The roles of B and plasma cells in the pathogenesis of inflammatory bowel disease (IBD) are largely unrevealed. Data on the characteristics of IgG4 in patients with IBD are scarce. In this case-control study, serum IgG4 levels were comparable between patients with IBD and healthy individuals, whereas patients with IBD had dramatically higher mucosal IgG4 counts than healthy individuals. In patients with UC, mucosal IgG4 counts were positively correlated with serum IgG4 levels, serum IgG4/IgG ratios, and the Mayo Index; serum IgG4 levels and IgG4/IgG ratios were associated with a history of intestinal surgery and medications. A significant mucosal IgG4 count was found in 33.3% of patients with IBD, whereas, elevated serum IgG4 levels were found in only 9.9% of patients with IBD. Lesions were more severe and extensive in IBD patients with high levels of serum and mucosal IgG4. High levels of serum and mucosal IgG4 decreased after treatment with glucocorticoids or other immunosuppressants. High IgG4 level may be a biomarker for a new subset of IBD. More studies are warranted to explore this new subset of IBD for personalized therapy in the future. Although the pathogenesis of IBD has not been fully elucidated, it is acknowledged that genetic susceptibility, immune dysregulation, gut microbiota, and environmental triggers are strongly involved. Given the dysfunction of the intestinal barrier in the susceptible population, various antigens and pathogens in the lumen of the gut could continuously induce an immune response leading to a disturbance of intestinal immune homeostasis, characterized by continuous or remittent inflammation. Immune lymphocytes are mainly classified as T cells and B cells. In the past 20 years, most studies regarding the pathogenesis of IBD have been focused on T cells. A huge amount of evidence has confirmed that T lymphocytes are a critical subset of immune cells in the pathogenesis of IBD. Nearly every subset of T cells, including Th1, Th2, Th17, and regulatory T cells (Tregs), has been reported to play a role in the pathogenesis of IBD8. In contrast, the role of B cell lineage in the pathogenesis of IBD has been overlooked for a long time and has only received attention since the past 5 years11. As another key subset of immune cells, what roles B cells play in the pathogenesis of IBD mainly remains unclear. Many B lymphocytes terminally differentiate into plasma cells characterized by the production of various antibodies. These antibodies are strongly involved in humoral immunity and may contribute to allergy and multiple autoimmune disorders. Excessive plasmacytic infiltration in the intestinal lamina propria is a remarkable pathological characteristic of IBD12, suggesting a potential pathogenetic involvement of B cell lineage in IBD. IgG4, which accounts for the smallest proportion of all IgG isotypes, used to be commonly ignored because it was regarded as a noninflammatory antibody. However, its role in various autoimmune disorders, and even in cancer, has become a research focus along with the identification and prevalence of IgG4-related disease (IgG4-RD)14.Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn’s disease (CD), is a series of nonspecific inflammatory conditions affecting the gastrointestinal tract. Some multicenter studies and meta-analyses have demonstrated an increasing prevalence of IBD in developing countries, including China15. Sporadic studies have shown high levels of mucosal and serum IgG4 in some patients with IBD. Shen et al.16 at the Cleveland Clinic investigated for the first time the characteristics of serum IgG4 in patients with IBD. In 2011, they reported one case of IgG4-associated pouchitis in a patient with UC16. Subsequently, this team found that elevated serum IgG4 level was associated with chronic antibiotic-refractory pouchitis among patients with IBD17. Rebours et al.18 compared the infiltration of mucosal IgG4+ plasma cells in intestinal tissues of patients with IBD and patients with autoimmune pancreatitis (AIP). This study demonstrated that patients with IBD had higher intestinal mucosal IgG4 counts than patients with AIP. Another study by Raina et al.19 focusing on mucosal IgG4 in IBD found that infiltration of mucosal IgG4+ plasma cells was associated with primary sclerosing cholangitis (PSC) and that mucosal IgG4 counts in patients with active UC were higher than those in patients with inactive UC. The above studies demonstrated that a rise in the level of either mucosal or serum IgG4 can be found in some sporadic patients with IBD, but there is a lack of matched case–control cohort studies comparing intestinal and serum IgG4 levels between patients with IBD and healthy individuals. To date, it has been well acknowledged that IgG4 is a critical marker for the diagnosis of IgG4-RD. In contrast‚ the role that the tissue IgG4+/IgG+ ratio of plasmacytic infiltration and the serum IgG4/IgG ratio play in diagnosing IgG4-RD remains controversial22. Besides IgG4, the characteristics of the serum IgG4/IgG ratio in IBD are also obscure and need to be identified.IgG4-RD is a multiorgan inflammatory condition primarily characterized by an excessive infiltration of IgG4-positive plasma cells in the affected tissues, elevated serum IgG4 concentrations, storiform fibrosis, and obliterative phlebitis, mainly affecting the pancreas, biliary tract, liver, and the retroperitoneum of the digestive tractBased on the above evidence, we hypothesized that IBD patients with significant level of serum and/or mucosal IgG4 may possess distinct clinical characteristics and that high level of IgG4 is a biomarker for a new subtype of IBD. Therefore, we conducted a prospective case–control cohort study characterizing serum IgG4 levels, IgG4/IgG ratios, and infiltration of mucosal IgG4+ plasma cells in Chinese patients with IBD and investigated their associations with clinical characteristics. In this study, we identified a small subset of patients with IBD exhibiting high levels of serum and mucosal IgG4, and these patients had their own distinctive clinical features. All the evidence suggests that high level of IgG4 may be a biomarker for a new subtype of IBD.23, including characteristic clinical, endoscopic, radiographic, and histological findings. Patients with IBD