Hepatocyte growth factor (HGF)-signaling via Met can induce mitogenic, morphogenic, and motogenic activity in various cell types. Met expression in the immune system is limited to cells with antigen-presenting capacities, including dendritic cells (DCs). Thus, it appears highly conceivable that Met-signaling impacts on adaptive immune responses. However, the mechanisms by which HGF imparts its effects on immunological responses are not yet fully understood. DCs possess unique functionalities that are critically involved in controlling both tolerance and immunity. HGF conveys immunoregulatory functions, which strongly correlate with that of DCs orchestrating the apt immune response in inflammation. Therefore, this review focuses on the current knowledge of Met-signaling in DCs with specific emphasis on the morphogenic and motogenic activities. HGF has been identified to play a role in peripheral immune tolerance by directing DC differentiation towards a tolerogenic phenotype. In skin immunity, Met-signaling was shown to drive mobilization of DCs by regulating matrix metalloproteinase activities. This is strikingly reminiscent of the role of Met for regulating a cell fate program during embryonic development, wound healing, and in tumor invasion known as epithelial-mesenchymal transition (EMT). Thus, the concept emerges that an EMT program is executed by Met-signaling in DCs, which will be also discussed. Hgf or Met in mice are embryonically lethal, emphasizing the role of HGF/Met-signaling for a proper development [Met, a high affinity transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF) was originally identified as proto-oncogene . HGF waselopment ,7,8. Latelopment ,10 and pelopment  regeneraelopment ,13,14. Melopment ,16. Simielopment ,18.The tissue regenerative function of HGF/Met correlates directly with immune regulation and inflammation . For exaWhile Met is predominantly expressed by epithelial cells, various hematopoietic cells are in addition able to acquire Met expression. In the hematopoietic compartment and therewith in the immune system, Met expression is limited to hematopoietic progenitor cells and their progenies with antigen-presenting capacities, including monocytes/macrophages, B cells, and dendritic cells (DCs). Met-signaling was found to play a role in monocyte-macrophage development  and in rDCs are the most professional antigen-presenting cells (APC) of the immune system. They are part of the innate immune system and are distributed in lymphoid and non-lymphoid organs throughout the body where they serve as sentinels of the immune surveillance system .DCs originate from self-renewing hematopoietic stem cells (HSCs) in the BM that develop into progenitor cells. To fulfill their function progenitor cells are obliged to migrate via the peripheral blood towards lymphoid and non-lymphoid tissues where they differentiate into immature DCs ,31. Uponversus-host disease following transplantations, and in infectious or autoimmune diseases [Hence, DCs possess unique functionalities that are critically involved in controlling and maintaining the balance between tolerance and immunity, thus developing an effective immune response and at the same time limit self-damage. This makes DCs attractive targets for immunotherapies, such as vaccines against cancer , treatmediseases .In this respect it is interesting that HGF mediates immunoregulatory activities, which correlate with that of DCs orchestrating an appropriate adaptive immune response. Moreover, although the mechanisms by which HGF/Met effects immunological functions are not yet fully understood an impact of HGF/Met-signaling on DC functions in splenic DCs , as wellet al. [In peripheral immune tolerance HGF has been identified to play a role by impairment of DC activation characterized by an obstructed antigen presenting capacity ,37. In tet al.  no detecet al. , was fouet al. ,40.In murine autoimmune models and in human monocyte derived DCs, HGF imparts DCs with suppressive competence resulting in effective induction of regulatory T cells (Tregs) ,39,41. SHGF treated DCs were found to be characterized by an increased expression of programmed-death ligand 1 (PD-L1) and IL-27, factors mediating DC-driven Treg generation . In addiMonocytes are a leukocyte sub-population with the capacity to differentiate into tissue macrophages and DCs under inflammatory conditions . In receHGF-induced migration of monocytes resembles the crucial motile property of DCs for executing their immune function. The motogenic capacities of activated DC are required for emigration form peripheral tissues and migration towards lymphoid organs where they instruct antigen-specific T cells. For this purpose, DC subpopulations in peripheral tissues, such as the skin, constitute a first immunological barrier to the external environment. In skin, dDCs and LCs were shown to express Met, and binding of its ligand HGF effectively activated Met-signaling ,28. ThisThese findings were supported using a conditional Met deficient mouse model where activated skin resident DCs failed to migrate towards the skin-draining lymph nodes despite an activated phenotype . This esHGF-induced DC mobilization and migration upon activation is associated with various phenotypic transformations that includes for example MMP activation and detachment from surrounding tissue. This is strikingly reminiscent of a Met-signaling driven mechanism seen during embryonic development, wound healing, and invasive growth of tumors known as epithelial-mesenchymal transition (EMT). In this process activated immobile epithelial cells convert transiently into a migratory mesenchymal phenotype ,16. ThusLCs in skin were shown to express E-cadherin and various other epithelial-like junctional proteins, including claudin-1, EpCAM/TROP1, TROP2, ZO-1, occludin, JAM1, and cytokeratins, which allows them to functionally integrate into the keratinocyte layer. Moreover, activation of LC was found to induce expression of the EMT regulators ZEB1 and ZEB2 and downregulation of E-cadherin and EpCAM, accompanied by upregulated expression of N-cadherin . TogetheAltogether this review summarizes the pivotal role of HGF/Met-signaling in DC mediated immunity . HGF/MetIn contrast, the role of HGF/Met-signaling for DC migration is less well established. Initial studies highlighted the importance of Met-signaling for control of DC motility and migration. First fundaments towards understanding Met-dependent mechanisms in DC migration were established by elucidating MMP regulation by Met-signaling. However, knowledge about intracellular signaling transduction pathways that control DC migration following Met activation is not yet fully exploited. Based on the notion that in wound healing keratinocyte and DC migration are regulated by the same Met-driven mechanisms, it is highly conceivable that similar protein signaling network dynamics exist in DCs and keratinocytes ,54,61.Met has been examined in numerous cancer-based studies as a critical factor for tumor invasion and metastasis and thus is highly considered as a drug target for tumor therapies. Accordingly, control of DC function by HGF/Met-signaling as a crux for DC-based immunity represents a potential starting-point for immune therapy against cancer and autoimmune diseases. However, means to target HGF/Met in order to favor immune tolerance might at the same time boost potential tumor cell towards invasive growth and metastasis. The other way round, blocking Met-induced migration of tumor cells in patients might alter their immune function towards development of autoimmune reactions. Nevertheless, this would be a matter of considering the pro and cons, depending on the required therapy and taking into account whether therapies are only temporarily applied.In summary, the knowledge of intracellular HGF/Met-signaling mechanisms in DCs is still incomplete and needs to be expanded by further research to allow save Met-based therapies in the future. Elucidation of downstream signaling pathways of Met involved in DC migration will also reveal whether a Met-driven EMT program is in fact operating in DCs.


Few studies have investigated the appropriateness of antibiotic use in postdisaster settings. We retrospectively evaluated clinical databases on health care delivered at clinics near shelters set up after the Great East Japan Earthquake, 2011. We defined appropriate, acceptable, and inappropriate antibiotic use for each diagnostic category, by applying and adopting precedent studies and clinical guidelines. From March to July, 2011, a total of 23,704 clinic visits occurred at 98 shelters with 7934 residents. Oral antibiotics were prescribed a total of 2253 times. The median age of the patients was 48.5 years old (range 0–97), and 43.7% were male. Of 2253 antibiotic prescriptions, 1944 were judged to be inappropriate (86.3% 95% CI 84.8%–87.7%). The most prescribed antibiotic was clarithromycin , followed by cefcapene pivoxil , levofloxacin , cefdinir , and cefditren pivoxil . The most frequent diagnosis for which antibiotics were prescribed was upper respiratory infection , followed by acute bronchitis , pharyngitis , traumatic injuries , acute gastroenteritis , urinary tract infections , and allergic rhinitis (5.1%). The majority of antibiotics prescribed at clinics after the Great East Japan Earthquake was inappropriate. Significant improvement of the use of antibiotics in postdisaster settings should be sought immediately in Japan. Studies had evaluated the appropriateness of antibiotic use in regular settings,–9 but ones for healthcare in postdisaster settings have not been evaluated.Natural disasters often result in the mass evacuation of people to shelters. The earthquake also led to mass evacuation with many people staying in shelters. To evaluate the appropriateness of antibiotic use there, we reviewed clinical data on the medical services provided at these shelters.The Tohoku area in Japan was hit by a massive earthquake with a magnitude of 9.0 and a subsequent tsunami on March 11, 2011, which resulted in more than 15,000 deaths (the Great East Earthquake).22.1 More than 50,000 people were evacuated to shelters following the tsunami, and more than 3800 people died or went missing. Japanese Red Cross Ishinomaki Hospital was the only designated disaster hospital in the Ishinomaki Medical Zone, and it was undamaged by the earthquake. The Ishinomaki Zone Joint Relief Team coordinated medical support from all over Japan, and medical workers provided care to people in shelters, typically rotating weekly. Many shelters and their aid stations lacked lifelines such as electricity, water, or sewerage systems, and medical care was provided without laboratory tests or imaging studies. Those who needed referral were transferred to Japanese Red Cross Ishinomaki Hospital, but transfer was difficult due to destroyed roads and lack of vehicles. The study was approved by the ethic committees both at Kobe University Graduate School of Medicine and Japanese Red Cross Ishinomaki Hospital.We extracted clinical data from database at Department of Health Crisis Management, National Institute of Public Health. The database was produced based on data from clinical charts used to provide care at 98 shelters or their accessory aid stations at Ishinomaki City, Miyagi, Japan. Ishinomaki was one of the most damaged areas following the Great East Japan Earthquake, mostly due to the massive tsunami.2.2We included patients who were prescribed oral antibiotics at each clinic. The use of intravenous, intramuscular, or topical antibiotics were excluded from the analyses. We collected age, sex, diagnosis, and prescriptions from the database, and evaluated the appropriateness of antibiotic use. which were based on clinical guidelines for appropriate antibiotics prescription by age group in the outpatient setting. For example, antibiotic therapy was not recommended for upper respiratory infections (URI) for both adults and children in this criteria.