Predominant evidence of non-alcoholic fatty liver disease (NAFLD) is the accumulation of excess lipids in the liver. A small group with NAFLD may have a more serious condition named non-alcoholic steatohepatitis (NASH). However, there is a lack of investigation of the accumulated lipids with spatial and molecular information. Raman microscopy has the potential to characterise molecular species and structures of lipids based on molecular vibration and can achieve high spatial resolution at the organelle level. In this study, we aim to demonstrate the feasibility of Raman microscopy for the investigation of NAFLD based on the molecular features of accumulated lipids. By applying the Raman microscopy to the liver of the NASH model mice, we succeeded in visualising the distribution of lipid droplets (LDs) in hepatocytes. The detailed analysis of Raman spectra revealed the difference of molecular structural features of the LDs, such as the degree of saturation of lipids in the LDs. We also found that the inhomogeneous distribution of cholesterol in the LDs depending on the histology of lipid accumulation. We visualised and characterised the lipids of NASH model mice by Raman microscopy at organelle level. Our findings demonstrated that the Raman imaging analysis was feasible to characterise the NAFLD in terms of the molecular species and structures of lipids. The prevalence of NAFLD was increased worldwide and estimated to be 20–30% of the global population2. NAFLD increases the risk of cirrhosis and liver cancer, and it is a disease with poor outcomes and limited therapeutic options in advanced stages. Furthermore, the clinical burden of NAFLD is not only in the liver dysfunctions but affects extra-hepatic organs and regulatory pathways3. Therefore, the development of diagnostic methods at an early stage and the understanding of the pathogenesis of NAFLD has become a serious clinical concern worldwide.Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease associated with the accumulation of excess lipids in the liverNAFLD can be classified into two types, i.e., non-alcoholic steatohepatitis (NASH) that leads to liver-related complications such as cirrhosis and hepatocellular carcinoma, and non-alcoholic fatty liver (NAFL) that basically has benign prognosis. Although several hypotheses were proposed to describe the pathogenesis of the NAFLD, the pathogenesis of the progression to NASH or NAFL is still under investigation. Moreover, NASH and NAFL at the early stage exhibit similar evidence of the accumulation of excess lipids without apparent inflammation and fibrosis, making diagnosis difficult. In the current studies, NAFLD was predominantly investigated in terms of protein or gene activities in the liver. Although the predominant evidence of NAFLD is the accumulation of excess lipids, the pathological role of the lipids on NAFLD has not been clearly understood due to a lack of lipid-based investigation of NAFLD.5, imaging mass spectrometry7 and Raman microscopy9. Among these methods, Raman microscopy is a good candidate for the investigation of the accumulated lipids based on molecular species and structures of lipids. Raman microscopy measures Raman spectra that reflect molecular vibrations of intrinsic molecules. Since the molecular vibrations are sensitive to the species of atoms and chemical bonds of molecules, Raman spectroscopy provides information about the molecular species and structures via molecular vibrations. As visible and tightly focused excitation laser can be used for observation, high spatial resolution down to 500 nm or smaller can be realized. Furthermore, it has the potential to utilize in vivo evaluation owing to the visible light usage. Raman microscopy is, therefore, a powerful tool for the investigation of lipids in cells and tissues15. Although some researchers applied the Raman microscopy to NAFLD18, the efficacy of Raman microscopy for the investigation of NAFLD is still under investigation.To investigate NAFLD in terms of accumulated lipids, some visualisation methods of lipid droplets (LDs) were proposed, i.e., dye-based visualisation methodsIn the present study, we seek to provide a proof-of-principle demonstration of Raman microscopy in the investigation of NAFLD. Especially, we visualise and characterise the accumulated lipids in the liver of mice by Raman microscopy for the investigation of NAFLD in terms of molecular species and structures of lipids.19. By using the conventional polarization imaging with cross-Nicol configuration, the crystallinity of lipids in LDs can be identified as shown in Fig. Typical Raman spectra of liver tissues of NASH model mice were obtained to reveal the spectral features for Raman spectral analysis. We focused on macrovesicular LDs accumulated near a central vein, as shown in region A in Fig. −1 as shown in Fig. −1 were assigned to CH2-related molecular bonds, indicating the presence of lipid contents in this region. The Raman band at 1662 cm−1 was assigned to C=C double bond stretching mode at the acyl chain, which is an indicator of the unsaturation of the acyl chain of lipids. The Raman bands at 699 and 1677 cm−1 respectively assigned to steroid ring vibration mode and C=C double bond stretching mode at carbon ring, indicating the presence of cholesterol content. As a result, the Raman spectra at the crystalline lipid-rich LDs reflected the molecular features of the LDs, including the cholesterols. The presence of cholesterol in the crystalline lipid-rich LDs was well agreed with the results of the fluorescence imaging with a cholesterol-specific dye in the previous studies19.In the crystalline lipid-rich LDs, main Raman bands were found at 699, 1265, 1302, 1442, 1662, 1677, 1735, 2855, 2875, 2900, 2935, and 2964 cm−1 as shown in Fig. −1, indicating relatively low cholesterol content at the LDs in terms of Raman spectral analysis. This result is also agreed with the result of the fluorescence imaging in the previous studies19. Importantly, Raman microscopy additionally has the potential to quantitatively evaluate the relative content of molecular species and structures without staining by analysing the relative intensity of each Raman band.In the amorphous lipid-rich LDs, main Raman bands were found at 1265, 1302, 1442, 1662, 1750, 2855, 2875, 2900, 2935, and 2964 cm−1 as shown in Fig. −1 were observed in the hepatocytes, which might be derived by cytochromes present in mitochondria in hepatocytes or deposited hemoglobin. As a result, the Raman bands of LDs can be confirmed against that of hepatocytes, indicating the Raman spectral analysis of LDs can be performed in the liver tissues of the NASH model mice.In contrast, in the hepatocytes with non-apparent LDs, main Raman bands were found at 746, 1001, 1309, 1448, 1592, 1660, 2855, 2875, 2900, 2935 and 2964 cm20. Firstly, we investigated the spatial distribution of molecular contents of lipids in macrovesicular LDs accumulated in hepatocyte of liver tissues of NASH model mice. We obtained two-dimensional Raman images of macrovesicular LDs in region A in Fig. −1, as shown in Fig. −1 were also observed. Raman images of each typical Raman bands were shown in Fig. −1, clearly reflect the distribution of LDs, which are well agreed with the histology of the liver tissues of the NASH model mice confirmed by the HE-stain image. As the imaging analysis, each LD seems to be separated from each other. In contrast, the Raman bands at 1592 and 2935 cm−1 visualised the distribution of hepatocytes or heme proteins.We also investigated the molecular contents of lipids in terms of the size of LDs. The previous studies had shown that the size of LDs is closely related to the prognosis of NAFLD−1 against 2855 cm−1, as shown in Fig. −1 against 2855 cm−1. Notably, some particulate structures of the intensity ratio were found in the internal structure of LDs, as typically indicated by the arrowheads in Fig. To analyse the more specific molecular species and structures of LDs, we performed intensity ratio imaging by using lipid-related Raman bands. The intensity ratio calculation was performed at each pixel to obtain relative contributions of lipid species and structures with the normalization of the amount of lipids. Firstly, we obtained an intensity ratio of 1662 cm−1 against 2855 cm−1, as shown in Fig. −1 against 2855 cm−1 among LDs. In addition, some of the LDs exhibited a high-intensity ratio partially on the outer wall of the LDs. These results might indicate that the relative content of cholesterol was varied depending on the macrovesicular LDs, and the cholesterol distribution was unevenly distributed even in a single macrovesicular LD.We also obtained the intensity ratio of 1677 cm−1, as shown in Fig. −1 was much smaller than that of macrovesicular LDs. Raman images of each typical Raman bands were shown in Fig. −1, reflect the distribution of LDs, but interestingly, we found the connecting structure between LDs, which was not apparent in the HE-stained image. In contrast, the Raman bands at 1592 and 2935 cm−1 visualised the distribution of hepatocytes or heme proteins as similar to the result around the macrovesicular LDs.We also investigated the spatial distribution of molecular contents of lipids in microvesicular LDs accumulated in hepatocyte of liver tissues of NASH model mice. We obtained two-dimensional Raman images of macrovesicular LDs at the dashed squared region of Fig. −1 against 2855 cm−1, as shown in Fig. −1 against 2855 cm−1. We also obtained the intensity ratio of 1677 cm−1 against 2855 cm−1, as shown in Fig. −1 against 2855 cm−1 was also homogeneous. These results might indicate the unsaturation degree of microvesicular LDs, and the relative content of cholesterol could distribute homogeneously in microvesicular LDs.Intensity ratio imaging was also performed by using lipid-related Raman bands. We obtained the intensity ratio of 1662 cm−1 against 2855 cm−1 and the that of 1677 cm−1 against 2855 cm−1 of the central region of the 37 LDs in macrovesicular LDs and 21 LDs in microvesicular LDs. The intensity ratio analyses of these Raman bands were shown in Fig. −1 against 2855 cm−1 of the microvesicular LDs (0.438 ± 0.038) was significantly higher than that of the macrovesicular LDs (0.425 ± 0.034). This result shows the microvesicular LDs have a higher unsaturation degree of lipids than the macrovesicular LDs. This tendency was also observed by the other indicator of unsaturation degree17 (intensity ratio of 1265 against 1442 cm−1 and 3012 against 2855 cm−1), as shown in Supplementary Fig. −1 against 2855 cm−1 of the microvesicular LDs (0.308 ± 0.061) was also significantly higher than that of the macrovesicular LDs (0.218 ± 0.104). This result shows the microvesicular LDs have a higher cholesterol content on average. However, the highest intensity ratio of 1677 cm−1 against 2855 cm−1 was found in the macrovesicular LDs, showing the partial aggregation of a higher content of cholesterol.Finally, we investigated the difference of molecular species and structural features of LDs between microvesicular and macrovesicular LDs. We evaluated the intensity ratio of 1662 cmIn the present study, we evaluated liver tissues of NASH model mice in terms of the molecular analysis of LDs within hepatocytes. We analysed spectral features of the Raman spectra of LDs with crystalline and amorphous forms of lipids and hepatocytes. We also successfully visualised the LDs according to the molecular species and structural features of lipids in the microvesicular and macrovesicular LDs. The Raman microscopy reveals the complicated distributions of lipids in LDs, which cannot be identified by conventional histological imaging techniques. Our results provided a proof-of-principle for the investigation of NAFLD using Raman microscopy in terms of the molecular species and structures of accumulated lipids in NAFLD.23; accordingly, various researches on NAFLD were conducted in terms of genome-wide association studies24, molecular biological studies26, and medical studies in both the aspect of treatment and diagnosis28. As regarding lipids, abnormal lipid metabolism and the induction of inflammation by overproduction of free fatty acids have been widely investigated. However, the current studies related to lipids