that coexisted with other autoimmune diseases were also included. Patients who could not be differentiated between UC and CD (IBD unclassified) were excluded. Finally, the study included 232 patients with IBD (104 UC and 128 CD). A 5-ml sample of peripheral blood was collected from each enrolled patient for measurement of serum IgG and IgG4 concentrations. A second 5-ml sample was collected from patients with high levels of serum IgG4 for re-examination of serum IgG and IgG4 concentrations after various therapies. The serum IgG4 levels and IgG4/IgG ratios in patients with IBD were compared with those in 45 healthy control individuals . Among the 232 patients with IBD whose serum IgG4 levels were measured, biopsies of intestinal tissue during synchronous colonoscopy or enterectomy were obtained from 117 patients (58 UC and 59 CD) to assess the infiltration of mucosal IgG4+ plasma cells. The infiltration of IgG4+ plasma cells in patients with IBD was compared with that in 64 healthy control individuals  who underwent endoscopy with normal endoscopic and pathologic presentations. The healthy controls were age- and sex- matched to patients with IBD. Patient selection and the main study procedures are listed in the flow chart  and coexistence of other digestive autoimmune diseases (such as PSC and AIP) were also recorded. Inflammatory markers, including C-reactive protein (CRP: cutoff level at 5 mg/L) and erythrocyte sedimentation rate (ESR: cutoff level at 26 mm/h), were measured, and disease activity indexes were evaluated following the Mayo Index for UC and the Clinical Disease Activity Index for CD.Serum was extracted from fresh peripheral blood after centrifugation. Thereafter, serum IgG and IgG4 levels were assessed by an automatic immunology analyzer  following the method of scatter turbidimetry. The cutoff levels, which were set as the upper limits of the reference ranges for these markers in our institutional laboratory, were 1.50 g/L for serum IgG4 and 15.5 g/L for serum IgG. Furthermore, patients with high levels of serum IgG4 were longitudinally followed up, and serum IgG4 and IgG levels were re-examined after therapy.24.All biopsy specimens from endoscopy or enterectomy were formalin-fixed and paraffin-embedded. The hematoxylin-eosin–stained slides of gastrointestinal biopsy specimens were evaluated for histopathologic indicators of disease severity by two independent pathologists. As previously described, active inflammation was graded as mild (cryptitis), moderate (crypt abscesses), and severe (erosions or ulcerations)2O2 in PBS for 3 min, and counterstained with hematoxylin. Then two independent researchers quantified and recorded the numbers of infiltrated IgG4+ plasma cells under a high-resolution microscope. The presence of >10 IgG4 cells per high-power field  was considered significant, as previously described24.Immunohistochemistry for IgG4+ plasma cells was performed on formalin-fixed, paraffin-embedded tissue sections. The slides were immune labeled with monoclonal antibody against IgG4 to assess the infiltration of mucosal IgG4+ plasma cells (cytoplasmic staining) using a standard principle. In brief, specimens were fixed with 10% formalin and embedded in paraffin. For IgG4 staining, deparaffinized sections were rehydrated and incubated with protein block. Thereafter, sections were incubated with monoclonal rabbit antihuman IgG4  at 4 °C overnight. After washing with phosphate-buffered saline (PBS), they were treated with peroxidase-conjugated goat antirabbit polyclonal antibodies  for 30 min at room temperature, developed with 3,3-diaminobenzidine and HData were presented as the mean ± standard deviation (SD) or standard error (SE) for continuous variables and as counts or proportions for categorical variables. The statistical association between categorical and continuous variables was analyzed by independent samples t-test or Mann–Whitney test (comparing two classes) or by one-way analysis of variance (ANOVA) or Kruskal–Wallis test (comparing three or more classes), while the association between two categorical variables was analyzed by the χ2 test or Fisher’s exact test, as appropriate. Spearman correlation coefficient (r) was used to determine the correlation between two continuous variables. The statistical analyses were conducted using the SPSS version 20.0 . All statistical tests were two sided. P-values < 0.05 were considered to indicate statistical significance.The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.P > 0.05). The IgG4/IgG ratios did not differ significantly between patients with UC and CD (P > 0.05). The serum IgG4/IgG ratios in patients with both UC and CD were lower than in healthy controls . To be specific, the serum IgG4 levels and IgG4/IgG ratios in therapy-naïve patients with UC were higher than those in their post-therapy counterparts, and patients with UC taking 5-aminosalicylic acid (5-ASA) had higher serum IgG4 levels and IgG4/IgG ratios than patients taking glucocorticoids . Serum IgG4/IgG ratios were 1.50 times and 1.39 times higher in serum elevated subgroup than in serum normal subgroup for UC and CD respectively . Of course, both serum IgG4 and IgG4/IgG4 ratios in serum elevated group were higher in IBD regardless of UC or CD than in normal controls . There was no significant difference in serum IgG4 levels and IgG4/IgG ratios between patients with UC and CD in the serum elevated subgroup. One patient with UC in the serum elevated subgroup and two patients with UC in the serum normal subgroup had concurrent PSC.Of the 232 patients with IBD in this study, 23 (9.9%) had elevated serum IgG4 (the serum elevated subgroup), as compared with 209 (90.1%) patients with normal serum IgG4 . Generally, serum IgG4 levels in the serum elevated subgroup were 5.18 times and 5.60 times higher than those in the serum normal subgroup for UC and CD, respectively  not only had extensive lesions (E3) but also presented with moderate-to-severe inflammation, suggesting that this subset of patients with UC was more likely to have extensive and severe lesions Table . The serThirteen of 128 patients with CD had high levels of serum IgG4, they mainly exhibited ileocolonic (69.2%), inflammatory (61.5%), and moderately active (84.6%) lesions. Like patients with UC with elevated