–15 We judged the criteria were useful and applicable in the postdisaster setting in Japan.For the definition of appropriateness, we applied and adapted the criteria developed by Fleming-Dutra et al, we applied and adapted the Japanese Association for Infectious Diseases (JAID) and Japanese Society of Chemotherapy (JSC) guideline as the reference, which recommended no antibiotic therapy for mild acute gastroenteritis in children, but recommended clarithromycin, amoxicillin, fosfomycin, or norfloxacin for suspected Campylobacter infections. Although meta-analysis on antibiotic treatment for Campylobacter infection showed only limited effects, we allowed this antibiotic use for this condition, considering the significant damage to quality of life at shelters. We also accepted other fluoroquinolones such as levofloxacin as an alternative to norfloxacin being mindful of the possible lack of stock at each clinic. For muscloskeletal traumatic injuries, we applied Lane et al as a reference for recommendation, but allowed other antibiotics such as amoxicillin/clavulanate or even the use of fluoroquiolones for the same reason above. For skin and soft tissue infections (SSTI) such as cellulitis, we used the latest Infectious Diseases Society of America guideline.For acute gastroenteritis, which was the 2nd most common infectious disease after URI in a precedent study but no recommendation was made by Fleming-Dutra et al, Likewise, we did not include fosfomycin in our recommendation since the fosfomycin calcium which is available in Japan has much worse bioavailability than the fosfomycin tromethamine available abroad. For other miscellaneous infections, we used our expert recommendations as the basis for the appropriateness of the use of antibiotics. On the other hand, we allowed the use of cefaclor for the treatment of SSTI since it is usually regarded as “first-generation cephalosporin” in Japan, instead of 2nd-generation in other countries. Cefaclor has reasonable bioavailability, and we judged that it can be used as an alternative to cephalexin for the management of SSTIs. If there is an ambiguity between appropriate/acceptable and inappropriate, 2 infectious diseases specialists discussed the issue to resolve the issue (KI and TF). These recommendations were summarized in Table We categorized the use of antibiotics to appropriate, acceptable, or inappropriate. Acceptable antibiotic use was defined as the use of an antibiotic which was not necessarily ideal, but which can be used as an alternative to the first choice, particularly in disaster situations. For example, the use of fluoroquinolones for SSTI cannot be recommended routinely due to their broad spectrum nature, but may be acceptable in circumstances where no other antibiotics were available. We restricted the recommended antibiotics to those approved in Japan. We considered oral 3rd-generation cephalosporins not appropriate for use in most circumstances, due to poor bioavailability, their broad spectrum, and the existence of better alternatives.2.3Statistical analyses were performed using STATA 14.1 (STATA Corp). Ninety-five percent confidence intervals (CIs) were calculated for estimates.3From March to July, 2011, a total of 23,704 clinic visits occurred at 98 shelters with 7934 residents. Oral antibiotics were prescribed 2253 times. The median age of the patients who were given antibiotics was 48.5 years old (range 0–97), and 43.7% were male.Of 2253 antibiotic prescriptions, 156, 153, and 1944 were judged to be appropriate, acceptable, and inappropriate, respectively , followed by cefcapene pivoxil , levofloxacin 380, 16.9%), cefdinir , and cefditren pivoxil . For each antibiotic, most prescriptions were judged to be inappropriate , followed by acute bronchitis , pharyngitis , traumatic injuries , acute gastroenteritis , urinary tract infections , and allergic rhinitis (5.1%) . Although there are studies investigating the amount of antibiotic consumption in Japan, few studies have investigated their appropriateness. Our findings, even though criteria for appropriateness are slightly different, were much worse than those in the United States.Fleming-Dutra et al although, the rate of defined daily doses per 1000 inhabitants per day for penicillin was much smaller in Japan than in European countries, oral 3rd-generation cephalosporins, oral macrolides, and oral fluoroquinolones were consumed at high rates in Japan. Our study findings are consistent with them, and we found that not only these broad spectrum antibiotics were used frequently, but also they were used inappropriately.According to Muraki et al,Clostridium difficile infection.,37,38The overuse of 3rd-generation cephalosporins poses significant risk to society. Oral 3rd-generation cephalosporins generally have poor bioavailability, yet can kill Enterobacteriaceae in the intestine unnecessarily, which can lead to an increase in antibiotic resistant organisms and  Also, the use of macrolides were judged to be acceptable in treating selected cases of Campylobacter infection. Despite this allowance, most macrolides prescribed in our study were inappropriate. Inappropriateness might be even worse than our estimate, since most cases of pharyngitis are caused by viral infection, and many can be treated with penicillins even if caused by bacteria. Additionally, most diarrheal illnesses are not caused by Campylobacter. However, given the circumstances where few diagnostic tests were available, we had to accept the potential overtreatment with macrolides to a certain extent. Despite this allowance, macrolides, particularly clarithromycin, were overused and misused according to our findings.In our study, the use of macrolides was accepted for the treatment of pharyngitis and acute gastroenteritis. Macrolides are recommended for acute bacterial pharyngitis when penicillin cannot be used due to allergy.Mycoplasma pneumoniae, Streptococcus pneumoniae, and Streptococcus pyogenes.,40 In addition, the use of macrolides is associated with significant side effects such as cardiovascular death. Clarithromycin is known to increase the risk of rhabdomyolysis when coadministered with statins. Even though macrolides can be useful in certain infections, they should never be used unnecessarily, particular in postdisaster settings, where one may not be able to treat the complications of medication use promptly.The overuse of macrolides in Japan has resulted in an increase in macrolide resistant organisms, such as We are not trying to make an assertion that the health care workers that provided care after the Great East Japan Earthquake were incompetent or ignorant. We simply sought the appropriateness of antibiotic prescriptions. The inappropriateness could come from number of factors, such as the lack of antibiotic stock at each clinic, the need to deliver hope to devastated people at shelters, or the lack of diagnostic tools to name a few. Some volunteer doctors might be specialists of different areas and might not be used to prescribe antibiotics at such settings . In Japan, not every physicians received training on primary care, not to mention the field of “disaster medicine.” We were able to reveal “what happened” after the earthquake, but were not able to reveal “why” this happened. In-depth investigations such as qualitative studies might reveal the root cause of antibiotic misuse. Further studies are needed to evaluate to what extent inappropriate antibiotic use is due to unusual environment after disasters, or due to inappropriate antibiotic use in daily practice.We investigated the cases where antibiotics were used, but we did not investigate the cases where antibiotics were not used. For example, 2739 diagnoses of URI were made during the study period, and antibiotics were prescribed in 1040, suggesting URI were appropriately managed in more than half the cases (62.0%). This is similar to antibiotic prescription rate for URI at nondisaster setting according to a cross-sectional study in Japan (60%).There are several limitations inherent in our study. First, since we extracted data for the diagnoses and the names of the antibiotics used, we might be missing important contextual information on each practice. There might have been justifiable reasons to prescribe antibiotics in individual cases, for which we judged them inappropriate. On the other hand, since we were not able to obtain detailed information about patients, there might have been more antibiotic misuse than we identified, such as contraindications due to underlying health problems, interactions with other medications, or the presence of known allergies. Second, our criteria of appropriate, acceptable, or inappropriate antibiotic use might be seen as too austere. However, we applied precedent studies on appropriate antibiotic use and current clinical guidelines. We were also mindful of the extreme environment after disasters, allowing rather broad spectrum antibiotics for certain infections, such as fluoroquinolones for SSTIs. We are afraid that we might have been underestimating the proportion of inappropriate antibiotic use, but we do not consider that we overestimated it. and we will definitely experience other disasters with health care needs.In conclusion, we found a significant number of inappropriate antibiotic use at clinics held after the Great East Japan Earthquake. Japan suffered another large scale earthquake in the Kumamoto area in 2016,We believe that even refugees after disasters do have the right to receive appropriate medications, such as antibiotics for their health and environment. Further improvement in the use of antibiotics in such circumstances should be undertaken.The authors thank Dr Seiichi Kobayashi for invaluable comments and discussion. The authors also thank Dr Daniel Mosby for editing the manuscript to eliminate grammatical or spelling errors.