have been predominantly performed by ensemble-averaged analysis of lipids without spatial information or by morphological analysis of accumulated LDs without molecular information. Since the accumulation of excess lipids is fundamental evidence of NAFLD, the analysis of lipids with both spatial and molecular information must provide an essential perspective in elucidating the pathogenesis of NAFLD. Our results demonstrated the feasibility of Raman microscopy for the molecular analysis with spatial information, and the Raman microscopy may provide an effective means to investigate the pathogenesis of NAFLD based on the characteristics of accumulated lipids.NAFLD induces hepatocyte steatosis and liver dysfunction with glycolipid metabolic disorders based on insulin resistance. The pathogenesis is thought to be due to various factors such as oxidative stress, endoplasmic reticulum stress, autophagy, lipotoxicity owing to free fatty acids, and innate immune activation associated with bacterial intestinal abnormalities19. The unsaturation degree of lipids is also related to inflammation according to the difference in metabolic activity of lipids30. As a result, the complicated distributions of lipids in LDs may indicate the possible places where inflammation is likely to occur according to the distribution of cholesterol and unsaturation degree of lipids, possibly leading to the investigation of the pathogenesis of NALFD in terms of lipids. Furthermore, the potential of leading to liver-related complications can only be diagnosed after inflammation and fibrosis are well developed under the current pathological diagnosis. If the relationship between lipids and inflammation becomes clear, it will lead to the possibility of diagnosis at the earliest stage of NAFLD or before the onset of the inflammation and fibrosis. Thus, although further investigations are required, our findings will possibly provide the fundamental knowledge for elucidating the pathogenesis of NAFLD and establishing diagnostic methods in terms of lipids in the future.Our findings in this study are that the lipids in LDs have the potential for complex distributions in terms of cholesterol and unsaturation degree of lipids. It is known that the activation of inflammatory cells is possibly induced by intake of LDs containing cholesterol accumulated in hepatocytes2 structure, C=C double bond and steroid ring, Raman microscopy will be a powerful means to discuss the molecular species and structures of lipids such as cholesterol content via the C=C double bond and cholesterol content via steroid ring vibrations. Moreover, Raman microscopy has a potential for application in intraoperative use by using a surgical microscope or flexible optical fibre system31. Raman microscopy offers the advantages of fast, in situ, non-destructive and molecular vibration-based observation, enabling the diagnosis applications for NAFLD without any treatment such as fixation nor staining. Although further development of an intraoperative Raman microscopy system is required, our proposed approach may also provide a unique and powerful means for NAFLD diagnosis in the future.Conventional bioimaging methods focusing on lipids are dye-based visualisation methods and imaging mass spectrometry; however, the molecular selectivity of the dye-based visualisation methods in lipid analysis is low because of the difficulty of the application of such as an immune-staining method or similar method of fluorescent proteins in the same manner of protein imaging. Therefore, the dye-based visualisation of lipids for NAFLD analysis is limited to morphological analysis. The imaging mass spectrometry is sensitive and selective in terms of lipid molecules and can clarify the details of lipid species, the length of acyl chains, and the degree of unsaturation. However, it is challenging to analyse microvesicular LDs or internal structures of LDs due to its low spatial resolution (> 10 µm). By contrast, Raman microscopy can achieve very high spatial resolution up to about half of the excitation wavelength, while obtaining molecular information via molecular vibrations. Although the Raman spectrum provides the partial molecular information such as the presence of CHIn conclusion, we demonstrated Raman spectral imaging of the liver tissues of NASH model mice. Our results revealed the feasibility of the Raman microscopy for the analysis of NAFLD based on the molecular features of accumulated LDs. Although further studies are required, our proposed approach may provide new insights into the investigation of the pathogenesis of NAFLD and the new development for NAFLD diagnosis based on accumulated lipids in the future.32. NASH model mice were obtained by feeding six-weeks-old male TSOD mice  on a high-fat/cholesterol/cholate diet  and purified water for 26 weeks and housed under normal conditions. The liver of the NASH model mice was excised after euthanasia. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted, and ethical approval was obtained from the Institutional Animal Care and Use Committee of Tokushima University, Japan .In this study, we utilized a fatty liver of mice exhibited NASH for the evaluation of Raman microscopy in the investigation of NAFLD. The detailed induction protocol and histopathological characterisation of the NASH model mice used in this study were given in our previous studyThe excised liver was immediately embedded in 4% sodium carboxymethyl cellulose compound and snap-frozen in liquid nitrogen. The samples were stored at − 80 °C until cryostat sectioning. The frozen samples were sliced into 5-µm thick sections with a cryostat microtome . Since the compound-embedded liver was snap-frozen and kept frozen until the sectioning, the tissue embedding compound presumed not to penetrate to the evaluated area of the liver. Two serial sections were obtained for Raman and histopathological analysis. The sections for the Raman analysis were mounted on a slide glass without any fixation nor staining. The sections for histopathological analysis were fixed with 95% ethanol and were subjected to hematoxylin and eosin (HE) staining. The histopathological characterisation, such as the presence of LDs, hepatocytes and other histological features, was performed by a pathologist (K.T.), who is the co-author of this article. We obtained 12 sections from the livers of a total of two representative NASH model mice.https://scienceedge.com). A single-mode frequency-doubled Nd:YAG laser  operating at the wavelength of 532 nm was used as an excitation laser light. The excitation laser light was focused on a sample through a 10 × objective lens  or a 60 × objective lens . The back-scattered Raman signal was collected with the same objective lens and detected by a spectrometer  with a cooled CCD image sensor . Raman spectrum from -30 to 3588 cm−1 was simultaneously obtained with a single exposure. Two-dimensional Raman spectral images were obtained by scanning the laser focus. The excitation laser power and the exposure time were 10 mW on the sample plane and 0.1 s, respectively.Raman spectra and Raman spectral images were acquired with a home-built laser-scanning confocal Raman microscope with an imaging software . To extract the Raman spectrum from a broad fluorescence background, we applied a modified polynomial curve fitting methodSupplementary Information.


Neisseria gonorrhoeae (NG) treatment, with few alternatives available. AMR point-of-care tests (AMR POCT) may enable alternative treatments, including abandoned regimens, sparing ceftriaxone use. We assessed cost-effectiveness of five hypothetical AMR POCT strategies: A-C included a second antibiotic alongside ceftriaxone; and D and E consisted of a single antibiotic alternative, compared with standard care (SC: ceftriaxone and azithromycin).Widespread ceftriaxone antimicrobial resistance (AMR) threatens Assess costs and effectiveness of AMR POCT strategies that optimise NG treatment and reduce ceftriaxone use.The five AMR POCT treatment strategies were compared using a decision tree model simulating 38,870 NG-diagnosed England sexual health clinic (SHC) attendees; A micro-costing approach, representing cost to the SHC (for 2015/16), was employed. Primary outcomes were: total costs; percentage of patients given optimal treatment ; percentage of patients given non-ceftriaxone optimal treatment; cost-effectiveness .All strategies cost more than SC. Strategy B (azithromycin and ciprofloxacin (azithromycin preferred); dual therapy) avoided most suboptimal treatments (n = 48) but cost most to implement ). Strategy D  was most cost-effective for both cost per optimal treatments gained (GBP 414.67 (EUR 554.53)) and per ceftriaxone-sparing treatment (GBP 11.29 (EUR 15.09)) but with treatment failures (n = 34) and suboptimal treatments (n = 706).AMR POCT may enable improved antibiotic stewardship, but require net health system investment. A small reduction in test cost would enable monotherapy AMR POCT strategies to be cost-saving. Neisseria gonorrhoeae (NG) [Antimicrobial resistance (AMR) has developed to every class of antibiotic used for treatment of the bacterial sexually transmitted infection (STI) eae (NG) , with ineae (NG) . NG, theeae (NG) , is assoWorld Health Organization (WHO) guidelines  recommenRapid diagnostics have been identified as a key approach to tackling AMR . Rapid tChlamydia trachomatis (CT) detection [Rapid tests are already being used for NG in some sexual health clinics (SHC) . While letection ,17. Costetection .gyrA for ciprofloxacin; 23S rRNA and mtrCDE transporter for azithromycin), are relatively well-understood, and their absence predictive of susceptibility (particularly for ciprofloxacin). The development of molecular AMR POCTs for detection of these determinants are thus technically feasible and therefore more likely to be immediately available [In this analysis, we assessed the cost-effectiveness in English SHC of five hypothetical AMR POCT strategies for the treatment of NG, which enable use of ciprofloxacin and/or azithromycin, either alongside, or as an alternative to, ceftriaxone. Potential diagnostic resistance-determinants of these antibiotics are small in number , where the AMR POCT was used as a reflex test to inform antibiotic selection irrespective of which test was used to diagnose NG initially. The AMR POCT strategies were chosen to either facilitate optimised choice of a second antibiotic alongside ceftriaxone , or enable a single antibiotic alternative to ceftriaxone (monotherapy) (We compared standard care (SC) for NG treatment in the UK  with fitherapy) .BoxStandard careStandard care with dual therapy of intramuscular ceftriaxone (500 mg) and oral azithromycin (1 g single dose).Dual therapy, including ceftriaxoneA) AMR POCT for ciprofloxacin resistance only. Infections identified as not resistant to ciprofloxacin are given oral ciprofloxacin (500 mg) plus ceftriaxone (500 mg). Infections identified as ciprofloxacin resistant are given SC.B) Dual AMR POCT for azithromycin and ciprofloxacin resistance. If no azithromycin resistance is identified, SC is given. If azithromycin resistant, ciprofloxacin (500 mg) and ceftriaxone (500 mg) are given unless there is ciprofloxacin resistance, in which case ceftriaxone (500 mg) is given alone.C) Dual AMR POCT for ciprofloxacin and azithromycin resistance. If no ciprofloxacin resistance is identified, ciprofloxacin (500 mg) and ceftriaxone (500 mg) are given. If ciprofloxacin resistant, SC is given, unless there is also azithromycin resistance, when ceftriaxone (500 mg) is given alone.Monotherapy optimisationD) AMR POCT for azithromycin resistance. If no azithromycin resistance is identified, azithromycin (2 g) is given. If azithromycin resistant, ceftriaxone (500 mg) and ciprofloxacin (500 mg) dual therapy is given. If the AMR POCT incorrectly shows no resistance , it is assumed the treatment fails. The treatment failure would be identified in the test-of-cure and the patient would then receive 500 mg ceftriaxone.E) AMR POCT for ciprofloxacin. If no ciprofloxacin resistance is identified, 500 mg ciprofloxacin monotherapy is given. If ciprofloxacin resistant, SC is given. If the AMR POCT incorrectly shows no resistance, monotherapy is assumed to fail, the