serum IgG4, this subset of patients with CD may also have relatively extensive lesions Table . Surpris24. We further characterized serum IgG4 in the therapy-naïve subgroup of patients with IBD. In our study, 78 patients with IBD were not placed on any treatment before enrollment. In this subgroup, the number of patients with UC was much smaller than that of patients with CD, because most patients with UC had been treated in other hospitals before being referred to our hospital. This subgroup contained 13 patients with UC  and 61 patients with CD . In general, similar to the findings in patients with IBD regardless of therapy, no significant difference among therapy-naïve patients with UC, therapy-naïve patients with CD, and healthy controls was found in both serum IgG4 levels and IgG4/IgG ratios (p > 0.05), although serum IgG4 levels and IgG4/IgG ratios were significantly higher in therapy-naïve patients with UC than in their post-therapy counterparts described above , and the serum IgG4/IgG ratios decreased in five patients , and serum IgG4/IgG ratios decreased in 10 patients  and 64 healthy controls were included to investigate the infiltration of mucosal IgG4+ plasma cells by means of immunohistochemical staining. Suppl. Table p = 0.0006). Moreover, mucosal IgG4 counts were positively associated with serum IgG4 levels and IgG4/IgG ratios in patients with UC  was seen in 39 patients with IBD (33.3%). In contrast, as discussed above, 23 in 232 patients with IBD (9.9%) demonstrated increased levels of serum IgG4 (>1.5 g/L). Generally, infiltration of mucosal IgG4+ plasma cells in the mucosal elevated subgroup was 28 times higher than that in the mucosal normal subgroup among UC patients , and 88.9% showed moderate-to-severe inflammation under microscopy. Twenty-one patients with CD had high levels of mucosal IgG4; they mainly exhibited ileocolonic (76.2%), inflammatory (57.1%), and moderately active (66.7%) lesions. As in IBD patients with high levels of serum IgG4, we can speculate that this subset of patients with IBD with high levels of mucosal IgG4 may be at risk of extensive and severe lesions than others.Among the 117 patients with IBD, eleven patients (9.4%) with IBD (four with UC and seven with CD) consistently exhibited high levels of serum and mucosal IgG4. Among the 39 patients with elevated mucosal count, 28.2% of patients with IBD with significant mucosal IgG4 counts (>10/HPF) also had high levels of serum IgG4 (>1.5 g/L); in contrast, all 23 patients with IBD with high levels of serum IgG4 had significant mucosal IgG4 counts. Two patients with CD were re-examined by colonoscopy after treatment with GC. Immunohistochemical IgG4 staining found that the infiltration of mucosal IgG4 cells in both patients was dramatically decreased Fig. .This prospective case–control study demonstrated that a small subset of patients with IBD can be characterized by high levels of serum and mucosal IgG4. These patients are generally prone to more severe and extensive lesions. The levels of serum and mucosal IgG4 decreased after treatment with GCs or other immunosuppressants. These characteristics indicate that high level of IgG4 may be a biomarker for a new subtype of IBD.25. Most previous studies focused on the infiltration of mucosal IgG4 cells in IBD, and only a small number of patients were examined for serum IgG4. One of the largest studies exploring serum IgG4 in patients with IBD was conducted by Navaneethan et al.17, who reported that approximately 8% of patients with IBD presenting with pouchitis had elevated serum IgG4. This study showed that these patients were more likely to have an antibiotic-refractory phenotype. With a similar elevated ratio of serum IgG4 (9.9%) in patients with IBD in our study, we found that patients with IBD with elevated serum or mucosal IgG4 were more likely to have extensive and severe lesions. Moreover, we surprisingly found that the serum IgG4/IgG ratios in patients with IBD were lower than those in healthy individuals, in contrast to significantly higher serum IgG4/IgG ratios in IBD patients with higher serum and mucosal IgG4 levels compared to those in healthy individuals. Sporadic studies have indicated the diagnostic value of tissue IgG4+/IgG+ plasma cells and serum IgG4/IgG ratios in IgG4-RD. Lehman et al.22 at the Mayo Clinic indicated that a moderately increased tissue IgG4/IgG ratio was not specific for cutaneous IgG4-RD, while another meta-analysis by Deng et al.20 proposed a modest effect of the tissue IgG4/IgG ratio of plasmacytic infiltration in diagnosing IgG4-RD. Song et al.21 also found that compared with IgG4 alone, combined measurement of serum IgG4 and IgG could increase the diagnostic sensitivity of AIP. Therefore, we speculated that a small subset of patients with IBD, particularly those who are characterized by high levels of serum and mucosal IgG4, may show a predominant proportion of serum/mucosal IgG4 among all IgG isotypes, underlying a monoclonal/polyclonal expansion of IgG4-expressing B cells via an unknown pathway.Patients with IBD and healthy individuals had comparable levels of serum IgG4, whereas patients with IBD had dramatically higher mucosal IgG4 counts than healthy individuals. Among patients with IBD, not all patients with significant infiltration of mucosal IgG4 cells (>10/HPF) had serum IgG4 levels >1.5 g/L. For example, immunohistochemical study found that 39 in 117 cases (33.3%) exhibited increased numbers of mucosal IgG4 cells; in contrast, serum measurement revealed that only 23 in 232 patients (9.9%) with IBD had elevated serum IgG4 levels. However, in patients with UC, mucosal IgG4 counts were positively correlated with serum IgG4 levels although quite a number of patients with significant infiltration of IgG4 cells had serum IgG4 concentrations <1.5 g/L. These differences may result from an excessive infiltration of plasma cells, particularly IgG4-expressing plasma cells, in the inflamed intestinal mucosa in a subset of patients with IBD. Another issue to be noted is that the upper limit of normal (ULN) of serum IgG4 in our study (1.5 g/L) was higher than the values defined in other studies  due to different laboratory methodset al.24 at the Harvard Medical School reported that the rate of significant infiltration of mucosal IgG4 