The MTT assay depicted a high amount of cytotoxicity of GAR-NPs in B16F10, HepG2 and KB cells. A considerable amount of cell apoptosis was observed in B16f10 and KB cell lines. In vivo cellular uptake of fluorescent NPs on B16F10 cells was also investigated. Finally the GAR loaded NPs were radiolabeled with technetium-99m with >95% labeling efficiency and administered to B16F10 melanoma tumor bearing mice to investigate the in vivo deposition at the tumor site by biodistribution and scintigraphic imaging study. In vitro cellular uptake studies and biological evaluation confirm the efficacy of the formulation for cancer treatment.Garcinol (GAR) is a naturally occurring polyisoprenylated phenolic compound. It has been recently investigated for its biological activities such as antioxidant, anti-inflammatory, anti ulcer, and antiproliferative effect on a wide range of human cancer cell lines. Though the outcomes are very promising, its extreme insolubility in water remains the main obstacle for its clinical application. Herein we report the formulation of GAR entrapped PLGA nanoparticles by nanoprecipitation method using vitamin E TPGS as an emulsifier. The nanoparticles were characterized for size, surface morphology, surface charge, encapsulation efficiency and  Garcinol (GAR) is a polyisoprenylated benzophenone derivative isolated from the fruit rind of Garcinia indica known as Kokum. Like turmeric, this plant is also used as a garnish in cooking and has been extensively used to treat gastric disorders and skin irritation2. Studies by different groups have revealed the potential antioxidant, antiinflammatory and anticancer effects of GAR. The anti oxidative property is due to its polyphenolic structure3. In vivo studies in animal models have shown its efficacy in suppressing azoxymethane induced colon cancer4, 4-nitroquinoline-1-oxide induced tongue cancer5, and nicotine-induced human breast cancer6. In vitro studies in different cancer cell lines reveal the efficacy of the compound in modulating cell signaling pathways involved in apoptosis and cancer development7. Though a large number of in vitro and in vivo studies have been done to establish its wide array of pharmacological effects, little is known about its pharmacokinetic properties and toxicity parameters. A study report claimed that oral administration of 285.71 mg/kg GAR in a mouse model bearing MDA-MB-231 did not result in systemic toxicity and mortality7. The compound is a hydrophobic polyphenol exhibiting extremely low aqueous solubility whereas freely soluble in common hydrophilic or organic solvents (0.344–1 g/ml). This necessitates the development of potential delivery systems to enhance its stability and bioavailability.In recent years a great deal of attention has been given to identify novel pharmacophores from natural resources that can be used to suppress cancers as well as reduce the risk of cancer development8. Poly(lactic-co-glycolic acid) or PLGA is one of the most successfully developed biodegradable and biocompatible polymer approved by Food and Drug administration (FDA) and European Medical Association for parenteral administration9. PLGA nanoparticles are internalized in cells through pinocytosis and endocytosis10.So far no studies have been reported regarding encapsulation of GAR in polymeric nanoparticles to improve its bioavailability and anticancer potency. The choice for an ideal polymer for nanoencapsulation is vital as it regulates the essential properties such as solubility, stability, drug loading capacity and drug release profile11 and other inflammatory disorders13. Promising pharmacological effects of GAR have encouraged us to develop a novel garcinol nanoparticle system to improve its aqueous solubility as well as bioavailability and exploit its potential therapeutic efficacy. There are reports of using TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) as an emulsifier in nanoparticle formulation of anticancer drugs which resulted in high drug encapsulation and substantially high cellular uptake by cancer cells16.Nanoparticle therapeutics has attracted increasing attention in recent years as an emerging treatment modality in cancerin vitro drug release profile. The physical state of GAR in nanopaticles (NPs) was investigated using differential scanning calorimetry (DSC), fourier transform infra red spectroscopy and X-ray diffraction (XRD). In vitro efficacy assay in different tumor cell line was examined by MTT assay, apoptosis assay and DAPI staining. Cellular uptake and flow cytometric studies of FITC labeled NPs in B16F10 cell line were performed. Most importantly GAR loaded nanoparticles (GAR-NPs) were radiolabeled with technetium-99m to ascertain the biodistribution and tumor accumulation (scintigraphic imaging) in B16F10 tumor bearing mice model. The results are discussed in the following sections.GAR-loaded vitamin E TPGS-emulsified PLGA nanoparticles were prepared by nanoprecipitation method. The nanoparticles were characterized in terms of their particle size, polydispersity index (PDI), morphology, encapsulation efficiency (EE), zeta potential and GAR encapsulated nanoparticles were developed to achieve maximum solubility and bioavailability of entrapped GAR. Blank and GAR-loaded PLGA NPs were prepared through a nanoprecipitation technique using vitamin E TPGS as an emulsifier. Table The morphology size of the GAR-NPs were determined using field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). FESEM images Fig.  showed tThe free flowing powder of GAR-NPs obtained after lyophilisation was stored at 4 °C for 3 months for particle size measurement. The particle size was slightly increased  for 24 h and 48 h. Both free GAR and GAR-NPs exhibited concentration and time-dependent cytotoxic behaviour. Detailed analysis of cytotoxic effect  were therefore treated with blank-NPs, free GAR and GAR-NPs before DAPI staining. This revealed rounded and intact nuclei in untreated control as well as in the group exposed to blank-NPs, while the cells treated with free GAR and GAR-NPs had nuclei that appeared more brightly stained with condensed chromatin showing crescent shaped profiles around the periphery of the nucleus or separate globular structures (apoptotic bodies). The features appeared more prominent in the cells treated with GAR-NPs. It could be concluded that GAR-NPs induced apoptosis exhibiting pronounced nuclear condensation of B16F10 Fig.  and KB  cells.The hemato-compatibility of GAR-NPs in different concentration ranges (0.5 to 100 µM) was determined Fig. . The resIn vivo pharmacokinetic analysis of GAR encapsulated nanoparticles were performed in adult Sprague-Dawley (SD) rats and compared with that of the suspension of free GAR following oral administration  for 6 h during incubation at 37 °C with normal saline and freshly collected rat serum. A maximum of 10% of degradation occurred after 24 h of incubation showing substantially high in vitro stability of the nanoformulation. The level of free pertechnetate generated was about 6% following 8 h incubation with excess histidine, the value increasing to about 12% at 24 h indicating sufficient stability of the radiolabeled nanoformulation towards transchelation. From these stability studies . The tumor-to-blood ratio was not very high initially (0.31 at 2 h) but improved with time (1.58 at 8 h). The uptake in other organs was also low. The radioactivity accumulation in the stomach was not very significant advocating sufficient udy Fig. . Intratuudy Fig.  was alsoin vitro cytotoxicity experiments performed against different cancer cell lines demonstrated the comparatively high cytotoxic potentials of GAR-NPs than free GAR. The difference in cytotoxicity was observed for different cell line studied. IC50 values were considerably lower in the case of B16F10, HepG2 and KB cell lines. The results boost up the efficacy of nanoformulation in cancer treatment. The mechanism of induction of cytotoxicity in B16F10 and KB cell lines was further assured by apoptosis assay. Morphological changes of nuclei during apoptosis were observed following DAPI staining. The results revealed the inhibition of cancer cells proliferation through an apoptotic pathway. The pharmacokinetic studies revealed sustained release of GAR over a period of 36 h following oral administration of nanoparticulated suspension whereas plasma levels of GAR were not detectable beyond 8 h after administration of suspension containing free GAR. The significant increase in AUC value of the nanoparticulated formulation in comparison to free GAR suspension also indicates improved bioavailability of the nanosuspension.Available literature data explains the potent cytotoxic activity of GAR and its derivatives against different human cancer cell lines. GAR exhibits emerging potential as a chemopreventive agent in carcinogenesis. However, GAR is highly hydrophobic (extremely insoluble in water) this may raise the major hurdle for the success of chemotherapy with GAR. Whereas not much effort has been put forward to solve this issue. We first report a novel GAR loaded PLGA based nanoparticulated drug delivery system using vitamin E TPGS as emulsifier. Nanoprecipitation technique produced nanoparticles of approximately 88 nm in size which is optimal for the ability of NPs to be used for intravenous administration and passive targeting to tumors. The physical characterization followed by substantially high encapsulation efficiency ensured the successful development of GAR-loaded nanoparticulated formulation. The sustained release pattern observed substantiated the use of GAR-NPs for tumor targeting. Cellular uptake study conducted using both flow cytometry and confocal microscopy, GAR-NPs showed substantially high time dependent uptake in B16F10 melanoma cell line. Time dependent cellular internalization of the nanoparticulted formulation will increase the intracellular accumulation of anticancer drugs leading to enhanced chemotherapeutic effect. The 99mTc-labeled GAR-NPs revealed moderate accumulation in tumor region. Accumulation in the tumor appeared at 2 h post injection time period and gradually increased up to 8 h. Tumor to muscle and tumor to blood ratios gradually increased with time throughout the period of study. Results obtained were further corroborated by scintigraphic imaging studies under a gamma camera. The tumor region was clearly visualized at 4 and 8 h post injection time period. The images also showed high-level of background activity in kidney, liver, GI-tract and urinary bladder  and internalization  and 99mTcO4− was extracted from a 5 N NaOH solution of 99MoO4− by butan-2-one. ECIL gamma counter (Model LV 4755) obtained from ECIL was used for measuring tissue and organ radioactivities. Scintigraphic imaging studies of animals were done in GE Infina Gamma camera equipped with Xeleris work station.PLGA 50:50 (PURASORB PDLG 5010) was obtained as a gift from Purac Biomaterials. FITC and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich. Vitamin E TPGS NF Grade was gifted by Antares Health Products. HPLC grade acetone was purchased from Spectrochem . All other solvents and chemicals were of analytical grade and procured from Merck, India. Garcinia indica were extracted with hexane17 at room temperature for 48 h. The extract was filtered and then concentrated under reduced pressure at 45 °C. The above step was repeated four times to yield the crude hexane extract (200 g), which was subjected to silica gel column chromatography (12 cm × 180 cm). Elution with petroleum ether-ethyl acetate  yielded eight major fractions. Based on TLC, fraction 5 (80:20) was subjected to repetitive column chromatography over a bed of silica gel . Elution was done with petroleum ether-ethyl acetate (80:20) to yield pure GAR (5.5 g) as indicated by a single peak in HPLC  of  kg of Ga extract 00 g, whi20. Briefly, an organic solution of PLGA (50 mg) and GAR (5 mg) in acetone (10 ml) was added to an aqueous vitamin E TPGS solution  under magnetic stirring at room temperature. The solvent was allowed to evaporate overnight. The suspension obtained