patient returns and receives 500 mg ceftriaxone alone.AMR: amtimicrobial resistance; POCT: point-of-care test; SC: standard care.The rationale for dual therapy strategies is based on the assumption that combination therapy is more effective at preventing emergence or spread of AMR and thereby preserves the use of ceftriaxone. The rationale for the monotherapy strategies is that an AMR POCT enables effective treatment of the known resistance profile, sparing the use of ceftriaxone .Each strategy consisted of a series of intended treatment regimens, contingent on the results of the AMR POCT used. For example, in strategy B, the earliest intended treatment regimen was SC, where the AMR POCT indicated azithromycin resistance; the second intended treatment regimen was ceftriaxone and ciprofloxacin, where the AMR POCT then indicated ciprofloxacin resistance; the third intended treatment regimen was ceftriaxone monotherapy.A decision tree model was constructed using TreeAge Pro version 2017 ) to simulate a hypothetical cohort of 38,870 NG-diagnosed SHC attendees , 8,488 women and 8,467 men who have sex with women (MSW)), representing the total number of NG diagnoses in England SHC in 2015, obtained from the genitourinary medicine clinical activity dataset national surveillance data (GUMCAD) . Our assSupplementary Table S1.Key model assumptions include: 100% compliance with test protocols; all patients entering the model are NG true-positives; dual AMR POCT results are available simultaneously; and there is no ceftriaxone resistance data  for the cost per additional optimal treatment gained and the cost per additional ceftriaxone treatment avoided. This was chosen as the measure of cost-effectiveness rather than other measures, such as cost per Quality Adjusted Life Years (QALY), because little data exist on the consequence of optimal vs suboptimal NG treatment on long-term outcomes, such as mortality or lifetime costs.We aimed to assess the costs and effectiveness of these AMR POCT strategies to optimise treatment regimen choice and reduce selection pressure on ceftriaxone. The primary outcomes were the total costs (2015/16 GBP(EUR)), the percentage of people given optimal treatment, and the percentage of people given non-ceftriaxone optimal treatment. Optimal treatment was defined as one which cured NG and did not contain an antibiotic against which there was resistance. Model definitions are provided in A: standard care; B: antimicrobial resistance point-of-care test strategy; ICER: incremental cost-effectiveness ratio.ICERs were calculated for two types of effectiveness: optimal treatments and ceftriaxone treatments avoided.Secondary outcomes were the percentage of people given a missed earlier intended treatment regimen (MEITR), and the percentage of people failing treatment due to resistance. MEITR was defined as the use of a treatment regimen which cured NG, but where an earlier intended treatment regimen would have provided optimal treatment because susceptible infections had been misclassified as resistant by the AMR POCT. MEITRs were independent of treatment effectiveness.Supplementary Figure S1). The purpose of the work was to determine AMR POCT strategy for short-term clinical impacts, because these are the data used for sexual health service provisioning and decisionmaking for adoption into SHC [AMR POCT strategy treatment regimens were developed with input from three senior clinicians at St George’s University Hospitals NHS Foundation Trust, London, who outlined current and hypothetical AMR POCT patient pathways . It was assumed the AMR POCTs produced results in 30 min . Costs to those procuring sexual health services provision, or to health systems as a whole, were not considered. Costs were estimated by adapting an existing model  and inclce users ,32) and ce users . Antibioce users . InitialWe conducted one-way analyses using TreeAge Pro version 2017 (TreeAge Software) and R software version 3.5.0 , for each of the model parameters by varying them independently at the ends of their ranges to examine the effect on the primary outcome . These aThis report was written following the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist .As the only data included for this study were nationally published surveillance data from PHE, and no patient data were used, ethical approval was not required.Supplementary Tables S4, S5 and S6.Overall AMR POCT strategy costs, treatments used, and treatment outcomes compared with SC in all groups are presented in Supplementary Tables S4, S5 and S6).The cost of SC NG management was GBP 2,856,168  for the total cohort . All AMRSupplementary Tables S4, S5 and S6).All AMR POCT strategies provided more optimal treatments than SC, in all population groups. Strategy B provided most optimal  and least suboptimal (n = 48) treatments. Strategies A and E equally provided the least optimal treatments and the most suboptimal (n = 813) .A MEITR refers to a treatment regimen being used when an earlier intended treatment regimen would have provided optimal treatment. In all population groups, the fewest were in Strategies A and E (n = 265), and B (n = 267), and the most were in Strategy C (n = 912) .There were some treatment failures in each monotherapy strategy due to false-susceptible AMR POCT results: strategy D had 34/38,870 (0.09% of treatments) and Strategy E had 248/38,870 (0.64% of treatments) . There wThe cost-effectiveness analysis (CEA) results are presented in When avoidance of ceftriaxone use was considered, Strategy D was most cost-effective relative to SC, costing GBP 11.29 (EUR 15.09) per ceftriaxone-sparing treatment gained. These findings were consistent across all population groups.Supplementary Figure S2.In one-way sensitivity analyses, the following four parameters had the greatest impact on cost-effectiveness per optimal treatment gained for all AMR POCT strategies and across all population groups: prevalence of azithromycin resistance; AMR POCT sensitivity; prevalence of ciprofloxacin resistance; and the cost of single detection AMR POCT. In monotherapy strategies, the cost-effectiveness model was additionally sensitive to cost of clinical management (both with and without injection), cost of ceftriaxone, and AMR POCT specificity (for strategy D). The cost multiplier for a dual detection AMR POCT impacted on AMR POCT cost-effectiveness for Strategies B and C. Tornado plots from these analyses are presented in Supplementary Figure S3). These rises in ICER were least for strategies B and D. With the exception of strategy B where ICER were consistent for all population groups, these increases in ICER were most limited in women.For all strategies, variation of ICER in relation to azithromycin resistance prevalence was minimal until prevalence fell to or below 3%, at which point it increased  and minimum (90%) sensitivity (maximum difference of GBP 169.21 (EUR 226.28) per optimal treatment gained in women) compared with other strategies where the difference was in the thousands. For Strategy D, change in sensitivity had little impact on cost per optimal treatment gained, whereas when specificity decreased to below around 95.5%, the cost per optimal treatment gained started to increase exponentially. The sensitivity analyses are presented in Supplementary Figure S5). For Strategies A and E, as ciprofloxacin resistance increased from ca 20%, there was an exponential increase in cost per optimal treatment gained for women only.The prevalence of ciprofloxacin resistance had very little effect on cost per optimal treatment gained in Strategies B, C and D . Strategy B had lowest costs per additional optimal treatment for dual therapy strategies.For the three single target AMR POCTs , reducing the cost of the test had the greatest impact on cost per treatment gained. Monotherapy strategies became cost-saving (ICER < 0) for all population groups when AMR POCT cost was ≤ GBP 18 (24.07 EUR) for Strategy D, and ≤ GBP 16 (EUR 21.40) for Strategy E , including AMR POCT diagnostic accuracy - a necessity as these tests are currently in early phases of development [As with all mathematical models, several assumptions were made . This may limit the generalisability of our results as it is not possible to know future resistance profiles. However, the results should be generalisable to the ranges used in the sensitivity analyses. In addition, as AMR POCTs are still in development, some of the model’s other epidemiological parameters will have changed by the time the AMR POCTs are available for use in routine practice, which may further limit the analyses’ applicability in the longer term. This highlights the need to continually conduct analyses such as these, to enhance our ability to predict and understand future trends. Our anlayses are also limited to data from England, with results perhaps less generalisable to other countries. This will be exacerbated by the 2019 change to 1 g ceftriaxone monotherapy, further setting it aside from guidelines in other European countries [Our analysis had some limitations. We used the most recent NG AMR data available from GRASP at the time , but AMRountries . Our modountries , costs iStrategy B was most effective for avoiding suboptimal treatments but the most costly to implement. Strategy D was the most cost-effective for both effectiveness outcomes , but resulted in treatment failures, as well as nearly 15-fold higher suboptimal treatments compared with Strategy B. Both strategies B and D enabled the re-use of ciprofloxacin, previously abandoned for the treatment of NG in the UK .All AMR POCT strategies were more expensive than SC, with dual therapy AMR POCT strategies more expensive than monotherapy strategies, suggesting that short-term net financial investments in AMR POCT adoption are required to gain long-term antimicrobial stewardship benefits. The O’Neill review of AMR  noted thThe monotherapy strategies resulted in treatment failures due to false susceptible AMR POCT results, although minimal relative to SC. Since we assumed ceftriaxone treated 100% of NG infections, there were no treatment failures for SC or dual therapy strategies. The most recent GRASP data suggest that ceftriaxone resistance remains low  ≥ 0.03 mg/L in 2018 compared with 16.6% in 2017 -45. ThisMost MEITRs  were in Strategy C, and the least in Strategies A, B and E. Avoiding MEITRs is important because it maximises the ability to use ciprofloxacin , or reduces the need for ceftriaxone use (Strategies B and D). These numbers were small compared with actual patient numbers in whom a MEITR might be used if these AMR POCTs were available more generally. For example, using national surveillance data ,46, we eSince a MEITR is due to susceptible infections misclassified as resistant by the AMR POCT, test specificity is key. In sensitivity analyses of AMR POCT accuracy, Strategy D was the only strategy where cost per optimal treatment gained was affected by changes in specificity. In all other strategies, cost per optimal treatment gained increased as sensitivity decreased. This is because these strategies contained an AMR POCT that included ciprofloxacin testing, so resistance  was detThus, prevalence of resistance has important implications for AMR POCT accuracy requirements and ICER of optimal treatments gained. In the azithromycin resistance sensitivity analyses, ICER increased when resistance fell below ca 3% , primarFrom a population-level antimicrobial stewardship public health perspective, increasing the number of suboptimal treatments may eventually lead to an increased number of resistant infections . The relOnce developed, AMR POCTs could have wide-ranging implications for clinical decisionmaking globally, including the potential re-use of antibiotics previously abandoned for the treatment of NG, ensuring the right treatment is given to the right person at the right time (precision medicine). Although it may be necessary to accept net health system investment to enable longer-term societal pay-offs by reducing infection rates and maintaining effective NG treatments, a relatively small reduction in test cost could enable some AMR POCT strategies to be cost-saving.empirical study and modelling is required to optimise their use for public health benefit.


Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and Penaeus monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (> 15 parts per thousand (ppt)) compared to grow-out ponds with low salinities (< 5 ppt). Considering that P. vannamei is an euryhaline species, we were interested in knowing if EHP can propagate in P. vannamei in low salinities.P. vannamei were maintained at three different salinities  while continuously challenged using feces from known EHP-infected P. vannamei over a period of 3 weeks. The fecal strings, used as a source of EHP inocula in the challenges, was sufficient to elicit an infection in shrimp maintained at the three salinities. The infectivity of EHP in shrimp reared at 2 ppt, 15 ppt, and 30 ppt salinities was confirmed by PCR and histopathology. The prevalence and the severity of the EHP infection was higher at 30 ppt than at 2 ppt and 15 ppt.In this study, we described an experimental infection using fecal strings as a source inoculum. Specific Pathogen Free (SPF) The data suggests that fecal strings are a reliable source of EHP inoculum to conduct experimental challenges via the fecal-oral route. An EHP infection can occur at a salinity as low as 2 ppt, however, the prevalence and the severity of the EHP infection is higher at a salinity of 30 ppt. Enterocytozoon hepatopenaei (EHP) is a disease that has been reported in several penaeid shrimp including P. monodon and P. vannamei [Hepatopancreatic microsporidiosis (HPM) caused by vannamei , 2. EHP vannamei –6. Recenvannamei .The main clinical signs of EHP are growth retardation , 8 that In countries where EHP has been reported, such as India, China, Vietnam, and Venezuela, shrimp farming is carried out in a wide range of environmental condition including coastal marine areas, estuarine areas, and inland areas. For instance, in some states in eastern part of India such as Andhra Pradesh, the ponds are filled with borehole water mixed with estuarine water, which causes the salinities of the grow-out ponds to fluctuate between 0 and 30 ppt with an average salinity of about 10 ppt. In contrast, in states located in the western part of the country such as Gujarat, salinities in pond water may vary between 30 and 44 ppt . In VeneThe final survival at the end of the EHP challenge was high, ranging from 90 to 100%, in the three salinities in each of the two independent experimental challenges . EHP copy number in the fecal string in challenge #1 was 1.6 × 103 ± 2.1 × 103 copies /ng DNA compared to 1.1 × 106 ± 2.0 × 106 copies /ng DNA in the inoculum of challenge #2  of 4.3 (p = 0.037). The prevalence of EHP in shrimp exposed to high salinity (30 ppt) was higher than shrimp exposed to low salinities (2 ppt and 15 ppt combined). The different grades of severity in this study are shown in Fig. The prevalence of EHP at 2 ppt, 15 ppt, and 30 ppt salinities in challenge #1 was 25, 33.3, and 25%, respectively. In challenge #2, the prevalence of EHP at 2 ppt, 15 ppt, and 30 ppt was 33.3, 30.0, and 87.5%, respectively. The degree of severity was higher at a salinity of 30 ppt in the second experiment  salinities  EHP was detected. The hepatopancreas tissue collected from the negative control treatment animals reared at the 2 ppt, 15 ppt, and 30 ppt salinities tested negative for EHP using nested PCR.Penaeus vannamei is an euryhaline species which is raised in a wide range of conditions including high salinities (30 ppt), estuarine environments (10–20 ppt), and low salinities (2 ppt) [3 copies of EHP/ ng of total fecal DNA), the HPM prevalence was similar (i.e. 25%) irrespective of the salinities . Dose-dependent challenge has been well documented for other shrimp pathogens such as AHPND and Hepatobacter penaei [6 CFU/ml is required to provoke high mortality (> 90%) in immersion challenge methods. In contrast, a lower dose (2.0 × 104 CFU/ml) does not cause mortality nor histological lesions in the challenged population [3 copies of EHP/ ng of total fecal DNA) used in challenge #1 caused mild infections in the challenged shrimp. However, severe infections (Grades G3 to G4) and a higher prevalence occurred in the challenge #2 when the EHP copy number in the inoculum was higher .Since 2009, HPM caused by EHP has emerged as an economically important disease being highly prevalent in some of the major shrimp producing countries in SE Asia and, more recently in the western hemisphere in Venezuela . In SE-A (2 ppt) . In this (2 ppt) , 15, rev (2 ppt) , shrimp r penaei . In AHPNEncephalitozoon intestinalis, an obligate intracellular microsporidium that causes gastrointestinal diseases in immunocompromised and immunocompetent people. Differences in polar tube germination associated with changes in salinity have also been reported by other researchers. De Graff et al. showed an increase in germination of Nosema apis spores at 0.5 N NaCl concentration [Nosema algerae spores at 0.1 M NaCl concentration vs. 0.05 M NaCl was reported by other researchers [The histological lesions in shrimp maintained at 30 ppt salinity were more severe. A moderate- severe grade of infection (G3-G4) was found in 50% of the shrimp affected. In contrast, only 16% of shrimp reared at salinity of 2 ppt showed grade G3 level of infection and 0% of shrimp reared at salinity of 15 ppt showed grade G2-G4 level of infection, according to the Lightner’s scale . The difntration . Similarearchers . In our https://www.tucsonaz.gov/water/water-quality-reports-and-publications), vs. the marine water artificially prepared at 15 ppt and 30 ppt that was around 787 and 1575 mg/L respectively . It has been reported that calcium is an important second messenger that activates many cell events and calcium influx might be, in part, responsible for the activation of microsporidian spore discharge at higher salinities [Hardness is another variable that was different in the three salinities used in this study and could have been a factor that affected the spore germination. The hardness in low salinity (2 ppt) was about 240 mg/L , as observed during a shrimp disease survey in Andhra Pradesh in 2019 . Similar conditions were recorded in two major shrimp farming areas in Venezuela, i.e. Maracaibo lake where the salinity is around 4–6 ppt and in Falcon state where the salinity varies from 36 to 40 ppt. In Venezuela, shrimp farming is not fully integrated, and the movement of nauplii and post-larvae between Falcon and Maracaibo lake area is a common practice. This suggests that EHP-infected PL and / or broodstock have been moved between these two zones. However, EHP has only been detected in the Falcon area where the salinities are high. In the Maracaibo’s lake where the salinities are low, EHP has not been reported. One possibility that has limited the spread of EHP could be the difference in water salinity.This study demonstrated that fecal strings from known EHP-infected shrimp could be used as a reliable source of inoculum to conduct EHP experimental infections via the fecal-oral route. An EHP infection can occur at a low salinity (i.e. 2 ppt) although the prevalence and the severity of infection is higher at a salinity of 30 ppt. These findings have implications in disease management in EHP-endemic areas.Penaeus vannamei were obtained from a commercial vendor in Florida, US. The SPF population has been screened for the last 2 years at the UA-APL without presence of any of the OIE-listed and non-listed diseases including EHP. The bioassays were carried out in the Aquaculture Pathology Laboratory (UA-APL) of The University of Arizona. The American Veterinary Medical Association (AVMA) guidelines were followed while processing shrimp for histopathology analysis and for euthanasia by salt water/ice slurry at the end of the experiment .Specific Pathogen Free (SPF) P. vannamei population originating in Thailand. Two independent EHP challenges were conducted. In both challenges, shrimp were maintained at three different salinities of 2 ppt, 15 ppt, and 30 ppt. For each experimental challenge, six 90-L tanks were filled with artificial seawater  that corresponded to the three salinity levels with two replicates for each salinity treatment. Temperature was adjusted at 25 °C (±0.6) with measurements each morning with a NIST thermometer. pH was measured with a pH indicator strips (Baxter®) once a week with a range of 7.5–8.0. Salinity was adjusted by changing 3 parts of the salinity every hour from 25 ppt  down to 5 ppt. In order to achieve a salinity of 2 ppt from 5 ppt, the salinity part was changed every 2 h. Once set up, salinity was measured with a salt refractometer (Sper Scientific®) once a week over the length of the challenges. Ten (10) SPF P. vannamei (weights: 2.0–2.1 g) were stocked in each tank for the experimental infection, respectively. Three 90-L control tanks were set up for each salinity as negative control treatment.The EHP isolate used in this study was obtained from a Over the course of the two challenges, the EHP-infected shrimp were fed daily at 5% of the total biomass. Fecal strings were collected daily by siphoning the fecal strings from the EHP-infected tanks into a tube 1 h after feeding. In order to prepare the inoculum for challenge #1, the EHP-infected fecal strings were pooled from three tanks with 5 shrimp per tank (mean weight 12.5 g). For challenge #2, EHP inoculum was prepared by pooling fecal strings from one 1000 L tank containing 60 EHP-infected shrimp (mean weight 9.0 g). The salinity of the EHP-infected tanks used as inoculum sources were constant for both challenges at 25 ppt (±1.0). After siphoning the fecal strings from the tanks, it was weighed and aliquoted in seven equal parts. Six of the aliquots were used as the inoculum for the experimental infection of each treatment tank. Each of the six aliquots were added to the treatment tanks by mixing the tube contents into the water of the treatment tanks. The remaining aliquot was preserved in 95% ethanol for EHP detection by PCR following a previously published protocol .Starting 1 h after the inoculation process, shrimp were fed once a day at 2% of the total biomass with a commercial pelleted feed . This process forced the shrimp to eat first the inocula that contained EHP-infectious fecal strings. Survival was recorded daily from the start of the challenge. At the end of the challenge, shrimp samples from the challenge #1 and #2 from each treatment tank were fixed in Davidson’s (AFA) fixative . DetailsThe Davidson’s alcohol-formalin-acetic acid (AFA)-fixed samples were processed, embedded in paraffin, and sectioned (5 μm thick) in accordance with standard methods , 22. AftTwo different PCR methods were used in this study: Conventional nested PCR for EHP detection and real-time PCR for quantitation of EHP load. EHP detection was carried out by a conventional nested PCR that targets the Spore Wall Protein 1 (SWP1) gene. The primers for the first step are: SWP 1F/1R . The primers generate a 514 bp amplicon. The primers for the nested step are: SWP 2F/2R  which generates a 148 bp amplicon. The PCR protocol for the first PCR reaction consisted of a 5-min initial denaturation at 95 °C followed by 30 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 58 °C and extension for 45 s at 68 °C with a final 5-min extension step at 68 °C. For the second (nested) PCR step, the template consisted of 1 μl of the final reaction solution from the first PCR step. The PCR protocol for the second, nested PCR reaction consisted of an initial denaturation at 95 °C for 5 min followed by 20 cycles of 30 s denaturation at 95 °C, 30 s annealing at 64 °C and 20 s extension at 68 °C with a final extension for 5-min at 68 °C . For EHPP-value < 0.05 was considered as statistically significant. The association between salinities and E. hepatopenaei was determined by calculating the odds ratio (OR) and the statistical significance at α = 0.05 (n = 38) .The EHP copy number obtained from the quantification analysis was transformed to log base 10 value prior to carrying out statistical analysis using SPSS v16.0. A one-way ANOVA with Tukey’s multiple comparison were performed and