count (>10/HPF) in patients with UC was 38%, compared with 5% in patients with CD. Mucosal IgG4 counts were significantly higher in patients with UC than in patients with CD. Virk’s study focused on a cohort of therapy-naïve patients with IBD, whereas quite a number of patients in our study who were examined with mucosal IgG4 staining had received various therapies before enrollment, particularly patients with UC. However, differences in composition between the two studies cannot explain the divergent results. It remains unclear whether there exists an ethnic difference in the distribution of mucosal IgG4-expressing plasma cells between Chinese and Western patients with IBD, underlying a difference in genetic pathogenesis. Despite similar levels of serum and mucosal IgG4 in patients with UC and CD, it was detectable in our study that patients with UC possess some distinct characteristics, such as an association between serum IgG4 levels and a history of intestinal surgery or medication use, a positive correlation between mucosal IgG4 cell counts and serum IgG4 levels, IgG4/IgG ratios, and Mayo Index. All such characteristics were absent in subjects with CD.We also found that IgG4 serum levels, IgG4/IgG ratios, and mucosal IgG4 infiltration were comparable in patients with UC and CD regardless of therapy. In contrast to our results, a study by Virk et al.26 identified four risk loci associated with various clinical phenotypes of CD. The complicated pathogenesis of IBD may partially account for current problems in treatment. To date, therapy for IBD mainly consists of 5-ASA, GC, immunosuppressants, and biological agents. Although biological agents, such as the widely used infliximab, are considered among the most promising medications, they cannot be of benefit for all patients with IBD because of problems of primary or secondary loss of response. This strongly indicates that further classification of IBD based on differences in genetic and immune pathogenesis is warranted for individual decision making and switching therapy for a given patient with IBD. The roles of IgG4-expressing plasma cells in the pathogenesis of this subset of IBD are worthy of further research, and the related signaling may contribute to personalized therapy.The pathogenesis of IBD is complex, with multiple factors involved. A strong consensus has indicated that there are substantial differences between the immune mechanisms of the two IBD subsets. For example, CD is characterized by increased production of the Th1 cytokines interleukin-12 (IL-12) and interferon γ, whereas UC is characterized by increased production of the Th2 cytokines IL-5 and IL-3. Furthermore, accumulating evidence suggests that intricate etiological differences also exist among different individuals with either UC or CD. For example, Alonso 27. Early studies identified excessive Th1- and Th2-type repertoires in the lesions of IgG4-RD, giving rise to a debate on whether Th1 or Th2 immune responses are involved in the pathogenesis of IgG4-RD30. This argument was broken by a series of studies conducted by Stone et al. at the Harvard Medical School. Their team first demonstrated that only patients with a history of allergy could exhibit expanded Th2-type repertoires, and more importantly, clonal expansion of CD4+ cytotoxic lymphocytes (CTLs) were found to be involved in the pathogenesis of IgG4-RD32. The team further demonstrated that the pathogenesis of IgG4-RD was associated with lesional infiltration of CD4+ Granzyme A (GZMA)+ CTLs, which could secrete interferon γ, IL-1β, and transforming growth factor-β1, and the ratio of these CTLs was correlated with the numbers of involved organs33. Another study by this team identified an expansion of CD19+CD27+CD20−CD38hi plasmablasts, which could be depleted by anti-CD20 treatment, in IgG4-RD patients, and the re-emergence of the de novo distinct oligoclonal plasmablasts was correlated with relapse of IgG4-RD34. Therefore, it is possible that a collaboration between unidentified specific T cells and IgG4 plasma cells/plasmablasts is involved in the pathogenesis of IBD related to high levels of serum and mucosal IgG4.So far, it still remains unknown about whether and how IgG4-expressing B cells directly function in the pathogenesis of various inflammatory autoimmune diseases. It is speculated that these IgG4+ B cells play either a pathogenic or a protective role, or that they are just secondary bystanders in this subset of IBD. What we can know, however, is that high levels of mucosal and serum IgG4 are detected in a small subset of patients with IBD who are more likely to have extensive lesions, particularly patients with UC. Furthermore, the level of serum IgG4 or tissue infiltration of IgG4+ plasma cells could decrease along with the decline in disease activity after treatment. The above observations imply that IgG4+ B cells may be involved in the immune response in this subset of IBD via an unknown pathway. Although there has been a lack of studies exploring the role of IgG4+ B cells in the pathogenesis of IBD, and the relationship between IBD and IgG4-RD is still obscure, published findings, such as the following, on the pathogenesis of IgG4-RD and IBD may suggest a potential role of IgG4 in IBD. As the most abundant inflammatory cells in the lymphocytic infiltration in IgG4-RD lesions, T cells were considered to drive the pathogenesis of the disease in IgG4-RD35. This means that it is commonly thought as being a bystander and cannot directly play a role in the pathogenesis of IgG4-RD. However, accumulating evidence indicates that IgG4 can bind FcγRI (CD64), FcγRIIa (CD32), and FcγRIIIb (CD16)39. In 2013, Uo et al.9 at Keio University identified an expansion of CXCR4+ IgG-expressing plasma cells in the affected mucosa of patients with UC. This distinct subset of plasma cells could bind FcγRI and FcγRII, and they could further induce the activation of pathogenic intestinal CD14+ macrophages in patients with UC. As described above, IgG4 can also bind FcγRI and FcγRIIa, indicating a potential involvement of monocytes/macrophages in the pathogenesis of this subset of IBD with a high IgG4 level, inducing an IgG4-immune complex-FcγR signaling in the pathogenetic mechanisms. The antigens, whether autoantigens or microbes, that drive