was filtered (Whatman filter paper 1) to remove any precipitate and centrifuged at 18,000 rpm at 4 °C . The supernatant containing the free drug was discarded; the pellet obtained was washed 2–3 times with distilled water and lyophilised  for 48 h to get a free flowing powder.Nanoparticles were prepared by the nanoprecipitation methodDrug free nanoparticles were prepared according to the same procedure. Fluorescent nanoparticles were prepared by replacing GAR with FITC (5 mg).2 = 0.998). Nanoparticle yield, drug loading and encapsulation efficiency were calculated from following three equations.DL and EE of the prepared nanoparticles were quantified by measuring the absorbance at 356 nm using a SHIMADZU UV-1700 UV-Vis spectrophotometer. The freeze-dried nanoparticle samples (3 mg) were dissolved in ethyl acetate (10 ml) for spectrophotometric measurement. GAR solutions of various concentrations in ethyl acetate (5–50 µg/ml) were prepared, and the absorbance at 356 nm was measured at different concentrations to generate a standard calibration curve (RParticle size distribution (mean diameter and polydispersity index) was determined using a Malvern Zetasizer Nano ZS . Briefly, 1 mg/ml of GAR-NPs suspension was prepared in MilliQ water by sonication for 30 s. The above suspension (10 µl) was diluted to 1 ml with MilliQ water and analysis was performed. For each sample, the mean diameter of three determinations was calculated. Properly diluted samples from the above suspension were also used for the measurement of zeta potential at 25 °C using the same instrument. Values reported are the mean ± standard deviation of at least three different batches of nanoparticles.The surface morphology of GAR-NPs was observed using field emission scanning electron microscope . The lyophilised samples were sputter-coated (QUORUM Q 150T ES) with platinum (4–5 nm) at a current intensity of 40 mA for 40 s. The images were captured keeping the accelerating voltage between 1–5 kV.The shape of GAR-NPs was further characterized by AFM . A drop (10 µl) of GAR-NPs suspension (1 mg/ml) was placed on a mica sheet. The drop was allowed to air dry for 5–10 min. The sample was further mounted on the microscope scanner. The shape was observed and imaged in ACAFM mode with frequency 166.6 kHz and scan speed 0.502 µm/s.The internal structure of GAR-NPs was determined by TEM . A drop (10 µl) of GAR-NPs suspension (1 mg/ml) was placed carefully on a 300 mesh carbon coated copper TEM grid. The excess solution on the grid was removed using a fine piece of filter paper and the samples were air-dried for 10 h. The dried sample was then examined at 120 kV under a microscope.−1 in an inert atmosphere in an FTIR spectrophotometer  to understand the possible chemical interactions occurring between the drug and the polymer matrix.In the FTIR spectroscopic study, GAR, PLGA, TPGS, the physical mixture of GAR with the excipients, and the nanoparticulated formulation with or without drug were scanned over a wavenumber range of 4000–400 cmThe physicochemical compatibility between GAR and the polymers was evaluated using a differential scanning calorimeter . Accurately weighed samples (10 mg) of GAR, PLGA, TPGS, phy-mix, blank-NPs and GAR-NPs were sealed separately in a standard aluminium pan heated over a temperature range from 0 to 400 °C at a constantly increasing rate of 12 °C/min in an atmosphere of nitrogen gas at a flow rate of 150 ml/min.−1.X-ray powder diffraction patterns of pure GAR, polymer and GAR-NPs were measured in an X-ray powder diffractometer . The measurements were performed in the 2–50° 2θ range using CuKα radiation  as the X-ray source and the rate of scanning was 1° minin vitro release of GAR at various time intervals, accurately weighed samples of GAR-NPs (50 mg) were suspended in 5 ml of phosphate buffered saline (pH 7.4) and stirred on a magnetic stirrer (100 rpm) at 37 °C. At preselected time intervals, 1 ml of the release media was taken out and centrifuged ; the supernatant was collected for analysis. To the pellet containing nanoparticles with unreleased GAR, an equal volume of fresh release media was added. The mixture was transferred to the original lot and the process continued. The collected supernatants containing released GAR were then analysed by UV-vis spectroscopy to determine the percentage of released GAR from the nanoparticles. The process was repeated thrice for each time point and the mean value was calculated. The drug release data were plotted as cumulative percentage drug release against time.To determine the For assessing the long term stability of GAR-NPs prepared by nanoprecipitation method, these were stored at RT, 4 ± 2 °C and −20 °C for three months as per ICH guidelines. After these intervals, samples were analysed to determine particle size and zeta potential value as per the previously described method. Experiments were performed in triplicates.22. For flow cytometry experiments, B16F10 cells were cultured (DMEM medium) in 35 mm dish. After overnight incubation, the culture medium was replaced with fresh medium, FITC loaded NPs (50 µg/ml) were added, and incubated for 2 h, 4 h or 8 h. After incubation for specified time periods, the cells were washed thrice with cold PBS to remove unbound NPs if present, trypsinised, and centrifuged at 1500 rpm for 2 min at 4 °C. Finally cells were resuspended in 500 µl of PBS and analysed using a flow cytometer  using FACS Diva software (BD Biosciences). The increase of fluorescence in the cells treated with NPs relative to that in the untreated control cells was expressed as mean fluorescence intensity relative to control.The cellular uptake of NPs was evaluated using flow cytometry and confocal laser scanning microscopy as per reported methods5) were seeded (DMEM medium) on a cover slip placed inside a 35 mm dish and incubated for 24 h at 37 °C in humidified air containing 5% CO2. Following 80% confluency in each dish, the medium was removed and replaced with FITC-loaded NPs (200 µg/ml) and incubated for 2 h, 4 h or 8 h. After incubation the medium was removed and 1 ml of 70% chilled ethanol was added to each of the dishes which were kept at −20 °C for 15 min to fix the cells. The ethanolic solution was removed, washed three times with PBS, and the nuclei stained with DAPI solution for 5 min. The cells were viewed and imaged under a confocal laser scanning microscope . The images were processed using Leica application suite software.The cellular internalization of FITC loaded nanoparticles was measured by confocal laser scanning microscopy. B16F10 cells , whereas HepG2 was maintained in MEM medium. All cells were plated at a density of 1 × 104 cells per well in 96-well plates and cultivated at 37 °C in humidified air containing 5% CO2 . After 24 h, the respective media were removed and replaced with 200 µl of fresh media containing different concentrations of free GAR (5–100 µM for 24 h and 1–55 µM for 48 h) dissolved in minimum amount of DMSO and GAR-NPs (1–55 µM of GAR for 24 h and 1–40 µM of GAR for 48 h). After specified incubation period, media containing free GAR and GAR-NPs were removed, 200 µl of MTT solution (1 mg/ml) added to each well, and incubation carried out for another 4 h at 37 °C in darkness. After withdrawing the culture medium, 150 µl of DMSO was added to each well to dissolve the intracellular formazan crystals and absorbance was measured at 550 nm using a microplate reader . Cell viability was determined as per the equation given below, where Abstest cells represents the amount of formazan determined for cells treated either with free GAR or GAR-NPs and Abscontrol cells stands for the amount of intracellular formazan present in non-treated cells.The cytotoxic effects of free GAR and GAR-NPs to B16F10, Hela, MDA-MB-231, HCT-116 and HepG2 cell lines were analysed by the MTT assay6) were cultured in 60 mm tissue culture dishes to reach 80% confluency. The cells were treated separately with different concentrations  of free GAR and GAR-NPs  and allowed to incubate for 24 h at 37 °C, 5% CO2. Then the cells were washed with PBS and trypsinised. The detached cells were collected by centrifugation at 1000 × g, resuspended in 0.5 ml 1X cold binding buffer, treated with annexin V-FITC (1.25 µl), and incubated for 15 min at room temperature in the dark. The cells were then centrifuged (1000 × g) at room temperature for 5 min. The supernatant was removed and the cell pellet was resuspended in 0.5 ml cold 1X binding buffer. A solution (10 µl) of propidium iodide was added to the cell suspensions. Apoptotic/necrotic and living cells were detected by a flow cytometer (BD FACS) and data were analysed using FACSDiva Version 6.2 software.B16F10 melanoma cells (1 × 1024. B16F10 and KB cells were grown separately on cover slips placed in 35 mm dish for 24 h at 37 °C in humidified air containing 5% CO2. The cells were treated with blank NPs (100 µM), free GAR (15 µM) and GAR-NPs (containing 15 µM GAR) for another 24 h under the same condition as mentioned above. Then they were washed twice with PBS and fixed in 70% ethanol at −20 °C for 15 min. After washing thrice with PBS, cells were treated with DAPI solution (4 µg/ml) for 5 min at room temperature and imaged under confocal laser scanning microscope . The images were processed using Leica application suite software.This study was done to understand the apoptotic effect of blank NPs, free GAR and GAR-NPs on cell morphologyg for 5 min and the optical density (OD) of the supernatant was measured at 570 nm. The value was compared with the OD of the supernatant (positive control) where full lysis occurred and the % lysis was calculated. The experiments were performed in triplicate to obtain the average value.Freshly collected blood samples from male Sprague-Dawley rats were taken in heparinised tubes and centrifuged (5 min at 1000 g) at 4 °C. The supernatant was discarded and erythrocytes were washed three times with PBS (pH 7.4). The suspension (2%) thus obtained was used for hemolysis study. In order to examine the hemolytic effect, 190 µl of the suspension was added to each well of a 96-well plate and treated with 10 µl of GAR-NPs . The negative control was prepared by adding 10 µl of PBS to 190 µl of erythrocyte suspension, whereas 10 µl of TritonX-100 (10%) was added to the erythrocyte suspension in the positive control. Following incubation at 37 °C for 1 h with gentle stirring, the unlysed erythrocytes were separated by centrifugation at 10,000 In vivo pharmacokinetic studies of GAR-NPs and aqueous suspension containing free GAR were done in male Sprague-Dawley (SD) rats (weight ~250 g). The animals were divided into two groups (n = 5). Animals in group-1 and group-2 were administered with aqueous suspension of free GAR and GAR-NPs respectively at a dose of 25 mg GAR/Kg body weight by oral gavage. The blood samples were collected from the retro-orbital plexus at predetermined time points  in heparinized tubes, centrifuged  to separate plasma. To each plasma sample (0.1 ml) methanol (1 ml) was added and vortexed, the clear supernatant collected after centrifugation was filtered was filtered by passing through 0.22 µm syringe filter. Finally the samples were analysed by HPLC using X-bridge Tm C18 column  eluted with acetonitrile:water (80:20 v/v) at a flow rate of 1.0 ml/min. Detection of the drug content was carried out with waters 2489 UV/Visible detector. All the pharmacokinetic parameters were determined using Kinetica 5.1 software (Thermo Fischer Scientific). The area under the curve (AUC) was calculated using the Trapezoidal method.99mTc (technetium-99m) by direct labeling method using stannous chloride dihydrate as a reductant. Briefly, 200 µl of aqueous sodium pertechnetate  was added to 200 µl of GAR-NPs suspension  followed by the addition of 25 µl of freshly prepared stannous chloride dihydrate (20 mg in 10 ml nitrogen purged water containing 100 µl of 6 N HCl) and incubation for 15 min at room temperature. The radiolabeled solution was stored at room temperature in shielded vials for subsequent studies. The radiolabeling efficiency was determined