hydrate with the filling effect and can optimize the internal pore structure of concrete, making the hardened cement paste and concrete stronger and denser. The hydration reactions can also strengthen the hardened cement paste and concrete to be more resistant to external deformation. Under low-magnification electron microscopy, the inner holes of the ordinary concrete are more numerous and are larger, and the surface morphology is not smooth or loose. Instead, there are few holes in the concrete containing 10% CANCP, and its surface is flat and dense.2, Al2O3 and Fe2O3 for 800 °C CANCP and 600 °C CANCP reached 88.76% and 83.37%, respectively, which are both greater than the 70% specified in the ASTM standard.The results of chemical composition analysis indicate that the total contents of SiOIt was shown via two methods (the lime absorption method and the strength index method) that CANCP has pozzolanic activity.The strength contribution rate of the pozzolanic effect of mortar CANCP is positive and increases with age. CANCP has a favorable effect on the strength of the mortar system from an early age.The compressive strength of concrete is maximized at 10% CANCP, and the early strength increases quickly. The compressive strength of concrete containing 20% CANCP is similar to that of the reference concrete without CANCP. When the content of CANCP is above 20%, the compressive strength of concrete begins to decrease, and the early strength decreases less.The compressive strength of concrete with various CANCP content increases with age, and early strength develops faster. Increased strength is generally consistent with age when the CANCP content is not more than 15%.The compressive strength of concrete containing CANCP with 800 °C calcination is higher than with calcination at 600 °C. CANCP with 800 °C calcination thus shows superior pozzolanic activity.CANCP can improve the strength of mortar and concrete mainly because it can accelerate the hydration of cement and exhibits the pozzolanic and filling effects. Accelerating cement hydration and the pozzolanic effect is important for improving the strength of mortar and concrete, to which the filling effect also contributes in a secondary manner.The results of experimental studies on calcined underground ant nest materials were presented, and the following conclusions were drawn:


Immunoglobulin G4- (IgG4-) related inflammatory abdominal aortic aneurysm (AAA) has been recognized as a manifestation of IgG4-related disease (IgG4-RD). We experienced one patient with multiple consecutive manifestations before and after endovascular stent grafting for IgG4-related inflammatory AAA (IAAA). A 71-year-old man was diagnosed with IgG4-RD due to increased IgG4 serum concentration, typical findings of parotid gland biopsy, and periaortitis in another hospital 2 years and 7 months before visiting our hospital. He came to our hospital because of abdominal pain and IAAA. He developed paraplegia after hospitalization and underwent endovascular stent grafting for the IAAA. About one month after stent grafting, he developed perforation of the sigmoid colon due to enteritis. He also had myocardial infarction. Finally, he died of intestinal bleeding. Here, we describe this case with rare, multiple, consecutive manifestations of IgG4-RD, some of which might be caused by IgG4-related IAAA or side effects of treatments rather than by IgG4-RD itself. We report this case because the clinical course seemed rare for IgG4-RD or IgG4-related IAAA. For treating IgG4-RD with IgG4-related IAAA, we should consider factors causing the symptoms and carefully select the proper treatment. Immunoglobulin G4-related disease (IgG4-RD) is a systemic condition with elevated serum IgG4 levels and infiltration of IgG4-positive plasma cells in organs such as the biliary tree, salivary glands, and retroperitoneum. IgG4-RD is commonly responsive to glucocorticoid therapy . In the A 71-year-old male patient visited another hospital due to abdominal pain 2 years and 7 months before visiting our hospital. He also had swelling of the lymph nodes in the neck and both inguinal regions. He had been diagnosed with peritoneal fibrosis, periaortitis, and bilateral parotitis based on a computed tomography (CT) scan  and physHe then visited our hospital due to abdominal pain since the last 2 months. A CT scan showed a 44 mm diameter AAA with thickened adventitia . It was Even after restarting PSL treatment, he had abdominal pain several times and we could not completely exclude the possibility of the IAAA impending rupture. We performed endovascular stent grafting for the IAAA on day 13 after hospitalization. Under general anaesthesia, the bilateral inguinal portion was incised. The main body of the Excluder®  was inserted and deployed through the left common femoral artery. Through the right common femoral artery, two stent graft limbs were inserted and deployed. Both internal iliac arteries were kept patent. Macroscopically, the adventitia of the right femoral artery was thickened. Microscopically, the intima and media showed atherosclerotic changes, without immunostaining of IgG4.On day 6 after stent grafting, he had strong pain in both lower limbs and systemic cyanosis. His systolic blood pressure increased to over 200 mmHg. PSL dose was increased to 50 mg/day, and intravenous treatment of prostaglandin E1 was started.On day 7 after stent grafting, he had bloody stools. Upper and lower intestinal endoscopy showed atrophic gastric mucosa, duodenal erosion, and ulceration and erosion from the sigmoid colon to the rectum. He was diagnosed with ischemic colitis. On day 34, he had strong pain in the abdomen and both lower limbs. A CT scan showed free air in the abdomen and emergent abdominal operation was performed. Macroscopically, he showed ischemic changes from the ileum to the upper rectum and perforation of the sigmoid colon. The intestine and peritoneum were strongly adhesive, and we could not observe some IgG4-RD related organs, such as pancreas and biliary tract. The lymph nodes were not swollen in the mesentery. The wide portion from the ileum to the rectum was resected and colostomy was performed. The pathology of the intestine showed cytomegalovirus colitis and ganciclovir was soon started. He was transferred for rehabilitation to another hospital on day 124 after admission and on day 111 after stent grafting.About one month later, he was transferred to our hospital again with acute myocardial infarction. Aspiration of the thrombus and stenting for the occluded left descending artery were performed . Three mWe reported the case of a patient with rare, multiple, consecutive complications of IgG4-related IAAA. IgG4-RD is a disease concept proposed by Hamano et al., based on the relationship between autoimmune pancreatitis and IgG4 . Its manThe manifestations or diseases that developed after he came to our hospital  seemed to be atypical of IgG4-RD. We cannot be sure whether they were related to IgG4-RD.Paraplegia is very rare with IgG4-RD, but there are some reports of paraplegia from sclerosing pachymeningitis , 13. We Myocardial infarction is also rare in IgG4-RD, but reports exist , 15. SakRegarding his enteritis, he had ischemic and cytomegalovirus enteritis. First, he was diagnosed with ischemic colitis with endoscopy, and, at the time of perforation of the sigmoid colon, the pathology showed cytomegalovirus enteritis. There was a report on cytomegalovirus enteritis along with ischemic enteritis , as wellOne of the most important factors that influenced atypical manifestations of IgG4-RD was PSL. Glucocorticoids are associated with an increased risk of myocardial infarction –22. As pAnother factor might be arterial spasms. He had high blood pressure, systemic pain rather than local pain, and systemic cyanosis when his condition worsened. We believe that his coronary artery, intestinal artery, limb artery, and spinal cord artery were spastic and caused the ischemia of the related organs. We administered nitric acid medicines, calcium antagonists, and prostaglandin E1, but the effects were not clear. We believe that the vascular spasms were related to his clinical conditions, although we could not find studies on the relationship between IgG4-RD and vascular spasms.Another strong possibility is that he had atheromatous embolization, which could explain his enteritis, myocardial infarction, pain in the bilateral lower limbs, and anterior spinal artery syndrome. IgG4-related IAAA, which does not have only features of IgG4-RD but also those of AAA, might have caused or been related to atheromatous embolization and some complications.μL, C-reactive protein level was 1.18 mg/dl (We thought of other differential diagnoses, but his conditions did not fit into any of them. When he came to our hospital, antinuclear antibody was negative, white blood cell count was 8840 18 mg/dl , and he Thus, we reported the case of a patient with IgG4-related IAAA who had parotitis, paraplegia, enteritis, and myocardial infarction before and after stent grafting. The clinical course seemed rare for IgG4-RD. We speculate that some symptoms or diseases were caused by atheromatous embolization which might be related to IAAA, corticosteroids, or arterial spasms rather than IgG4-RD itself. During the treatment of IgG4-RD, especially if complicated with IgG4-related IAAA, we should consider if the symptoms are caused by IgG4-RD or other factors and carefully select the proper treatment.