the expansion of specific IgG4+ B cells also need to be identified in further research.For a long time, IgG4 was thought to lack pathogenic activity because of its failure to bind complement and its low affinity for Fcγ receptors (FcγR)Our study made interesting findings that a small subset of patients with IBD showed an elevated level of IgG4, and they exhibited some distinctive clinical features. This study, however, had some limitations. First, considerable numbers of patients with IBD, particularly those with UC, were on medications before their inclusion in the study. Therefore, this study could not provide a therapy-naïve overview of IgG4 characteristics for all of patients with IBD. Second, the study was conducted at a single medical center, therefore, we only detected a small number of patients with IBD who had high levels of IgG4. More prospective studies from different medical centers are warranted to further characterize this subset of patients with IBD. Third, we characterized serum and mucosal IgG4 levels in Chinese patients with IBD and identified a subset of IBD patients with high IgG4 levels. IgG4 has been widely reported as just a marker for the diagnosis of IgG4-RD in the literature. Some studies, however, recently suggest a pathogenic possibility of IgG4 in the pathogenesis of IBD. More studies focusing on the role of B cells and plasma cells in the pathogenesis of IBD are warranted.In conclusion, this study characterized a new subset of IBD patients with high levels of serum and mucosal IgG4. These patients with IBD were more likely to have severe and extensive lesions, and the high levels of serum and mucosal IgG4 decreased after treatment with glucocorticoids or other immunosuppressants. These characteristics indicate that high level of IgG4 may be a biomarker for a new subtype of IBD. Although it remains unclear whether IgG4+ B cells play a pathogenic role in this subset of IBD, it is important to explore the related collaboration between IgG4+ B cells and other immune cells, such as T cells, monocytes, and others. Elucidation of the mechanism can contribute to personalized therapy for this subset of patients with IBD.Dataset 1


This paper discussed the effects of modified metakaolin (MK) with nano-silica (NS) on the mechanical properties and durability of concrete. In the first phase, trial mixes of concrete were prepared for achieving the desired value of the 28 days compressive strength, and the charge passed in rapid chloride permeability test (RCPT). In the second phase, statistical analysis was performed on the experimental results using the response surface method (RSM). The RSM was applied for optimizing the mix proportions for the required performance by exploiting the relationship between the mix characteristics and the corresponding test results. A blend of 10% MK + 1% NS as part of cement replacement exhibited the highest mechanical properties and durability characteristics of concrete; concrete mix showed that the 28-days compressive strength (CS) was 103 MPa, which was 15% greater than the CS of the control mix without MK or NS. The same mix showed more than 40% higher flexural and split-tensile strength than the control mix; also it resulted in a reduction of 73% in the rapid chloride permeability value. ANOVA technique was used for optimizing the nano-silica and metakaolin content for achieving maximum compressive strength and minimum RCPT value. Statistical analysis using ANOVA technique showed that the maximum compressive strength and lowest RCPT value could be achieved with a blend of 10% MK and 1.55% NS. Concrete is widely used construction material for more than a century for the rapid development of the built environment all over the world. New forms of concrete have been evolved through many advances via dedicated research and development over many decades. For example, in the first evolution cycle, researchers focused on increasing the compressive strength, and now the focus has changed to improve the functional characteristics and performance of concrete. For this current evolution in concrete technology, advances in the development and application of nano-materials have played a significant role. There are multiple reasons for the need of continuous evolution of concrete technology; one of them is the concerns on the greenhouse gas emission and depletion of natural resources for the mass scale production of cement; which is the primary constituent material of concrete. It is understood that the concrete structures can withstand well the harsh environmental conditions  that may result in structural damage . CorrosiMetakaolin (MK) is another kind of CRM, which is included in the CRM group two decades ago, because it offered some added benefits over the use of the other types of CRM ,9. MetakIn the continued efforts for advancement in concrete technology, researchers anticipated that the rapid developments in the nano-science might be useful for advancing concrete to the next generation development that is focused on achieving the desired functional characteristics and sustainability objectives. In this regards, some research studies investigated the effects of nano-silica on the performance of concrete and observed a significant enhancement in the compressive strength than the controlled mix made of 100% cement . Nazri aThe principal aim of this research study was to investigate the effects of modified metakaolin using nano-silica as partial replacement of cement on the mechanical properties and durability characteristics of concrete. For this purpose, a number of trial mix proportions were prepared, and samples were cast, cured, and tested for compressive, split tensile, and flexural strength tests. Durability characteristics were investigated using the rapid chloride permeability test, and porosity using mercury intrusion porosity tests. Experimental results of all trial concrete mixes were statistically analyzed using ANOVA technique for finding an optimum combination of metakaolin and nano-silica for achieving the highest desired compressive strength and the long-term durability.2 and the median grain diameter of 20 nm. The chemical composition of cement and metakaolin are shown in Materials used in this research were Type-I ordinary Portland cement (OPC) complying the requirements of ASTM C150, metakaolin (MK) and nano-silica (NS) were supplied by a local vendor in Malaysia. Nano-silica was comprising of 99.8% of SiOResponse surface methodology (RSM) is a combination of statistical and mathematical techniques is used for analyzing a function where several independent variables influence a dependent variable called a response. The principal objective of applying RSM is to achieve an optimum outcome of the desired response. The RSM is a four-step process: (1) The design of experiments; (2) conducting an experiment for obtaining the desired response; (3) modeling of factors and response, based on experimental results; and finally, (4) optimization and validation of the model . The mosIt is discussed in the literature that using high value-added materials (such as nano-silica) in the modern concrete mix proportion is to expect a high-performance of structure throughout the service life. The term high performance is referred to achieve the highest response  with the optimum mix proportions. For this study, the desired responses were; compressive strength, porosity, rapid chloride permeability, and chloride penetration depth, whereas, metakaolin and nano-silica were considered as the influencing factors . Level o3, details of all mixes are given in In this study, initial experiments were designed using face-centered central composite design (FCCCD) approach using the Design-Expert Software. In the trial mixing and testing program; nine mixes were prepared with constant water to binder ratio (w/b) of 0.35, and the total binder content (cement + MK + NS) was set as 500 Kg/mA 100 kg laboratory mixer was used for preparing concrete mixes; the mixing operation complied the requirements of BS EN 206-1 and complementary British standard BS 8500-2. Nano-silica was directly added to concrete constituents. However, special care was taken that it should be uniformly dispersed throughout the mix. Immediately after mixing of all concrete mixes, slump test was performed according to BS 1881: Part 102 to verify the achievement of the desired slump between 100 mm to 130 mm. 3 diameter and 200 mm long cylindrical samples complying with the requirements of BS-EN 12390-6. Similarly, flexural strength was performed at the age of 28 days on 500 × 500 × 100 mm3 samples, according to BS-EN 12390-5. Hardened concrete was tested for determining the mechanical properties and durability characteristics. Cube compressive strength, splitting tensile strength, and the flexural strength was determined for investigating the mechanical properties of concrete. For compressive strength, 100 mm cubes were tested at the age of 3, 7, 28, and 90 days according to the specifications of BS-1881-116 and BS-EN 12390-3. The splitting tensile strength test was performed at the age of 28 days on 100 × 100 × 100 mm3 was sprayed on the split half to distinguish the depth of penetration, the salt solution usually turn to silver color when exposed to AgNO3 solution. Porosity, pore size, and pore connectivity is an essential property of controlling the mechanical strength and durability of concrete. For this study, mercury intrusion porosimetry (MIP) was used to investigate the pores characteristic of concrete. The test was done at the age of 28 days; approximately 10 mm size mortar cores were used for testing. Samples were tested at the oven-dry condition; for that reason, samples were dried at 100 °C for approximately 48 hours [In the second phase of the hardened concrete testing program, durability characteristics were determined using chloride migration and pore structure analysis tests. For chloride migration, rapid chloride permeability test and immersion test for measuring chloride penetration depth were performed. Whereas, pore structure investigation was done using mercury intrusion porosimetry (MIP) that measured the total porosity and median pore diameter of the samples. The rapid chloride permeability test (RCPT) was performed at the age of 28 days according to AASHTO T277 or ASTM C1202, 100 mm diameter and 50 mm thick vacuum saturated concrete discs were used. The testing rig is composed of two cells; the first cell is filled with 3% NaCl solution, whereas, the second cell is filled with 0.3 M NaOH solution, the sealed concrete disc is placed in between the two cells. The RCPT is performed at 60 volts for 6 hours, during the test the current was measured at an interval of 3 minutes; at the end of the test time versus current plot was used to determine the total charge passed, which is represented in coulombs. According to ASTM, C1202-17 total charge passed is an indication of chloride permeability of concrete. Immersion test is a static test, which takes longer time if concrete pore structure is very tight. In performing the tests, concrete discs were immersed in a 3% NaCl solution until the age of testing. At the end of immersion, the discs were split into two halves, and AgNO48 hours .Field Emission Scanning Electron Microscopy (FESEM) analysis was performed to investigate the effects of nano-silica on the microstructural properties of mortar reaction product; the analysis also determined the characteristics of the interfacial transitional zone (ITZ) of the mortar samples cured for 28 days. For this study, FESEM model Zeiss Supra 55 VP instrument was used. The samples were cut using a diamond cutter, which was supposed to be conducive for full observation. Therefore, the samples were coated accordingly prior to FESEM analysis; they were coated with gold atoms in a sputter coater. The coated samples were then placed in the vacuum chamber inside the FESEM testing equipment.In the first phase, the effects of nano-silica and metakaolin were investigated on the mechanical properties . The experimental results are discussed in the following sections.Combination of MK and NS showed a reasonable increase in the flexural strength of concrete. Combination of 10% MK and 1% NS resulted in 43% and 32% increase in the flexural strength respectively than that was obtained for the control mix and mix (C-MK-10%). However, the addition of NS more than 1% showed a slight decrease in the flexural strength. A similar trend was observed for the tensile strength of NS/MK concrete. It can be concluded that a combination of 