by ascending instant thin layer chromatography (ITLC) using 2.5 × 10 cm silica gel coated aluminium sheet as the stationary phase and acetone as a mobile phase as per the previously reported procedure25.GAR-NPs were radiolabeled with 99mTc-labeled nanoformulation was ascertained in vitro as per the previously reported method25. Briefly, the radiolabeled nanoformulation (0.1 ml) was added separately to 0.9 ml of (a) normal saline, (b) serum, and (c) histidine solution (10−2 M). The resulting mixtures were incubated at 37 °C for 24 h. Samples were withdrawn from the mixture at various time intervals  and analysed by ITLC (described above) to check for any dissociation of the radiolabeled formulation.The stability of 99mTc-labeled GAR-NPs into B16F10 mouse melanoma cell lines was studied according to the method described previously with some modifications26. Briefly, 1 × 106 cells were cultured in a 35 mm dish with DMEM medium at 37 °C for 24 h. After the addition of about 37 kBq of 99mTc-labeled GAR-NPs to the medium, the cells were incubated at 37 °C in 5% CO2 for different time intervals . After desired time interval the medium was removed to stop cellular internalization and cells were washed with ice-cold phosphate-buffered saline (pH 7.2). The radioactivities in cell pellet  and the supernatant (unbound) were measured in a gamma counter . Cell surface-bound radioligand was removed by washing the cells with ice cold acidic buffer  for 5 min at 0 °C. The cell-associated radioactivity that was not removed by this procedure was taken as the internalized radioactivity. Finally, cells were treated with 1N NaOH at 37 °C for 10 min to detach them from plates and centrifuged. The supernatant was collected and the radioactivity was measured. The percent of internalized radioactivity was calculated according to the method described previously. All experiments were repeated thrice and the results were expressed as the mean ± standard deviation.The cell-binding and internalization of 99mTc-labeled GAR-NPs was studied in healthy male Sprague-Dawley rats weighing around 250–300 g (n = 3). To anesthetized and well-hydrated animals, 99mTc-labeled nanoformulation was injected through one femoral vein. At preset time intervals (ranging from 2 min to 8 h) blood samples (0.5 ml) were collected from the other femoral vein, weighed accurately and analysed in a gamma counter to measure the radioactivity in the sample. The percentage of injected dose/g of the blood sample (% ID/g) was calculated and plotted against respective time interval to generate the blood disappearance curve.Blood clearance of In vivo biodistribution studies were performed in B16F10 tumor bearing Balb/c mice (body wt 25 ± 1.5 g) as per the previously reported method25. Freshly collected B16F10 cells (2 × 107 cells) suspended in saline were injected intramuscularly to the thigh of the right hind leg of each mouse. After two weeks a palpable tumor (volume 1 ± 0.1 cm3) developed and the animals were used for biodistribution studies. To each of the well hydrated animals 99mTc-labeled GAR-NPs  were injected through the tail vein. At 2, 4, 8 and 24 h post injection, the animals (n = 4) were euthanized and blood sample was collected by cardiac puncture. Urine along with urinary bladder as well as other organs was also collected. All the samples were collected in pre-weighed scintillation vials and radioactive counts were measured in a gamma counter. Organ uptake was expressed as a percentage of the injected dose per gram of tissue (% ID/g) or per organ.All animal care and experiments were carried out as per guidelines laid down by the CSIR-IICB Animal Ethics Committee  registered under Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Government of India, New Delhi. All animal studies and experimental protocols were approved by IICB-AEC (Ref.: IICB/AEC-APP/August-Meeting/2013).27. Animals were sacrificed at 2, 4 and 8 h post injection (p.i.) time period. Tumors were dissected out washed thrice with PBS and stored in 10% formaldehyde at room temperature for 24 h. The tumor tissue was embedded in paraffin and cut into 5-µM-thick sections, and the nuclei were stained using DAPI. The intratumoral distribution of FITC labeled nanoformulation was imaged using fluorescence microscope .In a separate experiment, B16F10 tumor bearing mice were injected with FITC labeled NPs suspended in PBS at a concentration of 40 mg/kg body wt via tail vein99mTc-labeled nanoformulation  was administered through the tail vein, placed under GE Infinia Gamma Camera equipped with Xeleris Work Station, and static images were acquired at 2, 4, and 8 h post-injection.The tumor bearing mice were anaesthetized (intramuscular ketamine injection), All experiments were repeated for at least three times, each time on a different day. All mean values of animal experiments are expressed as %ID per g of tissues or organs ± SD. Statistical analysis was performed by one-way ANOVA. Graphs were prepared using GraphPad Prism 5 and ORIGIN 8.0.Supplementary info


External lipid membranes affect guest recognition properties of water-soluble hosts, enhancing molecular conformations and equilibria not observed in free solution. 19F or 13C nuclei that can be encapsulated inside the host, 2D EXSY NMR experiments can be used to analyze and compare the in/out guest exchange rates in aqueous solution, isotropically tumbling micelles, or magnetically ordered bicelles. These analyses show that embedding the deep cavitands in lipid bilayers slows the guest exchange rate, due to the lipids acting as a “compression sleeve” around the host, restricting guest egress. This effect also enhances guest conformations in the host that are not observed in free solution, such as axial cyclohexane conformers and ketone hydrates.The effects on the molecular recognition properties of water-soluble deep cavitand hosts upon embedding them in phosphocholine lipid bilayer environments have been studied by 2D NMR experiments. By employing suitable guests containing  Quantitation of the thermodynamics1H NMR spectroscopy. To maximize detection, these investigations are generally performed in controlled environments, in deuterated solvents and in the absence of NMR-visible additives and impurities.When analyzing the conformation and motion of molecules confined in small spaces, the predominant tool is NMR spectroscopy.1  spectroscopy of cavitand:supported lipid bilayer (SLB) aggregates1H NMR spectroscopy of these events has been limited to simple 1D experiments in fast tumbling micelles,Synthetic host molecules are capable of selective molecular recognition in far more complex and challenging environments than pure solvent, however. Hosts such as deep cavitand 1 2a,10 hav1 is soluble in water up to ∼20 mM, but is highly lipophilic, and is smoothly incorporated into a variety of lipid aggregates.3+) salts such as choline.2O solution, the association constants of guests such as 1-adamantanemethanol (Ka = 2.9 × 105 M–1), cyclohexanone (Ka = 1.6 × 105 M–1), choline (Ka = 2.6 × 104 M–1) and acetylcholine (Ka = 1.2 × 105 M–1) are relatively consistent, within an order of magnitude or so.5 M–1. The in/out kinetics of bound guests, as well as their motion while inside the cavity, have been extensively investigated in pure D2O by 2D NMR techniques.via an “SN1-like” mechanism, whereby the walls flex open to unfold the cavitand in the rate-determining step, followed by rapid guest exchange. In water, the energy barrier consists of three major components: the energy barrier to rotate around the C–O bonds in the cavitand walls–1), plus the energetic penalties of unfavorable solvation of the cavitand walls and the bound guest once unfolded. Depending on guest size, these barriers range from 16.0–17.2 kcal mol–1,–1 (adamantanol) to 14.6 s–1 (cyclohexane). All these investigations used 1H NMR for analysis, as the 1H peaks for bound guest are shifted strongly upfield, and are easily visible at negative ppm, unhindered by 1H peaks from the host or excess free guest.The tetracarboxylate cavitand What is not known is how embedding the cavitand in a lipid bilayer affects these molecular recognition properties. Other techniques have shown that host is capable of guest recognition in a bilayer, and that guest binding affinities are enhanced in some cases,13C, 15N or 31P. Unfortunately, observing individual proton signals via1H NMR analysis of the binding processes in those cases is complicated by the presence of multiple hydrocarbon peaks from the lipids. More sensitive NMR experiments are rendered impractical by line broadening, and so dynamic NMR experiments that are essential for analysis of guest motion and binding kinetics in synthetic cavity-containing hosts are challenging. Here we employ a variety of guest molecules with different detectable nuclei for molecular recognition in a deep, water-soluble host, and investigate the effect of embedding the host in biomimetic membrane environments on the guest dynamics, conformation and reactivity.NMR analysis of (bio)molecular structure and dynamics in membrane bilayers is well-studied,1H NMR analysis cannot be used, this introduces serious constraints on the nature of the guest. As such, we investigated a wide range of guest species . Also, the host:guest library must be paired with a suitable membrane environment for analysis. Obviously, a natural cell membrane is challenging to use, but a number of surrogates are known and used for NMR analysis of membrane-bound biomolecules. One of the most effective mimics is a magnetically oriented bicelle, which maintains the bilayer sheet form of natural membranes while aligning in the magnetic field to allow analysis by ssNMR techniques.NMR analysis of the molecular recognition of small molecules by deep cavitands in lipid environments is complicated by a number of factors. The guest must bind in the cavitand, and be detectable when bound inside the host in multiple different lipid environments. In addition, study of the in/out kinetics requires guests that are sufficiently soluble to display peaks for both free and bound guest. Study of internal motion or conformational bias requires guests that display multiple conformations or orientations while bound inside the cavity: if  species  for thei31P NMR analysis of the lipid system. The disordered micelles display only one averaged 31P phosphate peak , which are well-known to allow formation of both magnetically oriented bicelles and smaller disoriented micelles.31P NMR analysis of the lipid phosphate groups is an invaluable tool to confirm the structure of the lipid aggregates, but does not allow investigation of the host:guest properties of 1. We initially approached the host:guest studies using 1H NMR spectroscopy with the simplest, most optimal guest possible. Cyclooctane 2 is easily extracted into the cavity of 1, has an affinity >104 M–1 by NMR,1·2 in D2O was added to solutions of either DMPC:DHPC micelles (hereinafter denoted as PCm) or DMPC:DHPC bicelles (PCb) for a final [1] = [2] = 1.8 mM and the spectra acquired at 283 K and 308 K respectively  was applied.2 was only partially useful, we turned to guests 3 and 4, containing 19F nuclei. As 19F peaks are broadened in the solid state in a similar manner to 1H, we focused on fast-tumbling micelles in the 1·3/4 analysis, with a lower lipid concentration of 60 mg mL–1 for ease of measurement. To allow analysis of conformation and in/out guest exchange, the guest must contain 19F nuclei that are bound inside the cavity of 1, and display the characteristic chemical shift variations caused by the magnetic anisotropy of the host. Fortunately, both guests 3 and 4 are suitable guests for 1 in aqueous solution, albeit displaying weaker binding than the equivalent hydrocarbons. 