Staphylococcus aureus and Gram-negative Vibrio cholerae using XTT assay and growth kinetics assay. Results suggested higher efficacy of nanoparticles for Gram-negative, therefore further analyzed against Escherichia coli that confirmed its potency for the same. AuNPs-SL also signifies its efficiency at least metabolically active state; non dividing cells and biofilm of these microbes. Induced morphological changes were studied by SEM that revealed AuNPs-SL led to disruption of cell membrane and leakage of intracellular fluid to the surroundings. Inhibition of respiratory enzymes activity also plays a crucial role in bactericidal action as indicated by LDH assay. Synergy of AuNPs-SL with different antibiotics was also analyzed using checkerboard assay. These results suggested the possible use of AuNPs-SL as an antimicrobial therapy in the field of nanomedicine.Microbes develop several strategies to survive in the adverse condition such as biofilm formation, attaining non-dividing state, altering drug target or drug, thereby increases the burden of drug dosage. To combat these issues, nanoparticles have shown an alternative approach for new treatment strategy but synthesis via chemical synthetic route limits their application in biomedical field. Here, green method for the synthesis of gold nanoparticles using sophorolipid (SL) is discussed that is characterized by various techniques. Initially, the antimicrobial activity was checked against metabolically active state of microbes; Gram-positive  Among different survival strategies under adverse conditions, formation of biofilm and achieving dormant or non dividing phase are the privilege one2. A survey conducted by National Institutes of Health and Centre of Disease control suggested that most of the infection (nearly 65–80%) occurred by biofilm formation3. Biofilms are complex mixtures of bacteria, containing both states of bacterial existence; multiplying and non-multiplying bacteria4. In biofilm, cellular metabolic activity is reduced and cells are more resistant to drugs. A significant proportion of most human bacterial infectious disease (nearly 60%)5 is encompassed by non-multiplying bacteria and these are not easily killed by antibiotics6. Multiplying bacteria are killed easily in presence of antibiotics as compared to non-multiplying ones. Therefore, designing better therapeutic strategies, and/or modification and augmentation of the existing therapies are highly essential to combat these problems. As an alternative to the existing therapies, nanoparticles have shown greater potential to combat with the problem and are presently being used for several purposes such as; diagnosis of diseases, cancer sensing and therapy7, in vivo and in vitro cell labelling8, antimicrobial agents in implants9, cosmetics10, food11 and fabric industries12. Therapeutic efficacy and the mode of action for killing the microbes have given an edge to the nanotherapeutics. Nanoparticles  are synthesized either chemically or biologically, have shown promising results for antimicrobial activity against diverse spectra of microbes14. Among these, metal and metal oxide nanoparticles are shown to have a wide range of antimicrobial activity since the ancient time.In recent years, the emergence of antimicrobial resistance has been recognized as one of the most compelling problem in the field of biomedical sciences. Development of resistance has been reported even against the newly invented drug candidates. The reasons behind the emergence of bacterial resistance are complicated, but it can be discriminated as, partly due to the selection of bacterial mutants by antibiotics and partly due to reduced dosage intake by patients that lead to reduced levels of antibiotics which in turn, trigger bacterial resistance. Multiplying (logarithmic phase) and non-multiplying  are two different states of bacterial existence15, biomedical 14, electrical16, electrochemical energy applications17. Recently, gold nanoparticles (AuNPs) have gained profound attention due to high biocompatibility and nontoxic nature. AuNPs are used as carriers of biomolecules  for cellular imaging, molecular diagnostic, photothermal agents etc18. Synthesis of metallic nanoparticles can be achieved mainly by two methods; one is chemical synthesis and the other is green synthesis. Though chemical synthesis is the most frequently used method for nanoparticles synthesis, but involvement of various hazardous chemicals and solvents restricted their application in biomedical fields19. The final qualities of the synthesized nanoparticles are determined by the intrinsic nature of metal and capping agents apart from solvent system used for synthesis. Therefore, biological synthesis has emerged as a new strategy for reducing or/and stabilizing the nanoparticles meeting the requirement of clean, least toxic and environmentally benign and thus enhances the horizon of their applications in biomedical fields20. The most commonly used ideal alternatives for the greener synthesis of metallic nanoparticles are whole microorganisms (serving as nanofactories) or their products and plant extracts22. The nature of these extracts is generally starch, protein, lipids or their derivatives. Among Lipid, glycolipids are the most frequently used for greener synthesis of metallic nanoparticles.The unique characters of metallic nanoparticles like optical, electrical, physio-chemical and ease of surface fabrication with desired molecules, have widen the application window of nanoparticles in several fields such as biosensorsβ-D-glucopyranosyl-D-glucopyranose) linked β glycosidically to the hydroxyl group at the penultimate carbon of fatty acids23. Sophorolipids have been well established for biocompatibility and antimicrobial for diverse group of microbes such as bacteria, fungi, viruses24. Apart from these, SLs being biodegradable, least ecotoxicity, and cost-effective (as they can be produced using renewable resource substrates) provides an edge over other biomolecules25. The US FDA has also approved these biosurfactants/sugar esters for the use in food and pharmaceuticals26. Understanding the significance of SLs, it has been used as reducing/capping for the synthesis of different metallic nanoparticles27.Glycolipids are amphiphilic biomolecules having lipids as backbone and carbohydrate as the head part that is linked by a glycosidic bond, in which the carbohydrates moieties can vary from monosaccharide to polysaccharide. One of the important class of glycolipids is sophorolipids (SLs), in which disaccharide sophoroses  assay was performed. Different drugs having different mechanism of antimicrobial action were used to examine the synergistic effects with AuNPs-SL against these microbes.From the green chemistry prospect, the fundamental requirements for the preparation of nanoparticles are (i) the choice of solvent medium and (ii) non-toxic reducing and stabilizing agents. In the present work, gold nanoparticles were prepared using sophorolipid as reducing and stabilizing agents that were characterized using different techniques. Two pathogenic strains Gram-positive 4, represented as SBH). For this few drops of 100 mM sodium borohydride was used and thus, highly monodispersed sophorolipid capped gold nanoparticles reduced with NaBH4 (AuNPs-SL-SBH) were obtained.Synthesis of gold nanoparticles was performed using sophorolipid as reducing and capping agent. Various parameters such as pH of gold solution, SL concentration and temperature were optimized for synthesis. Finally 400 µg/ml concentration of SL and 80 °C were found to be best for sophorolipid capped gold nanoparticles (AuNPs-SL) synthesis. UV-visible spectrum of the synthesized nanoparticles was obtained by measuring the absorption in the range of 300–700 nm with maxima at 530–540 nm Fig. . MeasureThe UV-Visible spectra of AuNPs-SL-SBH Fig.  have sho29 (nearly 10% mass loss) and afterward due to sophorolipid30 (more than 20%). A similar pattern of thermal degradation was also observed by Niki et al.,(2013) where they found the multilayer decomposition of sophorolipids-functionalized iron oxide nanoparticles31. This data also suggest the compact packing of gold nanoparticles by sophorolipids.Capping by SL to AuNPs was also confirmed by thermogravimetric analysis (TGA). Figure −1), C = O group in carboxylic acid COOH (n = 1747–1738 cm−1), COH and CO of sophorose moiety  confirming the capping of AuNPs-SL by sophorolipids. All these details affirm the complexation of sophorolipids to gold nanoparticles and further matches with earlier findings32. Thus, capping of SL to AuNPs was confirmed by FTIR and TGA.Functional characterization of AuNPs-SL nanoparticles using FTIR indicated that the vibration peaks AuNPs-SL nanoparticles is almost similar to the sophorolipids Fig. . The folS. aureus and Gram negative V. cholerae by agar well diffusion method for their antibacterial activity. In this method, a particular volume of inoculum was spread over the agar plate. In which, the required number of holes were punched using borer for addition of antimicrobial compound and then plates were incubated for certain time interval. As the antimicrobial compound diffuses into the agar, growth inhibition of microbe is observed33. In this study, a clear zone of bacterial growth inhibition was observed in wells containing AuNPs-SL for both microbes after completion of incubation  described the MIC in the XTT assay as the lowest concentration of antimicrobial compound that prevents the colour change34. For S. aureus . Interestingly, MIC of AuNPs-SL against V. cholerae  and viability by XTT  assay. McCluskey eus Fig. , MIC valrae Fig.  was 25 µS. aureus and V. cholerae at a different concentration  for 15 h. Similar to XTT assay, the growth curve analysis also showed that sophorolipid has either equal or twofold lower activity than gold nanoparticles  