10% MK and 1% NS caused a significant improvement in flexural strength, as well as tensile strength, which can be termed as an optimum. Kim et al.  discusseIn the next phase of the study, the effects of nano-silica and metakaolin were studies on the durability characteristics of concrete. There were various tests conducted that can indicate the level of durability of concrete. In the following sections, results of various durability tests are discussed.Water absorption test gives an indirect indication of concrete durability; the faster and deeper penetration of water will be an indication of inadequate durability. Similarly, the combination of MK and NS also caused a significant reduction in water penetration depth. An addition of 1% and 2% NS with 5–10% replacement of MK has shown 27% and 54% reduction in penetration depth respective as compared to the penetration depth obtained in the control mix. Results showed that the addition of nano silica has higher compatibility with 10% of metakaolin on the reduction of water absorption in concrete.The rapid chloride permeability (RCP) test for all concrete mixes was carried out at the age of 28 days; Measurement of chloride penetration depth in concrete is a static test that represents the real-life situation. Chloride penetration depth determines the concrete cover requirements for achieving the desired service life of the structure. The chloride penetration depth, CPD was measured after three months immersion of concrete disc in 3% salt solution. CPD of the control mix was obtained as 11.83 mm, and for 5% and 10% MK concrete, it was measured as 5.80 mm and 9.00 mm respectively, the CPD 48% and 19% lower than the control mix. As discussed above that 1% and 2% dosage of NS with 10% MK caused a significant reduction in RCPT value, which trend is also shown in the reduction of CPD, which was reduced to 71% and 74% respectively with respect to the control mix CPD. This technique is preferred when assessing the porosity and pore size distribution of cement paste and concrete. The concrete pores are classified into three categories; pores in the range of 50> nm, 10–50 nm and <10 nm, are designated as “large capillary pores,” “medium capillary pores” and “gel pores” respectively. The difference in pore size leads to different effects on concrete strength and durability performance . The cap2 and at the same time acted as nucleation sites, leading to the better development of hydration products in such position [position .The effects of metakaolin with and without the combination with nano silica on the microstructure were investigated using FESEM. 2) is a statistical measure of how close the data are to the fitted regression line. R-squared is always between 0 and 1. In general, R-squared closer to 1 indicates the effectiveness of the model. They reported a good correlation was obtained between compressive strength and dry density, whereas, not very strong correlation found between compressive strength and water absorption (R2 was obtained as 0.63). Ramezanianpour et al. [2 was obtained as 0.814). To establish statistical correlation among various responses obtained from experimental tests is a widely used approach by the researchers for the appraisal of experimental findings ,43,45,46r et al.  investig2 was obtained as 0.93). Such correlation obtained in this study is found better than that reported in previous researches [2 is obtained as 0.82) and porosity versus depth of chloride penetration (R2 was determined as 0.88). The experimental program in this study included various concrete tests, the test results are used for a polynomial regression between various properties of concrete, and the related correlation was developed, searches . Similarp is a property of concrete attained from experimental results, i.e., compressive strength, RCP test, chloride penetration depth and porosity, MK and NS are independent linear parameters, (NS2) and (MK2) are square parameters, (NS×MK) is the interaction parameter, C is a constant, and A1 to A5 are the coefficients of equation.ANOVA technique was used to perform the statistical analysis of experimental data for onwards application in the response surface methods to determine the optimum mix parameters. The outcomes of ANOVA were optimized by widely accepted RSM techniques ,48. The The coefficients showed in In this research work, the chloride permeability, chloride penetration depth, and porosity were decreased with a higher content of metakaolin and its combination with nano-silica. The relationship between the content of MK and NS and their effects on chloride permeability, chloride penetration depth, and porosity are shown in It is not easy to get an optimal solution of all responses simultaneously; therefore, a multi-objective optimization technique was utilized for this study. It is a robust technique for the optimization of all responses at the same time. The optimization was carried out by maximizing the compressive strength while also minimizing the porosity, chloride permeability, and chloride penetration depth. As mentioned, the dependent variables are tested properties of concrete, while two independent variables are proportions of MK and NS. RSM was employed in order to find optimum ratios for maximizing the compressive strength and minimizing the porosity, chloride permeability, and chloride penetration depth. In order to optimize the response, a statistical RSM with multiple responses affected by multiple factors was conducted. The factors and responses are presented in Additional experiments were performed to verify the outcomes obtained from the theoretically obtained response. The primary reason to perform additional experiments is to recognize whether the optimum values could provide minimum chloride permeability, porosity, penetration depth, and the maximum compressive strength by considering the same concrete recipe. The specimen was prepared and tested at the age of 28 days. From the results and discussions, the following conclusions have been made: Modified metakaolin (5% to 10%) with 1% or 2% nano-silica content showed a significant enhancement of the mechanical properties of concrete. Increase in compressive strength was observed from 88.3 MPa to 103.6 MPa for the blending proportion of 10% MK with 