We are in the midst of the novel coronavirus (COVID-19) pandemic, the most significant global health event since Spanish influenza in the early 20th century. Increasingly draconian measures are being implemented worldwide to try to slow the spread of the virus. Antimicrobial resistance (AMR) has been cited as the most significant threat to the global health and global economy in recent years, but is now likely to be eclipsed by COVID-19 for some time. However, the emergence of COVID-19 also presents some important consequences for the development of AMR. This piece will highlight how managing the COVID-19 crisis could impact AMR in the clinic, beyond the clinic in the community, in the environment and in relation to public awareness. When civilization emerges from the other side of this global health emergency, efforts should be made to understand these potential effects on AMR, the other significant, and constant global health issue of our time.Healthcare systems around the world are under increasingly immense pressure. This is leading to several changes in practice that may have impacts on, or relevance to, AMR.For example, the UK government have published several documents relating to COVID-19 management in clinical settings. In the guidance for primary care, it is recommended that any room that has been used for a patient with a suspected SARS-CoV-2 (the causative agent of COVID-19) infection should remain closed and ventilation switched off until full sterilization has taken place .Use of antimicrobial soaps and disinfectant cleaners by members of the community and in the hospital will have increased hugely over the last few months. Higher usage is likely to continue, and may even remain high following the outbreak due to changes in infection and control policy or individual habits. As discussed above, these increased/improved hygiene practices may reduce the spread of AMR, which is a very positive outcome. However, there is also a potential negative impact that could arise from increased use of such products, as many of them contain biocides. Biocides are antimicrobials found in surface disinfectants and household cleaners  was first isolated in India  was also detected in China for the first time last year  will also have impacts for the spread of AMR. Movement of key AMR genes between countries in undeniable. For example, one of the key genes conferring resistance to last resort carbapenem antibiotics  WHO, . Media cPotential implications, both good and bad, of some of the current management practices and practicalities of managing the novel coronavirus outbreak in relation to AMR have been discussed. This is by no means a comprehensive list and without doubt, further impacts will become apparent as the situation rapidly progresses. This pandemic will be considered a significant event in human history. Both emerging infectious diseases and AMR are included in the UK government's National Risk Registry of Civil Emergencies (HM Government, The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Single-crystal X-ray analysis shows that the finely arranged triangular necklace 1 has two racemic enantiomers in its solid state with intriguing packing motif. The superior antibacterial activity of necklace 1 against both standard and clinically drug-resistant pathogens implies that the presence of Cu(I) center and platinum(II) significantly enhance the bacterium-binding/damaging activity, which is mainly attributed to the highly positively charged nature, the possible synergistic effect of heterometals in the necklace, and the improved stability in culture media. This work clearly discloses the structure-property relationships that the existence of two different metal centers not only facilitates successful construction of heterometallic triangular necklace but also endows it with superior nuclease properties and antibacterial activities.Sophisticated mechanically interlocked molecules (MIMs) with interesting structures, properties and applications have attracted great interest in the field of supramolecular chemistry. We herein report a highly efficient self-assembly of heterometallic triangular necklace  Precise assembly of heterometallic complexes is a challenge. Here, the authors design a heterometallic triangular necklace through a highly efficient threading-and-ring-closing approach driven by metal-ligand coordination, which shows strong bacterium-binding and cell wall/plasma membrane-disrupting capacity for killing bacterial cells. Molecular necklaces, as important members in MIMs family, are derived from catenanes, in which three or more side rings as molecular “beads” are threaded onto a central ring as molecular “chains”28. The early research of molecular necklace could be traced back to the work reported by Sauvage et al., who generated a mixture of molecular necklaces by accident in a very low yield and characterized their structures by using electrospray ionization mass spectrometry (ESI-MS)29. Moreover, Stoddart et al.32, Chiu et al.33, Grubbs et al.34, and Wu et al.35 also reported a series of molecular necklaces through covalent synthesis assisted by weak donor–acceptor π–π interactions or dynamic covalent chemistry, but the yield was also relatively low. Notably, Kim and coworkers realized the highly efficient assembly of molecular necklaces by taking advantage of coordination bonds and cucurbituril-based host–guest chemistry, which is definitely a big breakthrough38. Recently, Li, Stang, and co-workers have presented the highly efficient construction of the largest molecular necklace so far through hierarchical self-assembly involving coordination interactions and the subsequent host–guest chemistry40.Mechanically-interlocked molecules (MIMs) such as catenanes41. In particular, metals in molecular necklace would not only facilitate the threading process due to their template effect but also participate in the ring-closing process via coordination-driven self-assembly43. Moreover, the existence of metals will bring some interesting properties and applications of the resultant necklace. However, most of the documented metallic necklaces only involve one kind metal in their architectures, and no heterometallic molecular necklace has been reported so far. Therefore, there is a high demand for the design of functionalized molecular necklaces that possess heterometal centers and intriguing properties for applications.Generally, a well-established molecular necklace is self-assembled from three main components: molecular axis as chain, macrocycle as bead and molecular linker that joints them together Fig. . The pre44. Indeed, the biomedical applications of coordination-driven self-assembly of metallacycles and metallacages have been systematically investigated in last decade, and they can serve well as anticancer agents, drug delivery systems, biosensors, DNA intercalators, antibacterial agents and so on46. It is anticipated that molecular necklaces, the distinct type of metal coordination-driven self-assemblies, may also be developed for biomedical applications, especially when their biomolecule-interaction efficiency and stability are highly improved.It’s worth-noting that, though a major breakthrough in molecular necklaces regarding to their impressive structures as well as well-developed synthetic strategy has been achieved, the study of their properties and potential applications have persistently lagged behind expectations. To the best of our knowledge, the focus of the chemistry of MIMs, especially for molecular necklaces, has still stayed on molecular design and synthesis stage. Studies involving their applications are hardly seen at present in this area. The intrinsic nature of coordination-driven self-assemblies including their high dynamic metal–ligand coordination bonds, diverse metal ions, positively charged molecular nature, together with their highly tunable coordination geometries endows them with great merits in biomedical applications48. The bacterial DNA, cell wall, and plasma membrane are critical components of bacterial cells essential for survival and growth49. The negatively charged property of DNA, bacterial cell wall (displaying negatively charged lipopolysaccharides or teichoic acid), and plasma membrane (exposing negatively charged phospholipids)50 indicates that they are realistic targets of the positively charged metal coordinating complexes. The coordination-driven self-assembly of the positively charged metallosupramolecular complexes just provides a powerful platform to enhance the electric charges for severely disruption of the targeted DNA/cell wall/plasma membrane, and to improve the stability for prolonging the interaction time between the necklaces and the bacterial cells. Inspired by the recent development of biomedical and biochemical applications of coordination-driven self-assembly, we envision that molecular necklaces possessing multiple metals as well as the enhanced electric charges and stability may potentially serve as efficient DNA intercalators and bacterial cell wall/plasma membrane-disrupting agents, thus opening a promising window to their biomedical applications.The emerging drug-resistant bacterial pathogens have been becoming great threats to human health, and thus provoke the development of advanced antibacterial agents1 containing both Cu and Pt metals with strong antibacterial activity through a highly efficient “threading-followed-by-ring-closing” approach driven by metal ligand coordination  and platinum(II)–pyridine coordination motifs are particularly selected as they are mutually compatible, i.e., 2,9-disubstituted [Cu(phen)2]+ complex will not affect platinum(II)–pyridine coordination process52, probably duo to the steric hindrance53. The synthetic route to molecular necklace 1 is depicted in Fig. D1 consisting of two functional ligands of pyridine and phenanthroline was synthesized from the starting materials M1 and D2 according to a literature method54 with minor modifications  acceptor unit A1 in nearly quantitative yield. Notably, pseudorotaxane D1 could generated in situ without the further purification. Therefore, the heterometallic triangular necklace 1 was achieved in quantitative one-pot synthesis simply by mixing M1 and D2 with A1 2]+D5 and the corresponding diplatinum (II) acceptors A3 was proposed  spectrum indicated the unsuccessful self-assembly process –pyridine these two coordination motifs are not compatible, i.e., the controlled experiment demonstrated that 3,8-disubstituted [Cu(phen)2]+ is likely to interfere the platinum(II)–pyridine coordination process because of the potential competing coordination reaction between platinum(II) and nitrogen in 3,8-disubstituted [Cu(phen)2]+ 2]+ units was also synthesized by using