and OD590 was plotted in terms of percentage (%). In case of S. aureus,  that are adherent to each other and/or to a surfaceus, Fig.  nearly 9us, Fig. .Figure 3V. cholerae, similar pattern of result was obtained by CV and XTT assay  and completely inhibited at 50 μg/ml in V. cholerae whereas fourfold higher concentration of SL is needed to inhibit the non-dividing state cells.Another survival strategy of microbes under adverse condition is dormant stage or non-multiplying stage. bes Fig. . On compThe cellular effect of AuNPs-SL against these microbes was visualized by SEM Fig.  and TEM + to NADH and H+ during the oxidation of lactate to pyruvate. In the second step of the reaction, diaphorase uses NADH and H+ to catalyze the reduction of a tetrazolium salt to a highly colored product, formazan. It was found that most of the cellular inhibition occurred at 2 hours of incubation at higher concentration i.e., 60 μg/ml; whereas lower concentration has partial inhibition of cellular activity. This result also suggested that the inhibition of respiratory enzymes in concentration and time dependent manner. Thus, it is clearly demonstrated that the activities of respiratory chain dehydrogenases in both S. aureus . Here, AuNPs-SL induced interference in cell respiration was measured through LDH activity Fig. . In viabeus Fig.  and V. crae Fig.  were inhS. aureus, whereas with Amp it has shown additive effect. In the case of V. cholerae, AuNPs-SL has shown synergy with polymyxin, partial synergy with kanamycin and indifferent with ampicillin. The synergistic activities of AuNPs-SL in the presence of conventional antibiotics suggest that it might be possible to reduce the viability of bacterial strains at lower antibiotic concentrations. Most studies have been done on silver nanoparticles and similar types of interaction have also been reported by others39. This study demonstrates the synergistic effect of antibiotics and nanoparticles in improving their bactericidal property; it was suggested that nanoparticles can be effectively used in combination with antibiotics in order to improve their efficacy against various pathogenic microbes.Checkerboard microdilution method was used to evaluate the synergistic effects of AuNPs-SL with three conventional antibiotics having different mechanism of action against these bacteria and the effects were evaluated by determining the FICI. The results of the synergistic effect are presented in Tables et al., (2009) and they have shown its antimicrobial activity40. Although silver nanoparticles and silver itself are well reported for antimicrobial activity but its cytotoxicity limits its application to human cell lines41. However gold nanoparticles are reported to least toxic nanoparticles among other metallic nanoparticles. Considering these points sophorolipid capped gold nanoparticles were synthesized in single step procedure that imposes antimicrobial property. This biosynthesis method for synthesizing AuNPs-SL has a distinct advantage over chemical synthetic techniques such as high efficiency, biocompatibility, ecofriendly and low toxicity to the environment. As per the available knowledge, we are the first to report the formation of gold nanoparticles synthesized directly from sophorolipid. UV visible absorbance spectra in the range of 530–540 nm confirmed the surface plasmon resonance of biosynthesized gold nanoparticles i.e., AuNPs-SL. TGA and FTIR provided additional strong evidence of biogenic synthesis and capping of SL to gold nanoparticles. Biosynthesized AuNPs-SL was examined for antimicrobial potential against different phase of life cycle achieved by two pathogenic strains Gram positive bacteria S. aureus and Gram negative bacteria V. cholerae. Results suggested AuNPs-SL has pronounced antimicrobial potential against different important pathogenic microorganisms with especial efficacy against Gram negative bacteria. Previous antimicrobial studies by various groups on the activity of SLs suggested better activity against Gram positive bacteria as compared to Gram negative bacteria43. Similar to earlier finding, in case of V. cholerae, growth inhibition was not observed at the experimental concentration of SL. This difference might be due to their cell wall and cell membrane composition44. It is considered that the antibiotic agents are freely diffused through the cell wall of Gram positive bacteria. However, the permeability of the outer membrane determines the diffusion of a given antibiotic agent in Gram-negative bacteria. This outer membrane is composed of proteins (porins) and an asymmetric lipid bilayer, in which outer and inner layer is mainly composed of lipopolysaccharides and phospholipids respectively. In this way, the membrane structure allows diffusion into the periplasmic space mediated through different pathways. These porins are involved in the efflux of various compounds such as different groups of antibiotics. The alteration in porin composition in order to reduce the influx of compounds is also major threat towards antibiotic resistance imposed by Gram negative bacteria. These reasons might have led to differences in the zone of inhibition and MIC in these two pathogens upon treatment with AuNPs-SL and SL.Synthesis of nanoparticles using chemical and physical methods is potentially unfriendly to the human health and environment. Here, sophorolipid; a glycolipid was used to greener synthesis of gold nanoparticles. Glycolipid mediated metal nanoparticles (silver nanoparticles) synthesis was first reported by Singh Under environmental stress conditions, bacteria respond differently such as changing the morphology, changing cellular composition, forming biofilm and attaining non multiplying state. Biofilm and non multiplying phase of bacteria are imposing a major health problem in different aspects such as prosthetic valves, catheters and contact lenses. Since most of available drugs are based on the metabolically active cells therefore they fail to act against non-multiplying bacteria that ultimately leads to slow or partial loss of infected tissue. Apart from this repeated administration of drugs or antibiotics fails to act effectively. Here, results have shown the AuNPs-SL as promising bactericidal agent for non dividing cells too.With the use and misuse of antimicrobials, most of the pathogens develop resistance to multiple antibiotics thus reducing the effectiveness of drugs. Multidrug resistance (MDR) have emerged as major threats public health. Repurposing of drugs and combinatorial therapy are the main approaches that are adopted to combat this issue this issue. Metal nanoparticles have also shown new aspects to address this problem. Combinatorial therapy including the gold nanoparticles (here AuNPs-SL) with polymyxin and kanamycin have shown synergy against both pathogenic microbes. Polymyxin B binds to the cell membrane and alters its structure and makes it more permeable. AuNPs-SL exhibits synergy with polymyxin indicating the similar kind of bactericidal activity performed by these nanoparticles. Along with this, Kanamycin that affects the protein synthesis machinery, also exhibits synergy with AuNPs-SL. It reduces drug doses by eight fold in case of polymyxin for both microbes whereas fourfold and twofold for kanamycin in Gram positive and Gram negative respectively. In this way, it reduces the drug burden imposed by a single drug, hence improve the clinical outcomes. Since gold nanoparticles posses least cytotoxicity among other metal nanoparticles thus it is safe to use for medical applications.However the exact mechanism of antimicrobial activity exhibited by metal nanoparticles is unclear. But, synergistic effects shown by AuNPs-SL with polymyxin and kanamycin provide an idea towards the mechanism of its action. SL is known to be effective against the Gram positive bacteria but least for Gram negative bacteria. The uncapped gold nanoparticles do not exhibit antibacterial activity against the both of these microbes. However, capping of SLs to the gold nanoparticles mediates the antibacterial activity not only to the Gram positive but also Gram negative bacteria with higher efficacy for later one. SL mediates the formation of pores and disruption of cell membrane due to its surfactant like properties and also helps in the internalization of AuNPs-SL, which was also observed in TEM images. As evidenced by LDH assay, AuNPs-SL interferes with the metabolism upon internalization by binding to the enzymes and blocking its activity. Available reports (silver nanoparticles) of mechanism for antimicrobial activity suggests the binding ability with the cell wall and altering the cell membrane permeability, accumulation inside the cells and interaction to the respiratory enzyme are the major reasons or killing effect of microbes. We also observed a similar kind of findings as reported earlier. Thus, it can be said that AuNPs-SL exhibits its antibacterial properties by binding to cell membrane, disrupting cell membrane integrity, leakage of intracellular materials and interfering the enzyme activity.As the emergence of resistance imposed by Gram negative bacteria is more critical and hard to treat with the limited number of antimicrobial agents thus major concern to deal with this issue. AuNPs-SL is found to be very promising to handle this concern. AuNPs-SL can cross the barrier of biofilm and kills the residing cells effectively. Due to this property, it can prevent the emergence of resistance cells or persister cells. Thus this study provides the idea of using AuNPs-SL as antibacterial agents that have three major significant over existing antibiotic therapy (i) active against Gram positive and Gram negative bacteria both, (ii) have synergy with different antibiotics thus reducing drug burden, minimise side effects and (iii) disrupting the biofilm and non dividing cells