19F NMR spectra of the host:guest complexes shows that the bound 19F nuclei are upfield shifted, Δδ ∼ –2 ppm, indicating that the difluorocyclohexanyl group is oriented to the cavity interior. Presumably, the OH and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbdWZm6d" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O groups orient towards the external solvent to benefit from favorable H-bonding.As cyclooctane 3 and 4 show interesting and unexpected behavior when bound to 1, and provide excellent examples of the effect of embedding the host in the PC lipid environment. The 1H NMR spectrum of the 1·3 complex  separates the two fluorine peaks to reflect the distinct axial and equatorial positions (see Khyd(acetone) = 1.4 × 10–3, Khyd(fluoroacetone) = 0.11).4 in D2O in the absence of cavitand showed no obvious hydrate present. At the concentrations used, this indicates that <0.5% hydrate is present in solution. In contrast, when bound inside the cavity of 1, 13% of bound 4 exists in the hydrated form at 298 K. Embedding the host in PC lipids also biases the hydration equilibrium of 4, similar to the conformational bias seen in the binding of 3. When 4 was added to the 1·PCm system, 23% of the bound 4 was present in the hydrated form, compared to only 13% in 1·4 and <0.5% in free solution.Interestingly, the iol form . The 19F1 stabilizes normally unfavorable guest structures, and why this effect is enhanced when 1 is embedded in lipid aggregates. Cavitand 1 is well-known to display dual-mode recognition, whereby both the defined cavity and the upper rim groups can affect guest binding.1 than those with esters, ethers or ketones.3 evidently positions the OH group in closer proximity to the rim carboxylates, increasing the favorability of that conformer when bound. The hydrated gem-diol of 4 is capable of H-bonding with the carboxylates, whereas the ketone is not, hence the increased favorability of the bound hydrate , the same exchange conditions show only minimal crosspeaks for m1·3·PC  used to determine exchange rates; the full spectra are shown in the ESI.2OH axial. As such, the in/out exchange rates were determined by fitting the intensity of the four exchange peaks shown in τ ≥ 300 ms), the multiple methods of magnetization transfer in the system caused inaccuracies in the fitting, so the initial rate regions of the plot were used. For a detailed description of the fitting method, please see ESI.By taking the exchange spectra at multiple different mixing times, quantitation of the exchange rates was possible. The EXSY spectra of k–1 (or “koff”) for each guest, obtained at identical concentrations and temperatures for each guest in either aqueous solution (kfree) or in the micelle environment (kPC). Eyring analysisG‡. As expected, the rate is dependent on the nature of the guest, but most interestingly, it is also dependent on the external environment. The larger guest 3 shows a kfree = 4.2 s–1, comparable to that previously obtained for cyclooctanol.kPC = 1.8 s–1, corresponding to an additional 0.5 kcal mol–1 additional barrier conferred by the external environment surrounding the cavitand host. The same “compression sleeve” effect that enhanced the axial conformation of bound 3 slows the in/out exchange rate as well.4 showed that kfree = 8.7 s–1, similar to that to that previously obtained for cyclohexanone, as expected.kPC = 5.2 s–1. The retardation of exchange rate is slightly less in the case of the smaller guest 4, with a 0.3 kcal mol–1 additional barrier. Surprisingly, the EXSY spectrum of 1·4 allowed analysis of the in/out exchange of the hydrated gem-diol form of hyd4, as the crosspeaks were large enough to observe  = 5.7 s–1. Unfortunately, the equivalent crosspeaks in the PCm system were too small to accurately fit, so determination of kPC was unsuccessful in that case.EXSY analysis of guest  observe . The add1H to analyze the in/out exchange allows analysis of other, more biorelevant guests such as choline. While association constants for R-NMe3+ guests are easily determinable by ITC1 is susceptible to aggregation in free solution in the presence of lipophilic salts such as choline. Stable 1 : 1 complexes can be observed by NMR when substoichiometric amounts of guest are used,3+ salt causes aggregation and peak broadening, limiting analysis of the in/out rate by exchange NMR.5–8. 13C-enriched R-NMe3+ guests were easily accessed via reaction of the corresponding dimethylamino precursor with enriched 13CH3I. As cavitand 1 is capable of binding numerous R-NMe3+ salts in lipid environment, we investigated a series of guests 5–8 with variable upper rim functionality. The bola-type bis-NMe3+ guests 5 and 8 were initially targeted to investigate the possibility of slowed tumbling in the cavity of 1, as opposed to in/out guest exchange. Encapsulation in 1 slows the up/down interconversion rate of hydrocarbons such as trans-decalin, but quantitation is challenging due to peak broadening. As 5 and 8 are symmetrical, it was envisaged that exchange would be observed between the two conformers in the m1·5·PC system via13C–13C EXSY NMR analysis. Unfortunately, neither guest 5 nor 8 are bound by 1 in either free aqueous solution or in the PCm environment. Evidently, the nature of the upper rim has a large effect on guest recognition, so we turned to guests that can display favorable H-bonds with the carboxylate groups, choline 6 and the dimethylamino-variant 7.The use of guest nuclei other than 6 and 7 was far more successful, and representative examples of the 13C–13C EXSY spectra are shown in 3+ guest (6 or 7) overlaps with peaks from the R-NMe3+ group in the phosphocholine lipids, as would be expected. Even though 6/7 are 13C-enriched, a significant proportion of 13C-PC is present due to the excess of lipids in the sample. Despite the interfering peaks for free guest, 13C peaks for both bound 1·6 and 1·7 are easily observable, with the characteristic upfield shift observed. The relative change in 13C δ upon binding is proportionally smaller than that for 1H, with Δδ ∼ –2 ppm, but this is easily enough to allow exchange analysis via13C–13C EXSY.Analysis of the unsymmetrical guests 13C–13C EXSY spectra with varying mixing times were obtained for samples of m1·6·PC and m1·7·PC at 298 K with 5.8 mM 1, 16 mM guest and 60 mg mL–1 lipid, as usual. The exchange rates were acquired via fitting the crosspeak intensities extracted as slices from the 2D spectra. In this case, the diagonal peak corresponding to free guest overlapped with peaks from the NMe3+ groups in the lipids. As the concentration of the lipids was constant, the signal from the lipids remained constant in the low mixing time experiments, and the accuracy of the fit for the off-rate was not compromised. The rates are shown in kPC (6) = 3.0 s–1 and kPC (7) = 5.7 s–1. The bulkier 7 exchanges more rapidly than choline 6, in contrast to the results for hydrophobic guests, where larger guests showed slowed exchange. It is likely that positioning the extra steric bulk at the upper rim lowers the affinity of 7 for the cavitand, as has been observed for other R-NMe3+ species, and a more rapid exchange rate is seen. The results from kfree for 6 and 7: if the “compression sleeve” effect of the micelle environment is assumed to be constant, then kfree for 6 and 7 would be on the order of ∼6 s–1 and ∼10 s–1, respectively.13C-labeled guests 6 and 7 were amenable to EXSY analysis in isotropically tumbling micelles, we next employed these guests towards detection of in/out exchange of host 1 in the more challenging, yet more relevant magnetically ordered bicelles. The bicelles were formed as described above , loaded into a 4 mm Bruker solid state rotor, and solutions of cavitand 1 and guests 6 and 7 were added. As might be expected, the larger concentration of lipids (and their overlapping phosphocholine groups) made analysis via 1D NMR challenging, even with 13C-enriched guests. Fortunately, the magnetic alignment at 308 K was good, and the presence of exchanging guest 7 was observable by 13C–13C EXSY analysis  were performed on a Bruker Avance NEO 400 9.4 T spectrometer with a 5 mm Prodigy CPP BBO BB-H&F z-gradient cryo-probe or a Bruker 14.1 T (600.01 MHz 1H) Avance I spectrometer equipped with a 5 mm BBO Z-grad probe. Micelle (PCm) experiments  were performed on a Bruker Avance NEO 400 9.4 T spectrometer with a 5 mm Prodigy CPP BBO BB-H&F z-gradient cryo-probe, a Bruker 14.1 T (600.01 MHz 1H) Avance I spectrometer equipped with a 5 mm BBO Z-grad probe or a Bruker Avance III 700 16.44 T spectrometer with a 5 mm CP TCI H–C/N-D z-gradient cryo-probe. Magnetically ordered bicelle (PCb) experiments  were performed at 9.4 T  on a Bruker AVIII spectrometer equipped with a double resonance, 4 mm MAS probe. Proton (1H) and carbon (13C) chemical shifts are reported in parts per million (δ) with respect to tetramethylsilane . Phosphorus (31P) chemical shifts are reported in parts per million (δ), and referenced internally with respect to 85% H3PO4. Fluorine (19F) chemical shifts are reported in parts per million (δ), and referenced internally with respect to CF3COOH. Deuterated NMR solvents were obtained from Cambridge Isotope Laboratories, Inc., Andover, MA, and used without further purification. Mass spectra were recorded on an Agilent 6210 LC TOF mass spectrometer using electrospray ionization with fragmentation voltage set at 115 V and processed with an Agilent MassHunter Operating System. All other materials , and diheptylphosphocholine (DHPC)) were obtained from Aldrich Chemical Company, St. Louis, MO, or TCI, Tokyo, Japan and were used as received. Solvents were dried through a commercial solvent purification system . Molecular modeling  was carried out using the AMBER force field1 was synthesized according to published procedures:1· choline complex.1D NMR experiments (q = 3.2 (q = long chain lipid/short chain lipid) were formed by mixing together DHPC and DMPC dissolved in chloroform. The chloroform was evaporated off under a stream of nitrogen and then the lipids were lyophilized for 4 h before the micelles were prepared to remove any residual TFA and chloroform. The solid lipids were dissolved in HEPES buffer, pH = 6.5 for a combined lipid concentration of 290 mg mL–1. NMR samples were prepared by dissolving 100 μL of the lipid mixture in 400 μL D2O, to a final combined lipid concentration of 60 mg mL–1, 1 mM HEPES. Cavitand 1 and guests were added to the mixture and equilibrated for 1 h before NMR analysis.Mixed lipid micelles with q = 3.2 (q = long chain lipid/short chain lipid) by mixing together DHPC and DMPC dissolved in chloroform. The chloroform was evaporated off under a stream of nitrogen, the lipids were lyophilized for 4 hours before the bicelles were prepared to remove any residual TFA and chloroform, and the solid lipids were dissolved in HEPES buffer, pH = 6.5 for a combined lipid concentration of 290 mg mL–1. This solution was subjected to 5 minutes of vortexing, followed by 30 minutes in a water bath at 45 °C, followed by 15 minutes in an ice bath. This process was repeated five times until the lipids were clear and non-viscous at 4 °C and milky-white and solid at room temperature, indicative of bicelle formation. NMR samples were prepared by dissolving 250 μL of the bicelles in 200 μL HEPES buffer, pH = 6.5, and 50 μL D2O for a lock, to a final combined lipid concentration of 150 mg mL–1, 2.5 mM HEPES. This solution was added to a 4 mm Bruker solid-state rotor. Cavitand 1 and guests were added to the mixture and equilibrated for 24 h before NMR analysis.Magnetically ordered bicelles were prepared according to literature procedures.19F–19F or 13C–13C NOESY pulse sequence supplied with the Bruker software. Each of the 512 F1 increments was the accumulation of 6 scans (19F) or 32 scans (13C). Before Fourier transformation, the FIDs were multiplied by a 90° phase-shifted sine square function in both the F2 and the F1 domain. 1 K _ 1 K real data points were used, with a resolution of 1 Hz per point.The 2D NOESY spectra of the cavitand:guest exchange processes were recorded at 298 K at either 400 MHz or 600 MHz with the phase sensitive 1H-X double resonance 4 mm MAS probe with no spinning. The 2D EXSY spectra of the cavitand:guest 7 exchange process in bicelles were recorded at 316 K at 9.4 T with a NOESY pulse sequence. Each of the 128 F1 increments was the accumulation of 128 scans with a relaxation delay of 3 s. Before Fourier transformation the FID was multiplied by a 90° sine square function in the F1 domain and an EM function in the F2 domain. 