the same protocol  for comparison  NMR, two-dimensional correlation spectroscopy, diffusion-ordered NMR spectroscopy (DOSY), nuclear overhauser effect spectroscopy, and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) -bis(phenanthroline)s (+ unit55. In its packing structure, multiple intramolecular and intermolecular interactions were observed. Specifically, one pyridyl face-to-face packed with intramolecular phenanthroline with a centroid-to-centroid distance of 3.813 Å, while the other pyridyl stacked with phenoxy groups of the neighboring enantiomer with centroid-to-centroid distance of 4.183 Å. Intramolecular π–π stacking between phenoxy group and phenanthroline was also observed with a centroid-to-centroid distance of 3.533 Å. Consequently, two enantiomers alternatively packed together into a column along the a axis. Typically, it is very difficult to obtain good single crystals of large cationic macrocycles based on Pt(II)-N(pyridine) coordinative bonds, not to mention such complicated molecular necklace. Fortunately, single crystal of heterometallic triangular necklace with good quality was obtained by slow diffusion of ether into acetone solution -N cationic metallacycle. As shown in Fig. 1 on account to the orientation of [Cu(phen)2]+ unit. The size of the necklace is also very impressive with the exterior length of approximately 37 Å and internal length of approximately 15 Å, leading to a cavity with diameters of ~8 Å. Regarding to the packing motif, three [Cu(phen)2]+ units exhibited strong π–π stacking with the adjacent molecules with a centroid-to-centroid distance of 4.427 Å. As a result, every triangular molecule in solid state was arranged very closely to each other and stacked in a stagger packing mode, thereinto two enantiomers in each layer was alternatively aligned. The casual discovery of the chirality in molecular necklace would enrich the recent development of chiral chemistry in MIMs such as rotaxanes and catenanes57. The above results also highlight that the stoichiometry and position of Cu(I)-contained donor and the Pt(II)-contained acceptor unit can be precisely integrated into the well-defined molecular necklace in such coordination-driven self-assembly process, which is of high importance to the following biomedical applications.Single crystal suitable for X-ray crystallographic analysis was obtained for pseudorotaxane ion Fig. . A racemion Fig. . The com58. Investigations on DNA cleavage and developments of efficient chemical nucleases have attracted wide interest owing to their potential applications as promising therapeutic agents as well as diagnostic structural probes to analyze DNA information. Among all the chemical nucleases reported so far, transition metal complexes have obvious advantages because metal complexes with natural cationic character particularly favor the redox, hydrolysis and other photoreactions, thus leading to strong affinity to DNA60. For example, [Cu(phen)2]+ units have been widely studied as chemical nucleases for highly efficient DNA cleavage since the redox properties of the metal could not only promote the reactive oxygen species (ROS) generation but also have strong noncovalent interactions with DNA61. Recent studies have demonstrated that ditopic coordination compound containing copper and platinum centers displayed some intriguing nuclease properties with the enhanced DNA cleavage efficiency65. However, very few mechanically-interlocked molecules, especially molecular necklaces, have demonstrated their application prospects in this area67. On this basis, the study of the nuclease properties and application in DNA cleavage of the designed heterometallic molecular necklace 1 is of great interest. The DNA-cleavage of necklace 1 was then first systematically studied and compared with the above designed [3]catenane 2, metallacycles 3–6, pseudorotaxane D1 as well as di-Pt(II) acceptors A1 and A2. Indeed, DNA cleavage assay revealed that both the molecular necklace 1 and [3]catenane 2 had much higher DNA cleavage activity than the donor D1, the acceptors A1 and A2, and the metallacycles 3, 4, 5 and 6 -containing molecule D1, rather than the Cu(I)-free molecules , also led to obvious DNA degradation, but its DNA cleavage efficiency was much lower than the necklace -intercalating ability and antibacterial activity71. Considering the fact that molecular necklace 1 has combinational positive charges, π–π stacking ability, platinum(II)–pyridine coordination and [Cu(phen)2]+ units, we speculate that it may possess strong antibacterial activity. The positively charged molecular necklace 1 might possess antibacterial activity owing to its binding with the negatively charged components of the cell wall/plasma membrane, e.g., liposaccharides (LPS) and phospholipids. The binding ability of the molecular necklace to the bacterial cells was then investigated by both glass-adhering test and dynamic light scattering (DLS) analysis using the well-known bacterial pathogen Pseudomonas aeruginosa. Confocal observation of the 4′,6-diamidino-2-phenylindole (DAPI)-stained cells on the glass surfaces revealed that the glass surface coated with the necklace 1 could bind abundant bacterial cells, while the surfaces coated with [3]catenane 2 or the metallacycles 3–6 only had weak bacterial binding ability  than that of [3]catenane 2 (to 750–3548 nm), indicating much more severe cell aggregation caused by the binding of 1  from the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, the heterometallic necklace 1 caused drastic CF release  -carboxyl fluorescein (CF)-leakage model to evaluate the ability of the molecular necklace to damage the plasma membranein) Fig. , indicat1, we expected that it may severely kill the bacterial cells. Colony forming unit assays showed that, while [3]catenane 2 and the metallacycles 3–6, together with their donor D1, the Cu+ precursor Cu(MeCN)4PF6, and the acceptors (A1 and A2), only caused a decrease in cell viability to 30–65%, the heterometallic necklace 1 could kill almost all of the bacterial cells , the penicillin/tetracycline Escherichia coli strain (IC50 = 1.84 μM), and the multidrug-resistant Staphylococcus aureus (MRSA) strain (IC50 = 5.16 μM), indicating that it may be a superior candidate of antibacterial agents for fighting against multidrug-resistant pathogens.Owing to the strong bacterium-binding and cell membrane-damaging ability of necklace 1, 2, D1, and Cu(MeCN)4PF6) had higher antibacterial activity than the Cu(I)-free molecules  -contained donor and the Pt(II)-contained acceptor in the necklace 1 generated the highest cell wall/plasma membrane-binding/damaging and bacterial killing ability since the Cu(I)-free matallacycle 4 and the Pt(II)-free donor D1 exhibited much lower antibacterial capacity than necklace 1 -containing molecules  Fig. . In addiA2) Fig. . These re 1 Fig. .1 framework is also critical for efficiently killing the pathogens. To prove this contribution, a control experiment was carried out. The antibacterial activities of the ligand mixtures of D1 + A1′ or D1 + A2′ have been surveyed. Notably, the physical blends of D1 + A1′ or D1 + A2′ could not self-assemble into the necklace since Pt was protected by iodide or bromide moieties. It was found that, compared to the necklace 1, the mixtures of D1 + A1′ or D1 + A2′ exhibited much weaker bacterium-killing efficiency  double stained cells revealed that the necklace 1 induced severe cell aggregation and caused almost thorough cell death (indicated by the PI-positive cells with red fluorescence)   Fig. . In contath Fig. . Similarxes Fig. , confirm1 was further confirmed by cell surface distribution of the necklace 1 and ultrathin section observations of the bacterial cells , or tryptone  was needed for necklace 1 to be completely degraded. The enhancement of the stability towards LB medium for necklace 1 was probably due to its aggregation, which decreases its exposed molecular surface area and protects it from LB medium. The stability of coordination-driven self-assembly is essentially related to the intrinsic dynamic nature of metal-ligand coordination chemistry75. Based on large amounts of investigation and research on stimuli-responsive metal–ligand assemblies77, we believe that the degradation of necklace 1 probably stems from the ions such as chloride ion in LB medium  Cu(I) functions in bacterial binding to the cell wall/plasma membrane and disrupts their ultra-structures, together with in promoting DNA cleavage; (2) Pt(II) enhances the biomolecule-interaction capacity of Cu(I) to more efficiently disrupt targeted cell components, e.g., lipopolysaccharides, phospholipids, and DNA; (3) Stoichiometric Cu(I)-contained donor and the Pt(II)-contained acceptor can be precisely integrated into the architecture of necklace 1, which thus enhances the bacterial binding/damaging capacity and stability of the necklace.Together, these results indicate that Cu(I) and Pt(II) in necklace 1 was successfully synthesized through a “threading-followed-by-ring-closing” approach driven by coordination interaction. The crystal structure of 1 disclosed an elegant triangular necklace architecture containing a large [3 + 3] Pt(II)-N cationic metallacycle interlocked with three polyether phenanthroline macrocycles. The existence of two different metal centers not only facilitated the successful construction of necklace 1 but also endowed it with superior nuclease properties and antibacterial activities. Our studies revealed that the self-assembly of heterometallic necklace would significantly enhance its bactericidal activity. This enhancement might be mainly attributed to the synergistic effect of heterometals in the necklace, endowing it superior bacterium-binding and cell wall/plasma membrane-disrupting capacity for killing the bacterial cells. “Threading-followed-by-ring-closing” approach combining with coordination-driven self-assembly would allow us to further construct more complicated molecular necklaces in the future. And the promising DNA cleavage and antibacterial activities results obtained herein would also attract broad interests and provide directions for future chemical design in this field.In summary, a heterometallic triangular necklace The synthesis and characterization of new compounds present in this work, and the experimental details and additional data of DNA cleavage, bacterium-binding and antimicrobial tests are described in the Supplementary InformationPeer Review File