thus minimising the existence of persisters. Altogether it can be said that sophorolipid capped gold nanoparticles can be a promising agent for antimicrobial therapy  having different mode of bactericidal action.Gold (III) chloride hydrate (50790), Gold nanoparticles , ampicillin (A 9518) and kanamycin (60615), XTT (X4626) were purchased from Sigma-Aldrich. Luria-Bertini (LB) medium, yeast extract, peptone, dextrose and agar used for culture growth were purchased from Himedia.Stamerella bombicola (MTCC 1910) was used for SL production, and it was maintained on YPD plate . For inoculum preparation, S. bombicola was grown in YPD broth for 2 days at 30 °C with agitation (200 rpm) in orbital shaker. From this preparatory inoculum 2% inoculum was added to production medium and cultured for 7 days at 200 rpm at 30 °C as suggested by Faraz et al.45 provided in supplement.4, 100 mM) during the gold solution preparation.Sophorolipid mediated gold nanoparticles (AuNPs-SL) were synthesized by two methods. In the first method, 40 µl of sophorolipid (100 mg/ml) was added to 10 ml of chloroauric solution  mixed properly and kept at 80 °C till the appearance of characteristic red colour. Since this method took several hours of synthesis, therefore first method was modified by adding few drops of sodium borohydride  of purified powders of SL and dried AuNPs-SL was performed on a TGA-7 Perkin-Elmer instrument at a scan rate of 10 °C min−1.Initial characterization of SL capped gold nanoparticles (AuNPs-SL) was done using dual beam UV visible spectroscopy (Hitachi U-2900) by measuring the absorption in the range of 300–700 nm. Measurement of size was carried out using dynamic light scattering (DLS) method and surface potential was measured using Malvern Zetasizer. Further, morphological and topological analysis was performed using TEM (JEOL 2100) imaging for which sample was stained with 2% phosphotungstic acid (PTA). Fourier transform infrared  spectra of SL and AuNPs-SL was recorded five times per scan in the frequency region 4000–400 cmStaphylococcus aureus MTCC 1430 and Gram-negative V. Cholerae EL Tor strain N16961 were used as indicator organism for antimicrobial activity in this study. Bacterial strains were revived on LB agar (2%) plate media from glycerol stock (15%). For broth culture, strains were grown in LB broth from LB agar plate and kept at 37 °C, 200 rpm for overnight and then subcultured for 2–3 hours to obtain log phase cells48. Generally, cultures were diluted in ratio of 1:1000 in LB broth, from which 100 μL of culture was either spreaded on agar plate or added in microtiter plate for some assay else mention in the particular section.Two pathogenic strains, Gram-positive 49. 107 CFU/ml of cells was spread on LB agar media separately in three replicate plates. 6 mm diameter of wells was made on these agar plates using agar well borer and 100 μl of the following samples; AuNPs-SL (400 μg/ml), SL (400 μg/ml), Ampicillin (50 μg/ml) and AuNPs-Sigma were loaded separately into the wells. Plates were kept in incubator at 37 °C for overnight. The antimicrobial activity was calculated by measuring the diameter of zone of bacterial growth clearance on agar plate. Here, it is worth to write that all the concentration of gold nanoparticles mentioned here are in respect of SL concentration.Initially, the antibacterial activity of AuNPs-SL and SL was performed against indicated bacterial strains by agar diffusion method7 CFU/ml) was added to the plates, mixed well with testing compounds and incubated for 16 hours at 37 °C. Later on, bactericidal effect of AuNPs-SL and sophorolipid was investigated by colorimetric XTT reduction assay. The assay is based on the conversion of tetrazolium salt XTT to form water-soluble coloured formazan product by metabolic active cells that can be measured spectrophotometer50. XTT solution (0.5 mg/ml) was prepared in 1X PBS and menadione (1 mM) in acetone respectively. Prior to each assay, these solutions were thawed and mixed to give final concentration of 1 μM of menadione. From this, 30 μL of XTT was added to each well of microplate and incubated at 37 °C in dark for 2 hours. The change in colour due to viability of cells was measured using microplate reader  at 490 nm. Experiments were performed in triplicate and any inferences of nanoparticles in the measurement have been deducted from the absorbance imposed by samples and then average value is reported with ±SD.The AuNPs-SL and SL were taken in the range of 200–6.25 µg/ml with serially two fold diluted in 96 well plates. The concentration mentioned here is in terms of SL concentration. For controls, ampicillin (20 µg/ml), AuNPs from Sigma and media blank were taken. 100 µl of the log phase culture (107 CFU/ml of cells) was seeded with different concentration of the AuNPs-SL & SL  in 100 well honey comb plate. Negative (without any treatment) and positive controls (ampicillin) were taken properly as shown in the graph 2C and D. Uncapped AuNPs (AuNPs-Sigma) was also used for the study. The plate was kept at 37 °C with continuous shaking in the Bioscreener reader for 16 h and OD600 was measured at equal interval of 1 h. The experiment was done in triplicates for each sample set, average was calculated and graph was plotted.Growth kinetics studies of both the organism were performed in honey comb 100 well plate using Bioscreener reader. 100 µl of the log phase culture 48 and LB saline (2% NaCl)47 media respectively. V. cholerae is a halophilic microbe and under stress condition, biofilm formation is induced51. 100 μl of the diluted culture was put into each well of the 96 well plates containing different concentration of AuNPs-SL and SL  that was incubated for 24 h at 37 °C. SDS (125 μg/ml) treated and untreated cells of these pathogens were taken as control. Planktonic cells were removed after completion of incubation time and washed thrice with PBS (pH 7.2). The extent of biofilm formation was measured by CV and XTT assay as mentioned below. All the experiments were done in triplicate and the average value was expressed in extent of % biofilm that was normalized with untreated cells.Effect of AuNPs-SL on inhibition of biofilm formation and mature biofilm eradication was studied in 96 well microtiter plates by two methods; Crystal Violet assay and XTT assay. To study the extent of biofilm formation, the strains S. aureus and V. cholerae were grown overnight at 37 °C in orbital rotatary shaker that was further diluted to 1:100 into fresh LB sucrose (1% sucrose) and LB saline (2% NaCl) media respectively. 100 μl of the diluted culture was put into each well of the 96 well plates without any treatment to form biofilm. Planktonic cells were removed, aspired thrice with PBS, then treated with AuNPs-SL and SL  and incubated for another 4 h at 37 °C. Cells with SDS and without AuNPs-SL served as the positive and negative controls respectively. All the experiments were done in triplicate and the mean value was expressed in extent of biofilm in terms of percentage that was normalized with untreated cells.In case of mature (preformed) biofilm eradication study, the strains Crystal violet (CV) staining method: This was carried out as described by George A. O’Toole (2011) with slight modification52. Briefly, biofilm cells remaining were fixed using methanol for 15 minutes and dried at room temperature. Biofilm was stained with 0.1% CV for 15 minutes followed by washing with PBS to remove excess stain and stained biofilm was dried properly. Finally, suspension of the biofilm was done in 30% acetic acid followed by quantification by measuring the absorbance at 595 nm. The values obtained were considered as an index of bacteria adhering to the surface of well wall and extra cellular mass produced by them.XTT method: For XTT assay, aspired cells were resuspended into 1% glucose containing XTT-menadione solution and incubated for two hours at 37 °C in dark. Then measurement was taken at 490 nm in ELISA plate reader. Here the OD490 is reflection of viable cells residing within the biofilm.S. aureus cells was obtained following the method described by Hu et al.53. The culture was grown in LB medium at 37 °C, 200 rpm for seven days. Cells were centrifuged and washed twice with PBS (pH 7.2), resuspended in the same buffer and further incubated at 37 °C and 200 rpm for 7 days. Cell viability was checked by counting colony-forming units (CFUs) every 24 h. The culture showed a decrease in CFU/ml in the first 5 days, and remained constant thereafter, at 4 × 105. From this, 100 μl of cells were seeded into microtiter plate for study.Non-dividing state of V. cholerae was obtained using the method as described earlier55. V. cholerae was inoculated into alkaline peptone water for 16 hours at 37 °C in shaker then cells were collected by centrifugation, washed thrice with 1% artificial seawater and resuspended. The final concentration of cells was maintained as 108 CFU/ml in one litre flask containing 200 ml of 1% artificial sea water and incubated at 4 °C in the dark, without shaking. After one month, cell viability was performed by colony forming unit on LB agar plate for 3–5 consecutive days to know the constancy of viable cell numbers. Further, 100 μl of 4 × 106 CFU/ml was used for the activity analysis in 96 well microtiter plate.Non-dividing state of 490 of untreated cells, B = OD490 of treated cells.100 μl of non-multiplying cell suspension of both strains were mixed with equal volume of different concentration of AuNPs-SL and SL in concentration range of 25–200 μg/ml in separate microtiter plate and incubated for 16 h at 37 °C. An untreated sample, taken as control, was processed in the same way. Viability was checked by XTT assay and mean value was used for plotting graph viability.56. 