2 K (F2) _ 256 (F1) real data points were used with a resolution of 1 Hz per point.Bicelle samples containing guest 7 and cavitand were loaded in a 4 mm Bruker solid-state rotor, and experiments were performed on a Bruker AVIII spectrometer equipped with a 13C  dropwise. The reaction was stirred at ambient temperature for 16 hours, and the resulting precipitate was collected by vacuum filtration to obtain 220 mg of guest 5 as a white solid (88% yield). 1H NMR  δ 3.47 , 3.23  2.41 , 13C NMR  δ 62.3, 53.3, 17.4. ESI-MS m/z expected: 190.20, found [MH+] = 190.14.Tetramethyl-1,3-diaminopropane  was dissolved in 2 mL of acetonitrile at 0 °C followed by addition of iodomethane-13C  dropwise. The reaction was stirred at ambient temperature for 16 hours, and the resulting precipitate was collected by vacuum filtration to obtain 228 mg of a white solid (82% yield). 1H NMR  δ 3.92 , 3.39 , 3.07 . 13C NMR  δ 67.4, 55.6, 53.9. ESI-MS m/z expected: 105.16, found [M+] = 105.11.Dimethylethanolamine  dissolved in 1 mL of acetonitrile at 0 °C followed by addition of iodomethane-13C  dropwise. The reaction was stirred at ambient temperature for 16 hours and the resulting precipitate was collected by vacuum filtration to obtain 650 mg of white solid (76% yield). 1H NMR  δ 3.35 , 3.02 , 2.73 , 2.16 , 13C NMR  δ 53.8, 53.2, 44.1. ESI-MS m/z expected: 132.23, found [MH+] = 132.16.Tetramethylethylenediamine  was dissolved in 1 mL of acetonitrile at 0 °C followed by addition of iodomethane-N,N,N,N′,N′-Pentamethyl-1,2-ethanediaminium iodide-13C 7  was dissolved in 1 mL of acetonitrile at 0 °C followed by addition of iodomethane-13C  dropwise. The reaction was stirred at ambient temperature for 16 hours, and the resulting precipitate was collected by vacuum filtration to obtain 256 mg of a white solid (56% yield). 1H NMR  δ 4.09 , 3.35 , 13C NMR  δ 57.3, 53.7. ESI-MS m/z expected: 148.26, found [MH+] = 148.19.There are no conflicts to declare.Supplementary informationClick here for additional data file.


Debates about the benefits and harms of mammography continue despite the accumulation of evidence. We sought to quantify the disagreement across systematic reviews of mammography and determine whether author or design characteristics were associated with conclusions that were favourable to the use of mammography for routine breast cancer screening.We identified systematic reviews of mammography published between January 2000 and November 2015, and extracted information about the selection of evidence, age groups, the use of meta-analysis, and authors’ professions and financial competing interest disclosures. Conclusions about specific age groups were graded as favourable if they stated that there were meaningful benefits, that benefits of mammography outweighed harms, or that harms were inconsequential. The main outcome measures were the proportions of favourable conclusions relative to review design and author characteristics.From 59 conclusions identified in 50 reviews, 42% (25/59) were graded as favourable by two investigators. Among the conclusions produced by clinicians, 63% (12/19) were graded as favourable compared to 32% (13/40) from other authors. In the 50–69 age group where the largest proportion of systematic reviews were focused, conclusions drawn by authors without financial competing interests  and non-clinicians  were less likely to be graded as favourable. There was no trend in the proportion of favourable conclusions over the period, and we found no significant association between review design characteristics and favourable conclusions.Differences in the conclusions of systematic reviews of the evidence for mammography have persisted for 15 years. We found no strong evidence that design characteristics were associated with greater support for the benefits of mammography in routine breast cancer screening. Instead, the results suggested that the specific expertise and competing interests of the authors influenced the conclusions of systematic reviews.The online version of this article (doi:10.1186/s13643-017-0495-6) contains supplementary material, which is available to authorized users. Mammography is the most widely used screening technology for detecting breast cancers in asymptomatic women. Since its introduction, the relative harms and benefits of mammography have been the subject of ongoing debate. Both the age at which to begin breast cancer screening and the frequency of screening have been disputed. Conflicting recommendations persist despite decades of interventional and observational studies that are used as the basis for making recommendations –4, and cInconsistencies in the conclusions produced across systematic reviews may reflect the manner in which the reviews were designed and undertaken, or may be related to the expertise and competing interests held by the reviewers. Analyses in other domains have found that financial conflicts of interest can be associated with differences in the interpretation of the results when drawing conclusions and making recommendations –12. SpecBy measuring review design and author characteristics associated with certain conclusions in systematic reviews of mammography, we may be able to identify factors that are associated with inconsistent conclusions. Our aim was to quantify the degree of disagreement in the conclusions of systematic reviews of mammography for breast cancer screening and then measure associations between the conclusions, design characteristics of the reviews, and the professions and financial competing interests of the authors.We examined systematic reviews of mammography for breast cancer screening published between January 2000 and November 2015. The PubMed database was searched to identify relevant studies using the following search strategy: “(((((guideline*[Text Word]) OR recommend*[Text Word]) OR review[Publication Type])) AND (((((“mass screening”[MeSH Terms]) OR (“early detection of cancer” [MeSH Terms]) OR screen*[Text Word])))) AND (((mammogr*[Text Word]) OR (breast cancer screening[MeSH terms]) OR (mammography[MeSH Terms])))”. We additionally hand-searched the citations of included reviews after screening to identify any other relevant literature.Review articles were included in the analysis if they met all four of the following inclusion criteria: (a) the reviewers specified a search criteria and the databases in which the search was conducted; (b) the review was focused on mammography for breast cancer screening; (c) at least two primary studies addressing the harms or benefits of mammography were cited; and (d) the reviewers made conclusions about the harms or benefits of mammography for breast cancer screening in relation to the evidence. Outcomes related to benefits included breast cancer survival , and cost-effectiveness of screening for quality-of-life. Outcomes related to harms included over-diagnosis, false positives, unnecessary treatments, radiation cancers, anxiety or worry, and pain or discomfort. Reviews that examined only diagnosis endpoints without considering survival or harms were excluded, as were reviews that only considered high-risk populations or populations of women who had previously been diagnosed with breast cancer. Articles were also excluded if they were guidelines, no longer archived or accessible online, not peer reviewed, or were in a language other than English.Two investigators independently screened article titles and abstracts against the inclusion criteria, and then examined the full text of articles against the inclusion and exclusion criteria. Discrepancies were resolved by discussion at both stages.The review design characteristics extracted included patient age ranges covered in the evidence, the types of primary studies analysed, the set of outcome measures examined, the presence or absence of a meta-analysis, and the year the systematic review was published. Patient age ranges were assigned to one of four categories: 49 or under, 50 to 69 years, 70 years or older, and one other group for reviews that considered all ages or did not specify an age range. These age groups were selected to correspond with the common age ranges used in the most recent guidelines. The age group for women aged between 50 and 69 in particular is where there has been substantial disagreement about how often women should undergo mammography, and this was a focus of our study. Where age ranges differed from the four groups, we identified the group with the largest overlap , and four were labelled as non-favourable . A third investigator read any reviews for which there was a disagreement to produce a final grading. For each conclusion, we also extracted supporting conclusion statements from the systematic reviews, as well as any statements that made recommendations about who should undergo screening by mammography and how often it should be done.Some of systematic reviews examined evidence for different age ranges or frequency of screening separately, producing conclusions for each. These conclusions were considered separately. This means that systematic reviews may be represented in the analysis with more than one conclusion and those conclusions may differ.A linear regression was used to check whether favourable conclusions became more or less common over the period of study, testing whether changes in the primary evidence influenced the likelihood of a favourable conclusion in systematic reviews. Where appropriate, we performed chi-square tests to test the association between favourable conclusions and each of the review design  and author  characteristics extracted from the systematic reviews.The search returned 2726 publications, from which 50 systematic reviews met the inclusion criteria Fig. . Five syAmong the 59 conclusions, 42% (25/59) were graded as favourable. The statements that best characterised the grading are provided as Additional files The greatest number of conclusions were for women aged between 50 and 69 (22 conclusions), and for which there was no restriction on age range in the evidence cited or where no age range was specified . Of the remainder, 17% (10/59) considered women under 49, and 8% (5/59) considered women aged 70 and older . Another 41% (24/59) considered both randomised controlled trials and observational studies, 34% (20/59) considered only observational studies, and 5% (3/59) were based on cost-effectiveness studies. Mortality outcomes were used in 64% (38/59) of the conclusions. Meta-analyses supported 27% (16/59) of the conclusions.Most conclusions were associated with author groups that had a public health, epidemiology, or biostatistics 68%; 40/59) background. Among the 32% (19/59) of corresponding authors from clinical specialties, 6 were from oncology, 4 from radiology, and 10 from other medical specialties .The number of systematic review conclusions produced over the period peaked between 2004 and 2007, and increased again from 2010  were graded as favourable  were graded as favourable; compared to 29% (7/24) of conclusions based on studies of both controlled trials and observational studies; 55% (11/20) of conclusions based only on observational studies; and 67% 2/3) of conclusions based on cost-effectiveness studies . When corresponding authors declared no financial competing interests, 20% (2/10) of the conclusions were graded as favourable compared to 80% (8/10) when authors declared a financial competing interest or did not include a disclosure statement . Analyses in other age groups did not indicate a significant difference , the 20 conclusions were produced by different authors and we were able to more reliably test the association between the observed characteristics and favourable conclusions  and the set of all age groups identified in the systematic reviews are labelled by their reference number and age range (grey rectangles). (PDF 374 kb)Additional file 2: Table S5.Authors’ professional roles; . (PDF 602 kb)Additional file 3: Table S6.Systematic review conclusion and recommendation statements. (PDF 433 kb)Additional file 4:Included systematic reviews. References for all included systematic reviews. (PDF 229 kb)Additional file 5: Table S3.Associations between systematic review characteristics and conclusions in 5 conclusions of studies that included women aged 70 years and older. (PDF 193 kb)Additional file 6: Table S4.Included systematic review conclusions in detail. (PDF 774 kb)Additional file 7: Table S1.Associations between systematic review characteristics and conclusions in 22 conclusions of studies that did not specify age group or specified all ages. (PDF 193 kb)Additional file 8: Table S2.Associations between systematic review characteristics and conclusions in 10 conclusions of studies that included women aged up to 49 years. (PDF 193 kb)