Vibrio vulnificus (V. vulnificus) hemolysin (VVH) is a pore-forming cholesterol-dependent cytolysin (CDC). Although there has been some debate surrounding the in vivo virulence effects of the VVH, it is becoming increasingly clear that it drives different cellular outcomes and is involved in the pathogenesis of V. vulnificus. This minireview outlines recent advances in our understanding of the regulation of vvhA gene expression, the biological activity of the VVH and its role in pathogenesis. An in-depth examination of the role of the VVH in V. vulnificus pathogenesis will help reveal the potential targets for therapeutic and preventive interventions to treat fatal V. vulnificus septicemia in humans. Future directions in VVH research will also be discussed.The  V. vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Human infections usually occur following the consumption of contaminated seafood or via an open wound exposed to a contaminated water source  toxin is also likely to be critical to the success of infection. Supporting this, Gavin et al. showed that the MARTX toxin is essential for bacterial dissemination from the intestine  revealed that VVH has a similar cytolysin domain and a lectin-like domain of VCC (V. vulnificus septicemia.Although the VVH belongs to the cytolytic pore-forming family of toxins (PFTs), all of which cause cytolysis in a variety of mammalian cells, VVH as a virulence factor is under debate. An earlier study has shown that disruption of hemolysin gene ty model . Howeverlnificus , 11. Forpression –14. Fan the mice . Moreovethe mice . These rlnificus , 18. How strains , 20. Morlnificus . Besidesreviewed –23. Likereviewed –27. Molen of VCC . HowevervhA expression, and possible roles in pathogenesis. Future directions in VVH research was also discussed in this review. This in-depth evaluation of the contribution of the VVH to V. vulnificus pathogenesis may aid in the development of novel therapies aimed at treating and preventing sepsis in humans.In this review, we explore the features of VVH in its biological activity, regulation of vIn vitro studies have illuminated the effects of the VVH in various host cell types, including human epithelial cells, human umbilical vein endothelial cells (HUVECs), mice macrophages and lymphocytes  treatment  nor bovine serum albumin (BSA) affected activity . Blood lactivity . It was activity . Jin-Woootectant . Consequ of mice . Additioth NCF-1 . While pκB-dependent mitochondrial cell death via lipid raft-mediated reactive oxygen species production by the distinct activation of PKCα and ERK/JNK in intestinal epithelial cells  showed that infection with low doses of recombinant VVH protein induces necrotic cell death and apoptosis. The study further demonstrated that (r)VVH induces NF-al cells . Besidesfections .V. vulnificus has been shown to produce sufficient VVH in the small intestine to accelerate invasion into the bloodstream -primed splenocytes with formalin-inactivated VVH toxin, while co-expression of T-cell-polarizing cytokines -12, and IL-4) was detected in the cell culture supernatant , which was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kB activation , it increases extracellular VVH secretion through increased transcription of pilD, which encodes PilD, a component of the type II general secretion system responsible for extracellular VVH secretion  is one of the most important cellular regulatory cascades. QS is responsible for cell–cell communication and is mediated by a small diffusible molecule called autoinducer 2 (AI-2). LuxO is a central response regulator of the QS circuit in V. vulnificus, with disruption of luxO shown to increase the expression of smcR, crp, and luxS, which encodes the autoinducer 2 synthetase  pili are reported to play critical roles in evasion of the host innate immune system by resistance to complement-mediated killing  model is a mouse model of chronic and binge ethanol feeding, which mimics acute-on-chronic alcoholic liver injury in patients (in vivo, and the underlying mechanisms that contribute to acute infections by V. vulnificus in liver disease patient.ividuals . Althougnfection . Hepcidinfection . Therefonfection  could bepatients . This siYY contributed to the research of the literature and the writing and revision of the manuscript. ZF and JW contributed to the revision of the manuscript. All authors contributed to the article and approved the submitted version.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.


Established intravascular NIRF imaging targets include macrophages, cathepsin protease activity, oxidized low-density lipoprotein and abnormal endothelial permeability. Structural and molecular intravascular imaging provide complementary information about plaque microstructure and biology. For this reason, integrated hybrid catheters that combine NIRF-IVUS or NIRF-OCT have been developed to allow co-registration of morphological and molecular processes with a single pullback, as performed for standalone IVUS or OCT. NIRF imaging is approaching application in clinical practice. This will be accelerated by the use of FDA-approved indocyanine green (ICG), which illuminates lipid- and macrophage-rich zones of permeable atheroma. The ability to comprehensively phenotype coronary pathobiology in patients will enable a deeper understanding of plaque pathobiology, improve local and patient-based risk prediction, and usher in a new era of personalized therapy.Despite exciting advances in structural intravascular imaging [intravascular ultrasound (IVUS) and optical coherence tomography (OCT)] that have enabled partial assessment of atheroma burden and high-risk features associated with acute coronary syndromes, structural-based imaging modalities alone do not comprehensively phenotype the complex pathobiology of atherosclerosis. Near-infrared fluorescence (NIRF) is an emerging molecular intravascular imaging modality that allows for  In 1989, Muller et al. . This dein vivo atheroma burden and some high-risk features reflecting plaque vulnerability  and optical coherence tomography (OCT), assess rability . Despiter stress –9. To adr stress , 11 or pr stress , 13 for The underlying pathobiology of atherosclerosis provides a roadmap for molecular imaging targets and imaging agent synthesis . AtherosAnother high-risk plaque feature is microcalcification. Calcification is an active process that occurs during all stages of atherosclerosis. Macrocalcifications, which are detectable by CT scan due to high radiodensity [>400 Hounsfield unit (HU)], may be associated with more stable plaque phenotypes . In contPositive remodeling is thought to contribute to plaque instability by induction of neovascularization of vasa vasorum in the adventitia that eventually invade the intima and the fibroatheroma . These nThe goal of detecting high-risk plaques is to identify rupture-prone plaques and intervene in a way  to prevent future ACS. Very recently, the COMPLETE trial showed that in patients with STEMI and multivessel coronary artery disease (CAD), intervening on non-culprit lesions reduced a composite of CVD death and myocardial infarction , demonstin vivo visualization of molecular process that contribute to plaque vulnerability. The primary intravascular molecular imaging modality nearing clinical translation utilizes near infrared fluorescence (NIRF) detection of targeted NIR fluorophores, discussed in detail next.To tackle the limitations of anatomical intravascular imaging modalities, intravascular molecular imaging has been developed to allow for in vivo pathobiological processes (Near-infrared fluorescence (NIRF) is an emerging translational, intravascular imaging modality that has the ability to capture a wide range of rocesses . NIRF morocesses . After irocesses . Comparerocesses , 37.in vivo plaque inflammation in rabbit iliac arteries (1.5–2.5 mm in diameter) through blood  are amongst the most widely studied proteases. Cysteine cathepsins are mainly found in lysosomes and have been in implicated in atheroma-blood vessel remodeling through elastolytic and collagenolytic activity –46. ProS−/− mice when compared to wild type (WT)  and wide area of distribution to the liver, kidneys, and spleen, limiting the plaque target-to-background ratio (Oxidized LDL (oxLDL) particles are often found within atheroma lipid-rich necrotic core and have been explored as targets for NIRF imaging. Khamis et al.  designedype (WT) . LO1-750nd ratio .in vivo endothelial dysfunction-based inflammation in a rabbit model. The group demonstrated that within atheroma plaque, CLIO-CyAm7 deposited in macrophages and endothelial cells in areas of impaired endothelial barrier underlying areas of triggered plaque thrombosis , Stein-Merlob et al.  developerombosis  55). Th. Thin viin vitro using human plasma clots and in vivo by binding to murine thrombi using non-invasive NIRF imaging  is impractical for clinical use. For these reasons, hybrid intravascular molecular-structural catheters have been developed to enable concurrent co-registration of molecular and anatomical images, and accurate distance-based correction/quantification of the NIRF signal , 2 64, , 64, 66.in vivo rabbit iliac arteries after balloon injury  but with relatively low depth of penetration (1–2 mm) . When con injury . Similarn injury  designed, a new contrast-free autofluorescence-based method . In the in vivo experiment on rabbit aortas, Wang et al.  is an emerging imaging modality that is a natural extension of IVUS, and has the potential to detect certain fluorophores and nanomaterials that could enable IVPA-based molecular imaging. IVPA is a novel structural and molecular imaging modality that uses multiple wavelengths to illuminate the tissue of interest and identifies the acoustic waves generated by the thermoelastic expansion of the environment surrounding absorbing molecules . In an ig et al.  showed tistology .ex vivo NIRF imaging, ICG was found to accumulate in atheroma macrophages and was detected in vivo rabbit atheroma  is the only atherosclerosis-targeted NIR fluorophore approved by the US Food and Drug Administration (FDA) for human use, facilitating the use of NIRF catheter in human subjects once approved. The recognition that ICG, a blood flow imaging agent available for 50 years, could target atherosclerosis was somewhat unexpected. In 2011, Vinegoni et al. recognized that the amphiphilic properties of ICG might allow it to have an atheroma targeting profile. Using intravascular and atheroma , 81. Subpatients . The autin vivo plaque inflammation and macrophage accumulation. This finding provides evidence that DES may be a bio-stabilizer of high-risk, inflamed plaques , we anticipate that the only difference for NIRF-OCT or NIRF-IVUS will be the administration of a targeted molecular imaging agent at the start of PCI. Following PCI, completion NIRF-OCT or NIRF-IVUS will be performed, allowing assessment of non-culprit coronary artery segments proximal and distal to the target lesion that was stented. This will allow the generation of an integrated molecular-structural atheroma score reflecting the degree of pathobiology for the culprit artery. In addition another artery could be potentially imaged with the same goal. Ultimately, a NIRF-based pathobiology score will identify high-risk lesions, arteries, and patients, allowing the ability to more precisely target newer atheroma medical therapies  to those at highest risk. Overall, integration of NIRF pathobiology assessment will enable personalized medical therapy for patients with CAD, instead of a one size fits all approach as currently practiced worldwide .Both authors contributed to the writing of this review article.FAJ has received sponsored research grants from Canon and Siemens; he is a consultant for Boston Scientific, Abbott Vascular, Siemens, Acrostak, and Biotronik. Massachusetts General Hospital has a patent licensing arrangement with Canon, and FAJ has the right to receive royalties. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The separation and purification of specific chemicals from a mixture have become necessities for many environments, including agriculture, food science, and pharmaceutical and biomedical industries. Electrospun nanofiber membranes are promising materials for the separation of various species such as particles, biomolecules, dyes, and metals from liquids because of the combined properties of a large specific surface, light weight, high porosity, good connectivity, and tunable wettability. This paper reviews the recent progress in the design and fabrication of electrospun nanofibers for chemical separation. Different capture mechanisms including electrostatic, affinity, covalent bonding, chelation, and magnetic adsorption are explained and their distinct characteristics are highlighted. Finally, the challenges and future aspects of nanofibers for membrane applications are discussed. In recent years, membrane technologies have obtained great popularity for chemical separations based on their high separation efficiency, simple operational process, and relatively low production cost. A membrane acts like a barrier between two phases and allows a substance to be selectively transferred from one side to the other . It can Nanofiber membranes with a superb surface area to volume ratio, high porosity/tortuosity, and high permeability are an excellent choice for the fabrication of membranes. Several techniques such as nanolithography, phase separation, self-assembly, electrospinning, melt-blowing, and template synthesis have been used for the production of nanofibers. Among them, electrospinning is of particular interest because of its simplicity, versatility, flexibility, and the ease of processing . This teElectrospinning is a fiber formation method in which an electrical field is applied to polymer solutions or polymer melts to produce micro- and nanofibers. A typical electrospinning process comprises three main components: a feeding section, a high voltage power supply, and a collector section . The feeThe effect of the electrospinning conditions on the morphological and structural characteristics of the nanofibers have been discussed in the literature ,8,11. ThNanofiber membranes require several properties to be used for chemical separation. Depending on the application, they need to have a high porosity, appropriate pore size, as well as a high mechanical, chemical, and/or thermal resistance, as they may experience different temperatures, pH, stresses, and vibrations in a liquid flow. In addition, a nanofibrous membrane often needs to have a good selectivity and reusability to increase the separation performance and operational flexibility, and to reduce the material and labor costs. Nanofibrous fabrics can separate chemicals and impurities by different capture mechanisms including electrostatic, affinity, covalent bonding, chelation, magnetic, and size exclusion/filtration. The application of electrospun membranes for liquid filtration and oil/water separation have been reviewed by other authors ,12. As s3+, –NRH2+, –NR2H+, –NR3+, –PR3+, –SR2+) or negatively charged groups  in their chemical structures [Ion exchange membranes (IEMs) have been widely used for the separation/removal of dissolved ions from a liquid solution . Electroructures . These fElectrospun IEMs have been used for the selective separation of a variety of molecules such as proteins, cells, antibodies, and metal ions. By regulating the pH, these chemicals obtain negative/positive charges and can be adsorbed on oppositely charged nanofibers. Ki et al.  fabricatIf a membrane does not have an appropriate functional group for chemical adsorption, its surface chemistry needs to be modified. Grafting is one of the most common methods for enhancing the surface functionality of electrospun nanofibers. Wang et al.  combined2 nanoparticles in a PAN spinning solution to fabricate carbon nanofibers with robust mechanical properties for protein adsorption. To activate the nanofiber surface with N-H groups, the emitted nitrogen gas from the PAN carbonization process was reused. The resultant SiO2@CNF membrane possessed a bovine serum albumin (BSA) adsorption capacity of 30 ± 0.9 mg/g due to the surface roughness of the nanofibers, electrostatic attraction, and hydrophobic adsorption between the carbon and BSA protein.The incorporation of nanowhiskers and nanoparticles into nanofibers is another way to improve a membrane’s surface chemistry ,24,25,26E. coli) cells [Moreover, the charged chemical groups can be induced to the nanofiber surface by cationic and anionic polymers. Our research group, previously ,28,29,30i) cells , for exai) cells  incorporIn addition, appropriate functional adsorption groups can be introduced into the nanofibers by chemical treatments. Chiu et al. treated electrospun PAN nanofibers with sodium hydroxide (NaOH) to transform the cyanide functional groups (–C≡N) to hydrophilic carboxyl functional groups (–COOH) . They rev/v) nanofibers with air dielectric barrier discharge (DBD) plasma for acetylcholinesterase (AChE) enzyme immobilization. They reported that a 6 min plasma treatment induces NH3+ polar groups on the membrane surface which can immobilize negatively charged AChE (IP ~5.87) at pH 7.4. Although they did not report the enzyme capture capacity of the membrane, the activity of the enzyme immobilized on the plasma-treated membrane was about 12% more than that of the unmodified membrane. Furthermore, Najafi et al. [Plasma treatment can also be used for the surface functionalization of nanofiber-based IEMs. Doraki et al.  modifiedi et al.  recently2/g) make these materials excellent adsorbents for the separation of heavy metals [6O4(OH)4(COOH)6(BTC)2 and Fe3OF–(H2O)2(BTC)2·nH2O (MOF 808 and F300)) based on iron (III) and zirconium (IV) in polyacrylonitrile (PAN) and polyvinylidene fluoride (PVDF) electrospun nanofibers (−) of the MOF at pH > 5 and the positive charge of the heavy metals [Metal–organic frameworks (MOF) can also be used for the fabrication of IEMs for liquid purification. MOFs, also known as porous coordination polymers, are multifunctional materials consisting of metal ions or clusters and organic linkers. The extraordinary porosity, tunable pore sizes, and high surface area to mass ratios  of proteins/enzymes to a nanofiber surface [Covalent attachment is based on the binding of amino acid residues  of chitosan. Moreover, Xiao et al. [2 composite nanofibers via sol-gel electrospinning. A viscous PVA/gel gel was obtained by adding a percentage of PVA solution to a silica gel containing 3-Mercaptopropyltrimethoxysilane . They reported a large adsorption capacity of 489.12 mg/g for heavy metal ions thanks to the nanofibers’ large surface area of 290 m2/g. Such adsorption was attributed to the electrostatic attraction and complex formation between the metal ions and the thiol groups. Several researchers have used the chelation mechanism for capturing chemicals on nanofibers. For example, Haider and Park  examinedo et al.  examinedo et al.  producedo et al.  produced2OH) and sodium carbonate (Na2CO3) with the PAN nanofiber nitrile group. Adsorption capacities of 52.70 and 263.45 mg/g were reported for Cu(II) and Pb (II), respectively, due to the chelation between the available oxime sites on the fibers and the heavy metal ions. The difference between the metal ion adsorption capacities was attributed to the different number of chelating groups of the metals. Cu(II) prefers a 1:1 chelate complex, while Pb (II) prefers to form a complex with 2 amidoxime groups. In another study [2–NH2) for the removal of metal ions from water. They reported adsorption capacities of 114 and 217 mg/g for Cu(II) and Pb (II), respectively. If a membrane does not have a chelating group, its surface chemistry can be modified by chemical treatment methods. Kampalanonwat and Supaphol  modifieder study , they moInclusion complexation is another way of capturing chemicals from liquids. The capture mechanism is based on the noncovalent entrapment of a target/guest compound into a chemical compound/host containing cavities. Cyclodextrins (CDs) are well known hosts for the formation of inclusion complexes. CDs are natural cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic central cavity, containing α-linked glucopyranose units  60,61,6,661,62. −2 h−1). In the literature, there are many studies on the CD functionalized nanofibrous membranes for liquid filtration applications ,65. For Affinity membranes can capture targeted molecules based on highly specific binding interactions rather than molecular weight, size, or charge. An affinity adsorbent is made by immobilizing a ligand onto a substrate. The immobilized ligand is then contacted to the solution containing a molecule that binds specifically to that ligand. Electrospun nanofibers are an ideal material for the fabrication of affinity membranes because they can provide a high specific surface area and a highly tortuous porous structure for capturing ligands. In this part, we review the common ligands for the production of electrospun affinity membranes.Cibacron blue F3GA (CB) is an excellent ligand for the purification of many enzymes and blood proteins because its anthraquinone compound  mimics Biotin, or vitamin B, is another ligand for the production of affinity nanofibrous membranes. The high affinity of biotin to proteins such as avidin and streptavidin has been utilized in many protein and nucleic acid purification and detection methods. These proteins can bind up to four biotin molecules, which are normally conjugated with enzymes, bioreagents, and proteins . The bon2/g), a high membrane porosity of 71%, and a wide pore size distribution. Moreover, Zhu and Sun [Proteins A and G are other affinity ligands capable of specifically reacting with the antibody IgG. Protein A is cell wall protein component—a single polypeptide chain—containing four binding sites that specifically capture the Fc region of IgG. Protein G is a bacterial cell wall protein containing two high affinity IgG binding sites as well as albumin binding domains. Recently, protein A/G has been developed by combining four Fc-binding domains from protein A with two domains from protein G with the albumin binding site of protein G eliminated. Compared to A and G, Protein A/G has a stronger affinity effect, lower nonspecific binding, and higher stability in a broader range of pH values ,69,75,76 and Sun  applied 3O4 carbon nanofibers based on 1D polybenzoxazine (PBZ) by using an in situ polymerization method. The complete adsorption of Methylene Blue (MB) and Rhodamine B (RhB) dyes was obtained in 10 and 15 min, respectively, due to the extremely high surface area (1885 m2/g) and high porosity (2.325 cm3/g) of the nanofibers  magnetic carbon nanofibers have attracted great interest because of their large surface area, low density, good chemical resistance, and excellent charge transport capability . The prinofibers . Moreove3O4/carbon nanocables with core–shell structures using coaxial electrospinning. The Fe3O4 was embedded in the core of the carbon matrix to provide the nanocables with electrical and magnetic properties. The nanocables with a specific surface area of 322.6 m2/g and a pore size of 33.6 nm demonstrated excellent magnetic separation performance and effectively captured Rhodamine B and Cu2+ ions from an aqueous solution. Additionally, Lue et al. [In addition, Han et al. ,80 fabrie et al.  fabricatElectrospun nanofibers have been proven to be an excellent material for the fabrication of membranes for chemical separation. These nanofibers can provide large surface area to volume ratios, tunable surface morphology, tortuous porous structure, low mass transfer limitation, and multiple sites for attachment or interaction . Despite