107 CFU/ml of cells in 5 ml of LB broth media was treated with AuNPs-SL-25 μg/ml and incubated for 2 hours in orbital shaker at 37 °C. Cells were then centrifuged and washed with fresh PBS thrice. In brief, treated and untreated cells were fixed on coverslip and incubated in 2% glutaraldehyde solution for half an hour. Washing with PBS pH 7.2 was followed by dehydration with 30%, 50%, 70%, 90% and 100% ethanol. Final dehydration was done with tertiary butyl alcohol and desiccated in lyophilizer. Samples were coated with gold sputter before imaging in SEM (Zeiss Evo 40).The morphological changes induced by nanoparticles against these strains were visualized by using Scanning Electron Micrograph (SEM) and Transmission electron microscopy (TEM). For SEM analysis, untreated and nanoparticles treated cells were prepared following established protocolFor transmission electron microscope (TEM), samples were prepared by adding a few drops of treated and untreated cells on carbon coated grid and incubated for 10 minutes. Remaining samples were removed using blotting sheets and then stained using 2% phosphotungstic acid for 10 minutes. Extra stain was removed similarly and grid was dried for half an hour before imaging.57. Overnight grown culture was subcultured to obtain log phase cells and diluted in such a way that 10 ml of media contains 108 CFU/ml cells. Different concentration of AuNPs-SL and SL  was mixed to cultures and incubated for 4 hours. At different time intervals (2 hours), 1 ml of samples were withdrawn, centrifuged, washed thrice with PBS (pH = 7.2) and LDH activity was measured as per manufacturer instruction. The plate was then incubated with gentle shaking on an orbital shaker for 30 min at room temperature and reaction was stopped by adding stop buffer solution. After incubation, the OD495 was measured and treated samples were compared with untreated assigning untreated cells activity as 100%. All the experiments were performed in triplicate and data are expressed as means ± SD.To investigate the oxidative stress induced damage on respiratory system by nanoparticles, LDH activity was checkedS. aureus and V. cholerae. In this assay, minimum inhibitory concentration (MIC) was determined, for antibiotics and AuNPs-SL individually and in their paired combinations58. The extent of synergy between antibacterial drugs is often expressed as fractional inhibitory concentration (FIC). Three antibiotics drugs, namely ampicillin (Amp), kanamycin (Kan) and polymyxin B sulphate (Poly) were used to investigate their synergy with prepared AuNPs-SL against these microbes. Bacterial cultures were grown as mentioned earlier and from this, 100 μl of was added to the same 96 well microtiter plate containing different dilution of drugs in combination of AuNPs-SL or separately drug and AuNPs-SL, incubated at 37 °C for 16 h in bioincubtor. Afterwards, XTT assay was performed to determine the MIC and FIC of antibiotics and AuNPs-SL. The lowest concentration at which no change in colour of XTT occurred was noted as the MIC value of the individual and combined test agents. FIC was evaluated from the MIC of antibacterial compound A and the MIC of antibacterial compound A in combination with test agent B. Therefore,A checkerboard microdilution technique was used to explore the synergism between the antibiotics and AuNPs-SL against these microbes bacteria i.e., FIC of antibacterial A = MIC of antibacterial compound A in combination/MIC of antibacterial compound A alone.In similar manner, FIC of antibacterial compound B was calculated and the ΣFIC index was done by adding these two FIC agents.59.The calculated FIC index was used as indicator of the nature of interaction between the two tested antimicrobial agents as suggested by American Society of MicrobiologyStatistically significant differences between the groups were determined using two-way ANOVA and multiple pairwise comparison procedures. Statistical analysis was performed using GraphPad Prism 6 software.Supplementary information


This notion highlights that the anti-MET therapy can be effective as the first line of intervention in only a few MET-addicted cases, while it is certainly more relevant to block MET in cases of advanced neoplasia that exploit the activation of the invasive growth program to promote dissemination in other body parts. Few data are instead related to the role played by RON, a receptor homologous to MET. We have already demonstrated an implication of MET and RON genes in brain metastases from lung cancer. On this basis, the aim of this work is to recapitulate and dissect the molecular basis of metastatic brain dissemination from lung cancer. The latter is among the big killers and frequently gives rise to brain metastases, most often discovered at diagnosis. Molecular mechanisms leading to tumour spread to the brain are mostly unknown and in turn these tragic cases are still lacking effective therapies. Based on previously published data from our group, we aim to summarise and analyse the pathogenic mechanisms leading to activation of the scatter factor receptor in brain metastatic lesions of lung primaries, from the point of view of replacing the currently used empirical treatment with a more targeted approach. The process of metastatic dissemination begins when malignant cells start to migrate and leave the primary mass. It is now known that neoplastic progression is associated with a combination of genetic and epigenetic events. Cancer is a genetic disease and this pathogenic concept is the basis for a new classification of tumours, based precisely on the presence of definite genetic lesions to which the clones are addicted. Regarding the scatter factor receptors MET and Recepteur d’Origin Nantais (RON), it is recognised that MET is an oncogene necessary for a narrow subset of tumours (MET-addicted) while it works as an adjuvant  Lung cancer is among the big killers and is frequently associated with brain metastases, most often discovered at the time of diagnosis. Lung carcinoma includes a series of different diseases which can roughly be divided into two groups based on clinical and histo-pathological features: non-small cell lung cancer (NSCLC), accounting for almost 80% of lung cancer diagnoses, and small cell lung cancer (SCLC), responsible for the remaining 20%. In 2015, the World Health Organization adopted the classification recently developed by the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society . GrowingPatients are usually asymptomatic in the early stages of the disease. This is related to the sparse pain–fibre innervation of the lungs and the significant respiratory reserve that both lungs provide. The lack of symptoms is particularly true for lung cancers that originate in the periphery of the lungs. Approximately 5–10% of lung cancer patients are asymptomatic at presentation . These cThe molecular mechanisms regulating metastatic spread to the brain are largely unknown ,11. CurrBurgeoning evidence indicates that invasive growth is executed by stem and progenitor cells and is usurped by cancer stem cells. The MET proto-oncogene, which is expressed in both stem and cancer cells, is a key regulator of invasive growth. MET encodes for the receptor tyrosine kinase (TKR) for the hepatocyte growth factor (HGF) or scatter factor. It is located on chromosome 7, band q31, and is constituted by 21 exons separated by 20 introns codifying for a trans-membrane tyrosine kinase receptor synthesized as a single–chain precursor which undergoes post-translational cleavage into two disulfide-linked α and β subunits. The extracellular domain comprises two main regions, both involved in ligand binding. The first is known as SEMA domain, due to its homology with semaphorins, and includes the α chain and the N-terminal portion of the β chain. The second is the “immunoglobulin-like domain,” containing four disulfide-linked loop structures. The intracellular domains of MET include three functional portions: (i) the juxtamembrane sequence, including the Ser975 residue, which, upon phosphorylation, downregulates kinase activity; (ii) the catalytic region, containing Tyr1234 and Tyr1235 residues, which, upon receptor dimerization and transphosphorylation, upregulate kinase activity; (iii) the carboxy–terminal sequence. The residues Tyr1234 and Tyr1235, located in the catalytic domain, are critical for receptor activation. After that, MET elicits intramolecular phosphorylation of the other two critical tyrosine residues (Tyr 1349 and Tyr 1356) at the C-terminal of the α-chain. These two sites and the surrounding amino acids constitute the so-called “multifunctional docking site”, a motif that, when activated after phosphorylation, induces a series of biological processes that leads to invasive growth. In distinct cells and tissues, MET-driven specific activities are fulfilled by dedicated signaling cascades, with some transducers dominating over others according to the context, timing, and biological complexity . ThroughWith respect to lung cancer, MET gene amplification occurs in about 4% of lung ADCs and 1% of SCC ,17. Accumetastogenes for many others. This notion highlights that anti-MET and anti-RON therapy can be effective as the first line of intervention in aforementioned addicted cases, whereas it is certainly more relevant to block MET and RON in cases of advanced neoplasms that exploit the activation of the invasive growth program to promote distant dissemination (https://cancer.sanger.ac.uk/cosmic). Notably, the mortality rate after brain radiotherapy was significantly higher in tumors carrying somatic MET mutations compared with euploid wild-type MET lesions (p < 0.008) (https://cancer.sanger.ac.uk/cosmic). In silico analysis suggested a damaging role of the changes found.The understanding of the molecular mechanisms underlying tumor spread to the brain is still in its infancy and, in turn, effective treatment for these tragic cases is yet to arrive. The concept