Antibiotic resistance is a global public health problem that requires our attention. Indiscriminate antibiotic use is a major contributor in the introduction of selective pressures in our natural environments that have significantly contributed in the rapid emergence of antibiotic-resistant microbial strains. The use of probiotics in lieu of antibiotic therapy to address certain health conditions in both animals and humans may alleviate these antibiotic-mediated selective pressures. Probiotic use is defined as the actual application of live beneficial microbes to obtain a desired outcome by preventing diseased state or improving general health. Multiple studies have confirmed the beneficial effects of probiotic use in the health of both livestock and humans. As such, probiotics consumption is gaining popularity worldwide. However, concerns have been raised in the use of some probiotics strains that carry antibiotic resistance genes themselves, as they have the potential to pass the antibiotic resistance genes to pathogenic bacteria through horizontal gene transfer. Therefore, with the current public health concern on antibiotic resistance globally, in this review, we underscore the need to screen probiotic strains that are used in both livestock and human applications to assure their safety and mitigate their potential in significantly contributing to the spread of antibiotic resistance genes in our natural environments. Since its advent, antibiotics remain as the major therapeutic strategy that is used to address numerous diseases of infectious etiologies both in human and veterinary medicine . HoweverIn the human clinical setting, these antibiotic-resistant pathogens have caused numerous treatment failures that eventually led to both hospital morbidities and mortalities . OverallHowever, microbes used as probiotics are not exempted from acquiring antibiotic resistance genes. Given their shared microbial environment in the gastrointestinal tract, a risk of pathogenic microbes acquiring antibiotic resistance genes from probiotic microbes exists, and vice versa . If imprThis review aims to describe the processes that govern the spread of antibiotic resistance in relation to antibiotic resistance genes. Antibiotic resistance gene transfer in the absence of probiotics is discussed first to elucidate the ongoing problem of the prevalence of antibiotic-resistant bacterial strains. Probiotic uses in both human and veterinary applications are then described and reviewed to reaffirm their beneficial use. Screening of probiotic bacterial strains for antibiotic resistance genes is then discussed to evaluate the safety of probiotic use. Finally, probiotic use in relation to the spread of antibiotic resistance genes is tackled to clarify the potential role of probiotics in propagating antibiotic resistance.Although the remarkable increase in the incidence and prevalence of antibiotic resistance were observed after the introduction and widespread use of antibiotics , antibioTaking advantage of these ecological interactions, we have learned to harness the use of antibiotics for a variety of applications . We haveHowever, with rampant antibiotic use, the natural way of developing antibiotic resistance has also changed in both manner and rate. Due to different human applications, multiple avenues for microbes to encounter antibiotics have put immense selective pressure on microbes to develop antibiotic resistance . In turnJust like any evolutionary response, adaptive changes of an organism are passed on to the next generation via the organism’s genetic material. Some microbes possess antibiotic resistance genes that confer their ability to survive exposure to antibiotics . AccordiIn the study of In addition, As mentioned earlier, antibiotic resistance genes are vertically transferred from one generation to another and favor the survival of resistant microbes . HoweverBacteroides spp. infection is specifically attributed to conjugative transposons . Since 2006, all EU countries have prohibited the use of antibiotics for the sole purpose of growth promotion in agricultural livestock industry . In addiWith the current paucity of clear regulatory policies in many countries, a global effort is needed in order to consolidate an effective strategy in controlling antibiotic resistance. Without a unifying regulatory guideline to follow, the problem on antibiotic resistance will most likely persist given the interweaving ecological interactions that govern the spread of antibiotic resistance. Furthermore, the spread of antibiotic resistance genes through human travel or trading of animal products between nations may make instituted regulatory policies of some countries less effective.As the development of antibiotic resistance strains continues, the use of probiotics as a substitute for antibiotics is becoming more popular in both the medical field and livestock agriculture . ProbiotMany farm animals are considered to be adapted in a symbiotic relationship with microbes as shown by specialized gastrointestinal organs that enable microbial fermentation . The capSalmonella spp. infections through accelerated establishment of what is considered normal and healthy microbiota for the aforementioned birds , the Assessment and Critical Evaluation of Antibiotic Resistance Transferability in Food Chain (ACE-ART), and the Joint International Organization for Standardization-International Dairy Federation Action Team on Probiotics (ISO-IDF) have individually contributed to address the issue . In evalIn the paper of Given the separate approaches in screening probiotic bacterial strains, multiple independent studies have demonstrated several methods in screening different probiotic strains for antibiotic resistance genes . In placLactobacillus and Enterococcus. Both genera are currently extensively screened for species that can be used as probiotic bacteria (The most common group of bacteria used as probiotics belongs to the group of lactic acid bacteria . Includebacteria .Lactobacillus probiotic strains are capable of improving digestion, absorption, and availability of nutrients in both livestock animals and humans (Helicobacter pylori, a pathogen regarded as the major cause of gastritis and peptic ulcers, and is a risk factor for gastric malignancy in humans (Lactobacillus is considered negligible at less than one case per million individuals (d humans . They arn humans . In addiividuals .Enterococcus probiotic strains are also known to be effective in reducing recovery periods of acute diarrhea in both animals and humans (Lactobacillus, the genus Enterococcus has member strains that are considered opportunistic pathogens and are sometimes the etiologic agents of some human nosocomial infections, such as bacteremia and infective endocarditis (d humans . Howevercarditis .It has also been reported in several studies that some species of lactic acid bacteria have intrinsic resistance to bacitracin, kanamycin, teicoplanin, vancomycin, and beta-lactams . Given tin vivo transfer of the vancomycin resistance gene, a plasmid encoded gene, was successfully demonstrated between lactic acid bacteria Enterococcus faecium strains and Lactobacillus acidophilus probiotic strains during digestive transit in mice. The results highlighted the risk of probiotics being a conduit for the spread of antibiotic resistance (Due to horizontal gene transfer, concerns are still raised particularly in lactic acid bacteria strains that carry mobile genetic elements such as plasmids . In the sistance .In the study of Bifidobacterium (Bifidobacterium is a known major constituent of the gut microbiota of both animals and humans. Bifidobacteria have the capacity to metabolize non-digestable host dietary carbohydrates such as plant-derived dietary fiber.Another group of bacteria commonly used as probiotics belong to the genus acterium . BifidobBifidobacterium are known to inhibit adherence of enterotoxigenic E. coli, enteropathogenic E. coli, and C. difficile to intestinal epithelial cells (Lactobacillus, Bifidobacterium was shown to alleviate side effects of H. pylori eradication therapy as some Bifidobacterium strains also suppress H. pylori-induced genes in human epithelial cells (Bifidobacterium strains are also known to alleviate infectious diarrhea as well as inflammatory bowel disease (Lactobacillus, infectious diseases due to Bifidobacterium are extremely rare (As a probiotic, strains of al cells . In combal cells . In addi disease . Like Laely rare .Bifidobacterium strains are known to have intrinsic resistance against ciprofloxacin, nalidixic acid, mupirocin, streptomycin, and aminoglycosides (Bifidobacterium strains distributed in the Japanese market. A total of 23 Bifidobacterium strains were isolated and tested for 15 antibiotics. Results confirmed the intrinsic resistance of Bifidobacterium against aminoglycosides. However, Bifidobacterium animalis subsp. lactis was found to be resistant to tetracycline and was in fact harboring a tetracycline resistance gene in its chromosome. The study concluded that although there is no risk factor for safety found in Bifidobacterium strains distributed in the Japanese market, the presence of the tetracycline resistance gene stresses the need for future evaluation (ycosides . Howeverycosides . In the aluation .The problem with the absence of a unified world-wide health authority that assumes responsibility for regulating probiotic use is exemplified by the case mentioned in the paper of In addition, given the established ecological nature of the emergence of antibiotic resistance problem, it is deemed logical to deduce that regulating probiotic use should also involve an ecological approach. It should be noted that studies involving the regulation of probiotic use in animals are lacking when compared to those of human applications. We should be reminded that numerous studies have already shown the connection between antibiotic resistance genes in animals and those in humans . WithoutFigure 1. Therefore, although probiotics are currently generally regarded as safe, we think that it is imperative to implement proper regulation on their use in both livestock and human applications globally to effectively mitigate their potential contribution in the spread of antibiotic resistance genes in our natural environments.The emergence of antibiotic resistant pathogens through the spread of antibiotic resistance genes is an ecological problem that is exacerbated by the widespread indiscriminate use of antibiotics in livestock agriculture, and in veterinary and human medicine. The use of probiotics in lieu of antibiotics to control some diseases in animals and humans may reduce the antibiotic selective pressures on microorganisms in our natural environments and contribute in reducing the problem of the rapid emergence of antibiotic resistant pathogens. However, probiotic bacterial strains used in both animal and human applications also have risks in becoming conduits themselves in spreading antibiotic resistance genes. We conclude that the use of probiotics to address the global problem of emerging antibiotic resistant microorganisms is a “double-edged” sword – with both beneficial effects and associated risks as depicted in II and JI conceptualized and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.