that cancer mutated kinases molecularly mark druggable targets has led to intensive efforts to survey the kinome across a wide spectrum of human tumor types for mutations  and to tmination  On this < 0.008) b; (ii) RE168D change being the most frequent one. As discussed above, the SEMA domain of MET, which is shared with semaphorins, plexins, as well as the RON receptor, consists of a highly conserved variant form of the seven-blade β-propeller fold, defined by a set of cysteine residues, which form four disulphide bonds to stabilize the structure [Moreover, the vast majority of MET mutations found in metastatic lesions affected the extracellular SEMA domain of the receptor, with the tructure . While ttructure  and has tructure . Indeed,anoikis. Through a mechanism known as epithelial-mesenchymal transition (EMT), cancer cells acquire a metastatic phenotype [Although preliminary, these study results do not disprove the initial premise, since it is conceivable that changes affecting the SEMA domain sequence may be reflected in structural alterations of the extracellular portion of the receptor. This could, in turn, affect the physical interactions between metastatic cells and the surrounding microenvironment, thus facilitating their highly invasive properties. Contrary to expectations, preliminary data pointed out that cell plasticity characterizing SEMA-mutated clones does not rely on increased deformability and adaptation to foreign environments. The motile behavior of SEMA-mutated cells seems to be unrelated to matrix stiffness. This finding was quite surprising, as a high migration ability, as well as an increased elasticity and deformability, were hypothesized to be involved in dissemination of metastatic cells to the brain. Overall, the SEMA mutants displayed: (i) an altered Young’s module (tension/deformation ratio); (ii) activation of interatomic interactions between cell surface and surrounding stroma when the cell was deformed; (iii) an altered extracellular matrix (ECM)/cell stiffness balance. It is well known that morphogenesis and metastases seem to arise from the same genetic program that instructs cells to undergo a biological process named henotype . Metastahenotype . Cancer henotype . Our preThis crucial point may open new avenues for research focused on a deeper understanding of tumor progression with relevant diagnostic and therapeutic implications. Overall, the rationale of targeting MET in NSCLC has a controversial history. MET activation is responsible for about 20% of cases of resistance to EGFR inhibitors, and there is increasing evidence regarding the sensitivity to anti-MET inhibitors in the absence of concurrent EGFR mutations or MET ex 14 skipping variants ,6. TogetTo successfully target brain metastatic lesions, therapeutic molecules have to be able to pass across the blood–brain barrier (BBB), which exists between the blood microcirculation system and the brain parenchyma. Its integrity is relevant in protecting the brain from systemic toxins, in assuring adequate nutrient levels, as well as in maintaining local homeostasis. As a net effect, it renders the brain an anatomical sanctuary site. It is anatomically and functionally distinct from the blood–cerebrospinal fluid barrier and the choroid plexus . Blood fwww.ClinicalTrials.gov, identifier: NCT02143466). Capmatinib, an orally available highly potent and selective inhibitor of MET, demonstrated a manageable toxicity profile in treatment–naïve patients with NSCLC and MET exon14 mutation. Preliminary data reported that capmatinib is able to pass the BBB and it is active in brain. In in vivo models, the combination of capmatinib and the pan-EGFR inhibitor afatinib completely suppressed tumor growth in mice orthotopically injected with cells derived from brain metastasis from NSCLC patients [In patients . All in patients  and, henpatients . Altiratpatients . More repatients . It is apatients ,63,64,65patients , althougOver the past few years, the improved knowledge of the biological, genetic, and molecular heterogeneity of tumors, together with the development of improved pharmacological technologies, has allowed the identification of molecular targets for novel therapeutic strategies. This fast process has led to the overall reconsideration of the biological and genetic peculiarities that can make each tumor a pathology of its own. The identification of patients likely to respond to specific treatments according to the presence of relevant molecular targets  needs clinical studies focused on a constant and productive interaction among the professionals with a significant background in the various disciplines. The process of metastatic dissemination begins when malignant cells start to migrate and leave the primary mass. Cancer is a genetic disease and this pathogenic concept is the basis for a new classification of tumors, based precisely on the presence of definite genetic lesions to which the clones are addicted. Growing evidence suggests that cancer cells inappropriately execute the MET-driven invasive growth program which is actively involved in tumor onset and progression. The presented data sheds new light on MET, and its sibling RON, as promising candidate genes in both onset and therapy of secondary brain metastases. Further mechanistic validation is needed to test the presented hypothesis. If experimentally confirmed, future therapies could take advantage of novel MET inhibitors specifically designed to cross the BBB and could possibly enhance the radio-sensitivity of brain lesions.


We report an uncommon case of immunoglobulin gamma 4-related ophthalmic disease (IgG4-ROD) presenting as meningitis and panuveitis. A 35-year-old male with no prior ophthalmic history presented with headaches, altered mental status, and fever of unknown origin. A lumbar puncture (LP) revealed an elevated white count with lymphocytic predominance, confirming a suspected meningitis. After an extensive work-up, he was discharged on oral acyclovir to cover for presumed aseptic meningitis. The patient initially improved, however, bilateral eye pain, redness, and photophobia 2 weeks after discharge prompted his first visit to the ophthalmology clinic. Exam at that time was consistent with bilateral anterior uveitis for which he was given topical prednisolone and cyclopentolate. In addition to the preceding work-up, quantitative immunoglobulin serology including IgG4 levels was added. At follow-up, he was found to have increased ocular inflammation with vitreitis, nerve head edema, and subclinical macular thickening. Visual acuity (VA) had decreased in both eyes. Serology titers for IgG had resulted in a significant elevation in IgG subclass 4 (IgG4). Optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) confirmed posterior retinal involvement. The patient was diagnosed with presumed bilateral panuveitis secondary to IgG4-ROD. IgG4-RD can be a serious condition that requires careful consideration and intuition to diagnose. This report serves to encourage ophthalmologists to consider IgG4-ROD in cases of idiopathic systemic inflammation with ophthalmic involvement. Immunoglobulin G4-related disease (IgG4-RD) is a recently discovered inflammatory condition characterized by IgG4-mediated fibroinflammation and enlargement of the affected organ . While tA 35-year-old male with no prior medical or ophthalmic history presented with headaches, altered mental status, and fever of unknown origin. An extensive work-up for infectious, inflammatory, and neoplastic causes was noncontributory. Labs revealed marked elevations in both erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), indicating an underlying inflammatory process. Computer tomography (CT) scan of the head showed diffuse inflammation and thickening of the meninges . Lumbar The patient initially improved; however, two weeks after discharge, bilateral eye pain, redness, and photophobia prompted his first visit to the ophthalmology clinic. Slit lamp exam was notable for grade 2 anterior chamber cells with flare in both eyes. The right eye showed posterior synechiae with pigment on the anterior lens capsule  while thAt follow-up, his symptoms persisted with a decrease in vision from 20/20 in both eyes to 20/25. He was found to have increased ocular inflammation with persistent anterior chamber cells and subsequent vitreitis and bilateral optic nerve head edema in both eyes Figures  and 5. OSerology titers for IgG revealed a significant elevation in IgG subclass 4 (IgG4) at 251 mg/dL. A presumed diagnosis of bilateral panuveitis secondary to IgG4-ROD was made. The patient was offered a vitreous biopsy to further confirm the diagnosis, but he declined. He was treated with high dose oral prednisone at 60 mg/day with full resolution in anterior chamber cells, optic nerve head edema Figures  and 10, IgG4-RD is an increasingly recognized immune-mediated condition that can yield destructive inflammation and fibrosis in a number of organs . When thHere, we describe a rare case of probable IgG4-ROD presenting as meningitis and progressive panuveitis that required an extensive work-up to identify. While IgG4-RD is typically diagnosed by rheumatological specialties, we advise ophthalmologists to consider IgG4-ROD and to add IgG serology to the work-up in cases of idiopathic inflammation. When the presentation varies beyond the expected, the risk of misdiagnosing the condition heightens, which can be detrimental to the patient's capacity for recovery. A condition like IgG4-ROD can offer an opportunity for elevated collaboration between ophthalmology and rheumatology for a final diagnosis. Despite favorable responses to steroids, long-term management of relapsing IgG4-RD and igG4-ROD patients can be challenging, further supporting the need for awareness and multidisciplinary cooperation as it may require immunomodulatory therapy.


Immunoglobulin subclass G4‐related disease (IgG4‐RD) is characterized by an abundance of IgG4 antibodies in the serum and tissue. Glycosylation status of antibodies can impact on immune effector functions and disease pathophysiology. We sought to establish glycosylation patterns in a prospective cohort of patients with IgG4‐RD and the relationship with disease activity and response to treatment.n = 22), disease controls with primary sclerosing cholangitis (PSC) (n = 22), and healthy controls (n = 22). Serum IgG and subclasses were quantified using ELISA. Fc and Fab glycosylation were analyzed by mass spectrometry and lectin affinity chromatography, respectively. Disease activity, organ damage, and response to treatment were assessed using the IgG4 Responder Index.We assessed IgG Fc‐tail and Fab‐arm glycosylation status in patients with IgG4‐RD (P = 0.01), with a preferential increase in IgG4‐specific Fab sialylation, which was independent of IgG4 Fab‐arm exchange. There was a reduction in IgG1‐specific Fc bisection and hybrid structures in IgG4‐RD (P < 0.01), which recovered upon steroid treatment and correlated with disease activity. Overall, IgG Fc galactosylation was reduced in both IgG4‐RD and PSC (P < 0.01), with a preferential reduction in IgG1‐specific sialylation and enhancement of IgG4‐specific bisection in PSC. IgG4 fucosylation and IgG1/2/3 hybrid structures negatively correlated with complement C3 and C4 levels in IgG4‐RD (P < 0.01), but not PSC.Immunoglobulin G Fab sialylation was increased in IgG4‐RD compared with PSC and healthy control (We report the first study showing unique antibody glycosylation status in a prospective cohort of IgG4‐RD and PSC patients, which may determine modulation of the immune system and contribute to disease pathophysiology. This is the first study to investigate Fc and Fab antibody glycosylation patterns in patients with IgG4‐RD and investigate the relationship of disease activity, organ damage, and treatment with glycosylation status.n = 22 with biliary disease, and other organ involvement), disease controls (DCs) (n = 22 with primary sclerosing cholangitis [PSC]), and healthy controls (HCs) (n = 22) were prospectively recruited from the John Radcliffe Hospital, Oxford, UK, a tertiary referral center for IgG4‐RD and PSC. Patients enrolled in this study had serum collected at diagnosis before treatment (IgG4‐RD and PSC) and after 6–8 weeks of corticosteroid treatment (IgG4‐RD) and were prospectively followed‐up. Ethical approval for the study was obtained from the Research Ethics Committee Oxfordshire (10/H0604/51), and the study was registered on the UK National Institute of Health Research portfolio (10776).Immunoglobulin subclass G4‐RD patients (The diagnosis of IgG4‐RD was made using the HISORt criteria (2006/7) for IgG4‐related pancreatic and biliary diseaseDisease controls were patients with PSC, diagnosed in accordance with the EASL guidelines for cholestatic liver disease.https://igg4rd.oxnet.nhs.uk for IgG4‐RD and the Oxford ACCESS database for PSC patients. Data recorded in IgG4‐RD patients included baseline demographics, serological evaluation at diagnosis, remission and relapse, imaging characteristics, histological morphology and immunostaining, organ involvement, disease activity and organ damage, treatment and response, disease relapse, malignancy, and mortality.Clinical data were entered onto IgG4‐RD Registry database Immunoglobulin subclass G4‐RD activity, organ damage, and response to treatment were assessed using the IgG4 Responder Index (IgG4‐RI).Serum IgG and subclasses were measured at diagnosis and during treatment (remission and relapse). Comparisons between IgG4‐RD patients, DCs, and HCs were made at diagnosis, before steroid initiation. Total serum IgG and IgG4 were measured by nephelometry . Elevated serum IgG (≥ 16 g/L) and IgG4 (≥ 1.4 g/L) were defined by institution range .Complement levels were measured at diagnosis in IgG4‐RD patients and on recruitment in PSC patients. C3 and C4 levels were measured by turbidimetry. Reduced C3 (< 65 mg/dL and C4 < 14 mg/dL) levels were defined by institution range .Human IgG was quantified using an ELISA as described previously.Human IgG subclass ELISAs were carried out analogously to the IgG ELISA. Microtiter plates were coated with monoclonal mouse antihuman IgG1 or IgG4, incubated with samples, and detected with horseradish peroxidase hepatobiliary‐conjugated polyclonal rabbit antihuman IgG and tetramethylbenzidine substrate solution.Fc glycosylation was analyzed by mass spectrometry as described previously.Sambucus nigra agglutinin (SNA) western blot confirmed sialylation of Fab and Fc glycans in recombinant IgG4 antibodies to a degree similar or higher compared with polyclonal IVIg (data not shown). The N‐glycosylation site in the Fab arm of ADL mutant D86N was selected and introduced as described previously.Recombinant IgG4 antibodies  were produced using the serum‐free FreeStyle 293 expression system (Invitrogen) as described previously.Immunoglobulin subclass G4 ADL wild‐type (without Fab glycans) and IgG4 ADL mutant D86N (with Fab glycans) were incubated in a 4:1 ratio in 1‐mM glutathione for 24 h at 37°C while shaking to allow IgG4 half‐molecule exchange to take place.Fab glycosylation was analyzed by lectin (SNA) affinity chromatography as described previously.P = NS). Serum total IgG levels were higher in IgG4‐RD and PSC patients compared with HCs   only  , PSC , and HCs  were well matched   , using the IgG4‐RI. This was not the case for the other subclasses (data not shown).Serum IgG4 levels fell with corticosteroid treatment in IgG4‐RD patients (01) Fig. a and cor05) Fig. b but notn = 12 with active disease), PSC patients (n = 10), and HCs (n = 10) using SNA affinity chromatography, whereby essentially only sialylated Fab glycans are retained. Total IgG Fab sialylation was enhanced in IgG4‐RD compared with PSC and HCs  compared with that of the other IgG subclasses (n = 22 with active disease) and controls  using mass spectrometry. Fc glycosylation patterns in HCs were in accordance with recently published data  were determined for patients (IgG4‐RD P < 0.01) .Immunoglobulin G Fc galactosylation was decreased in IgG4‐RD and PSC patients compared with HCs (P < 0.01)  Fig. c. In conn = 21), to investigate whether the observed differences in Fc glycosylation are recovered  and after (post 6–8 weeks) corticosteroid treatment    Fig. a. IgG2/301) Fig. b, indicaFc glycosylation patterns were assessed with respect to complement levels in IgG4‐RD and PSC. C3 and/or C4 levels were reduced in 5 of 22 (23%) IgG4‐RD  and in 1 of 12 (8%) PSC patients  when measured at baseline. The patient with hypocomplementaemia in the PSC group had co‐existent Sjogren's syndrome. IgG4 fucosylation and IgG1/2/3 hybrid structures negatively correlated with C3 and C4 levels in IgG4‐RD Fig. a–f. In PIn the first study to investigate IgG glycosylation in patients with IgG4‐RD and PSC, we report unique glycosylation patterns in patients with active disease and in response to treatment. Specifically, we observed enhancement of IgG and IgG4‐specific Fab sialylation in IgG4‐RD compared with PSC and HC. Furthermore, there was a decrease in IgG Fc galactosylation and IgG1‐specific Fc bisection in those with active IgG4‐RD compared with HC, with a recovery of IgG1‐specific bisection with treatment. There was also an increase in IgG4‐specific Fc fucosylation and Fc hybrid structures, the latter responded to treatment and correlated with disease activity.N‐glycans linked to N‐glycosylation sites that emerge during somatic hypermutation. IgG Fab glycosylation is an important process in immunity, with demonstrated effects on stability, half‐life, and binding characteristics of antibodies and BCR.Fab glycans are complex‐type biantennary Immunoglobulin glycosylation plays a crucial role in modulating antibody‐mediated responses and skewing the immune system toward a pro‐inflammatory or anti‐inflammatory direction. Reduction in total serum and antigen‐specific IgG Fc galactosylation is one of the most prominent changes observed in a wide variety of chronic inflammatory and autoimmune diseases, believed to be important in complement activation and antibody‐mediated phagocytosis.n = 12) compared with HCs (n = 8), with IgG4 non‐fucosylated N‐glycans decreased in those with hypocomplementaemia (n = 7).Agalactosylated N‐linked glycans (G0 N‐glycan) have been suggested to represent a more pro‐inflammatory phenotype, for example, with respect to activation of complement pathways. Hypocomplementaemia has been described in patients with active IgG4‐RD.emia n = .7Recent studies have demonstrated that abnormalities in IgG glycosylation play a major role not only in triggering but also in the activity and relapses in autoimmune disease.in vitro studies.Taking into account the central role of glycosylation, it could be considered an attractive target for the development of safer therapy in patients with IgG4‐RD and PSC. B‐cell depletion is an attractive therapeutic option in IgG4‐RD. Non‐fucosylated recombinant anti‐CD20 rituximab (BLX‐300) appears to be as efficient as classical fucosylated rituximab in target‐cell binding and apoptosis induction but is considered to be less toxic in Figure S1. Serum IgG4 levels with disease activity and corticosteroid treatment in IgG4‐RD. (a) Correlation plot of serum IgG4 levels with total disease activity calculated using the IgG4‐Responder Index. Spearman rank correlation, **P < 0.01. (b) Paired data of serum IgG4 levels pre and post corticosteroid treatment. Wilcoxen signed rank test, ****P < 0.0001.Click here for additional data file.Figure S2. IgG4‐Responder Index (IgG4‐RI) activity score with corticosteroid therapy and glycosylation status. (a) Paired data of IgG4‐RI activity score pre and post corticosteroid treatment. Paired t test, ****P < 0.0001. (b) Correlation plot of the IgG4‐RI activity score and IgG2/3 Fc hybrid structure glycosylation. Linear Regression Analysis, **P < 0.01.Click here for additional data file.Figure S3. Complement levels with glycosylation status in IgG4‐RD. Correlation plot of C3 levels with (a) fucosylation, and (b) IgG1 hybrid structures (c) IgG2/3 hybrid structures. Correlation plot of C4 levels with (d) fucosylation, and (e) IgG1 hybrid structures (f) IgG2/3 hybrid structures. Spearman rank correlation, *P < 0.05 **P < 0.01.Click here for additional data file.


A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography–ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera.A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples.Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value . Detection of guideline-recommended antibiotics in stool specimens did  and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes .Among 845 patients, 11% (90) were These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study. Infectious diseases research on how antibiotics influence infection and transmission of pathogens relies heavily on self-reported medication use that is rarely objectively confirmed . This prVibrio cholerae ) were azithromycin susceptible. Intermediate resistance was present for trimethoprim/sulfamethoxazole (81% [13 of 16]) and tetracycline (75% [12 of 16]). All isolates (100% [16 of 16]) were erythromycin resistant.Among patients from whom specimens were collected and cultured, 6.4% (16 of 249) were positive for Patients were asked whether medications had been taken for their current illness before admission, because they were unable to articulate antibiotic names. Among patients with paired urine and stool samples, 55% (24 of 44) reported taking medication before admission. There was no concordance between self-reported medication use and detection of medication by LC/MS ; among all patients with cholera, 48% (43 of 90) reported medication use . FindingP = .057; P = .001; P = .158; P = .041; P > .05, by the Mann-Whitney U test; Given that only 1 sample was negative for antibiotics as measured by analysis of paired urine and stool specimens, stool samples were analyzed for correlates of microbiota changes as a function of antibiotic detection in stool alone. Samples with lytic vibriophage or trace antibiotic detection were removed as potential confounders. Permutational multivariate analysis of variance (based on Bray-Curtis dissimilarities) was used to assess β diversity as a function of (1) detection (n = 59) versus nondetection (n = 9) of any antibiotic  and a decrease in V. cholerae load was associated with azithromycin (P = .042). Comparisons of the relative abundance revealed that metronidazole was associated with a decrease in Prevotella organisms (P < .001), which are associated with patients who recovered from cholera and healthy controls [Prevotella, Bacteroides, Blautia, and Ruminococcus organisms), metronidazole was associated with an increase in taxa associated with active cholera and/or the early phase of recovery from cholera , baseline chromatographic separation enabled unambiguous assignment. For analytes with complex metabolites , MS fragmentation data aided in metabolite identification and therefore parent confirmation.Findings of LC/MS were consistent. An artificial control mixture containing standards of all analytes was run with each batch and showed consistent elution times. Blank samples distributed throughout each run did not show the presence of analytes. Chromatographic separation was designed to detect both parent medications and their metabolites, which are generally more polar than the parent drug and therefore elute earlier. The method monitored full scan (MS), MSDifferent frequencies of detection between stool and urine specimens for some antimicrobials  were likely attributed to differences in sample collection time and conditions, matrix complexity, and recovery. When the 16-component standard mix was spiked into a stool sample, 50% analyte loss was observed in filtered samples, compared with the nonfiltered samples. Further optimizing stool preparation to minimize dilution and avoid filtration may increase sensitivity. Differences in excretion pathways, possible first-pass metabolism, and degradation may also affect detection frequencies.LC/MS poses benefit versus cost challenges. The advantages are that LC/MS identifies the antibiotic and has a higher sensitivity. In contrast, the bioassay offers a high sensitivity, specificity, and positive predictive value, but the negative predictive value is unacceptably low. The bioassay is tractable and less expensive, at $1–$2 per sample, compared with LC/MS (approximately $20–$160 per sample); however, the downstream cost of measurement bias without LC/MS is likely high.Prevotella spp.) provided an important example of the unanticipated consequences of inappropriate antibiotic use. Second, it supported azithromycin as a recommended agent for cholera, because of the reduction in the absolute and relative abundance of V. cholerae. Third, it revealed the lack of ciprofloxacin-associated suppression of V. cholerae despite in vitro detection of susceptibility by antimicrobial susceptibility testing. These data caution against the use of ciprofloxacin for cholera.The value of incorporating the LC/MS metadata into the microbiota analysis was 3-fold. First, it provided confirmation that metronidazole was associated with a relative reduction of expected taxa, such as anaerobic genera. In addition, the relative decrease of genera associated with disease recovery (eg, Blautia obeum and Paracoccus aminovorans, that are known to be associated with inibition in vivo and promotion of growth in vitro of V. cholerae, respectively [Escherichia organisms from other genera within the Enterobacteriaceae family. Enterobacteriaceae was grouped next to genera associated with the early phase of recovery from cholera, based published data on Escherichia species [These findings should be viewed within the context of the limitations of the study. First, the study was conducted at only 2 sites within a single district in rural Bangladesh. Second, the mass spectrometry results are presented as qualitative data. LC/MS can provide quantitative data. We decided on a qualitative method not only due to cost and logistical constraints, but also to enable analyte annotation and detection of metabolites, and because physiologic variation in free water volume in urine and diarrheal stool may devalue quantitation. Third, the medication-vendor survey and clinical study were in different districts, yet both were in rural settings. Fourth, the microbiota analysis was restricted by a small sample size and limited antibiotic-negative stool samples. This limitation negated the ability to adjust for age and sex, resulted in a lack of significance for some bivariate comparisons after adjustment for false-discovery rates, and caused insufficient representation of important taxa, such as ectively , 44. The species . DespiteThe findings suggest that there may be no true negative control when attempting to account for antibiotic exposure in settings like those in this study. Similar studies that incorporate LC/MS will contribute to a more complete understanding of the scale of antibiotic exposure. Collectively, these data will provide important clinical insights that will help guide our approach to combating AMR and encouraging better antimicrobial stewardship.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiz299_suppl_Supplementary_DataClick here for additional data file.


Hypostomus subcarinatus Castelnau, 1855 is rediscovered in the Lagoa da Pampulha, an urban lake in the Rio das Velhas basin (Rio São Francisco system) in the state of Minas Gerais, southeastern Brazil. Herein, H. subcarinatus is redescribed and diagnosed from its congeners based on characters such as blue-tan dorsal fin in live specimens, slender bicuspid teeth, dentaries angled more than 90 degrees, moderate keels along lateral series of plates, small roundish dark spots, one plate bordering supraoccipital, by having nuptial odontodes mainly on pectoral, dorsal and caudal-fin rays, and long anal-fin unbranched ray. The rediscovery of H. subcarinatus more than 160 years after its original description was an unexpected event, because the Lagoa da Pampulha is an artificial, silted and polluted urban lake. The lake is located in downtown Belo Horizonte, the third largest urban agglomeration in Brazil with a population exceeding 5.9 million inhabitants. Hypostomus subcarinatus was described by Castelnau . Lagoa da Pampulha, tributary of Córrego da Onça, Rio das Velhas, Rio São Francisco basin: ICT-UFMG 2885, 18, 110–226.0 mm SL, 8 Jan 2002, V. Vono, B. P. Maia & L. G. M. Silva. MCNIP 1103, 7, 158.1–249 mm SL, 19°50’30”S 43°59’38”W, 30 Jan 2014, A. A. Weber & D. Gontijo. NUP 20229, 7, 164.9–248.9 mm SL, 19°50’30”S 43°59’38”W, 23 Dec 2017, I. S. Penido & T. C. Pessali. MCNIP 1761, 7, 191.1–308.8 mm SL, 19°50’30”S 43°59’38”W, 15 Apr 2016, I. S. Penido, C.H. Zawadzki, F. M. Azevedo & T. C. Pessali.Hypostomus tapijara: Paraná State, Rio Ribeira de Iguape basin. NUP 863, 9, 85.9–251.3 mm SL. NUP 869, 25, 111.0–350.0 mm SL and NUP 2795, 3, 174.9–193.2 mm SL, Rio Capivari.Hypostomus unae: Bahia State, Rio de Contas basin. NUP 9811, 5, 78.9–53.7 mm SL, Rio das Pedras. NUP 9814, 81.5–102.7 mm SL, Rio Oricó. MCP 41473, 10, 80.2–126.5 mm SL, Rio Preto do Costa. Rio Pardo basin. MCP 41334, 3, 55.2–120.8 mm SL, Rio Panelinha.Hypostomus velhochico: Minas Gerais State. Rio São Francisco basin: MZUSP 73816, 1, 83.4 mm SL, paratype, municipality of Presidente Juscelino. NUP 12065, 92.6 mm SL, paratype, municipality of Pirapora, Rio das Velhas. NUP 12066, 1, 82.8 mm SL, paratype, municipality of Santana do Pirapama, Rio das Velhas. NUP 12067, 1, 80.2 mm SL, paratype, municipality of Santana do Pirapama, Rio das Velhas.Hypostomus watwata: French Guyana. MNHN A. 8919 , 194.5 mm SL, Rio Cayenne. Guyana. BMNH 1932.11.10.31 , 261.2 mm SL, Berbice River.Hypostomus wuchereri: Bahia State. BMNH1863.3.27.15, 1, syntype, 203.8 mm SL, exact locality unknown. BMNH 1852.13.12.8, 1, 127.3 mm SL, syntype, exact locality unknown.S1 TableHypostomus subcarinatus. For the proportion and percentage ranges the holotype data were not included.Individual measures in millimeters and counts for the holotype and 24 specimens of (XLSX)Click here for additional data file.