1% NS, hence, obtained as the best combination. Such combination also caused a significant improvement in the splitting tensile strength  and flexural strength  as compared to that of the control mix was observed.The modified metakaolin using nano-silica has tremendously increased the resistance against the penetration of chloride in concrete. The best combination (10% MK + 1%NS) showed a 74% lower value of the chloride penetration depth as compared to the controlled mix. The same combination resulted in the 56% less porosity, a 62% decrease in water absorption, and a 13% reduction in the median pore diameter when compared with the similar performance criteria of the control mix. From the FESEM analysis, the addition of MK and NS revealed the significant enhancement in the microstructure of concrete. ANOVA models of the compressive strength, charge passing, chloride penetration depth, and porosity for MK and NS concrete have been developed and statistically validated. All models were significant and ready to use for the prediction of concrete properties.Based on multi-objective optimization using RSM; the optimum combination of modification was obtained as 10% MK + 1.55% NS. In experimental validation, the optimum combination proved in resulting highest compressive strength and the lowest Porosity, chloride permeability, and chloride penetration depth with the mean error of 2.0%.


Results showed that the compressive strength of carbonation-cured concrete was more than 10% higher than that of moisture-cured concrete at the same age; a steel bar is stable in carbonation-cured concrete; and carbonation-cured concrete exhibited better abrasion resistance and chloride ion permeability than that of moisture-cured concrete. The optimization of pore structure and improvement in the micro-hardness are the reasons for the improved chloride ion permeability and abrasion resistance of carbonation-cured concrete.The research shows that carbonation-cured concrete has several mechanical and durability properties that are better than those of moisture-cured concrete. However, many properties of carbonation-cured concrete have not yet been studied. In this research, carbonation-cured concrete was prepared by pre-curing, carbonation curing, and then moisture curing. The compressive strength, CO In recent years, various forms of precast concrete have been used, for example as components in prefabricated buildings  and urba2, eventually becoming a silica gel and losing its binding capability in the process of natural carbonation. However, carbonation curing of concrete means that the concrete is cured in a high-pressure atmosphere of pure CO2, and the unhydrated cement minerals react with CO2 to solidify it in concrete in the form of carbonate. Hence, the hardening process of concrete is accelerated and its mechanical and durability properties are improved [Carbonation curing is a new method of curing that is different from the natural carbonation of mature concrete. As the main hydration product in mature concrete, calcium-silicate-hydrate (C-S-H), reacts with atmospheric COimproved ,13,14,15It is well known that the carbonation reaction lowers the pH value of concrete and may cause corrosion of steel . In ordeThis study aimed to examine the above problems, by investigating the pH value, chloride ion permeability and abrasion resistance of carbonation-cured concrete. By comparing the differences in pore structure and micro-hardness between carbonation-cured and moisture-cured cement paste, the enhancement mechanism of anti-chloride ion penetration and the abrasion resistance of carbonation-cured concrete was explained.The cement used in this study was ordinary Portland cement (OPC), and its chemical composition is shown in 2 in the concrete and reduce the degree of carbonization [2 with a purity of 99.9% for 12 h. The fourth step was conventional curing. The carbonation-cured sample was cured for 6 days or 27 days in a curing box with a temperature of 20 ± 2 °C and humidity of ≥ 95%. The carbonation-cured concrete on the 1st, 7th, and 28th day of aging were named C1d, C7d, and C28d, respectively. Moisture-cured concrete was used as reference, and the curing conditions were: temperature 20 ± 2 °C, RH ≤ 95%. They were named H1d, H7d and H28d according to their age.The water-cement ratio is a key factor affecting the workability and carbonation of concrete. When the water-cement ratio is relatively high, although the concrete has good fluidity, excessive free water will hinder the diffusion of COnization ,25. Whennization ,27. The In order to study the mechanism for enhancing the strength and durability of carbonation-cured concrete, paste samples were also formed. The sample size was 40 mm × 40 mm × 40 mm. The water-cement ratio of the paste sample was 0.35. The curing method of the paste samples was consistent with that of the concrete samples.The samples that were cured at a corresponding age were tested for compressive strength, which was taken as the average value of three test pieces. The strength values were tested using a WAY-2000 electro-hydraulic material testing machine .final is the mass of concrete after carbonation curing, minitial is the mass of concrete before carbonation curing and after initial curing, mevaporated is the water evaporated during the carbonation curing process, which was collected on the walls of the pressure vessel through the absorbent paper after the carbonation curing was completed, and mcement in sample is the mass of cement in the sample; the above parameters were in units of g.The carbon sequestration of carbonation-cured concrete was calculated using two methods. The first method calculated the carbon sequestration by the mass change in the carbonation-cured concrete, as shown in Equation (1):3 to produce CO2. Therefore, the equation for calculating the carbon sequestration of the sample is as shown in Equation (2):The second method calculated the carbon sequestration by thermal analysis. Due to the different degrees of carbonization at different depths of the sample, 50 g of mortar samples without coarse aggregate was taken from each of the five layers of the samples: 0–10 mm, 10–20 mm, 20–30 mm, 30–40 mm, 40–50 mm. The mortar sample with a total mass of 250 g was ground and heated consecutively to 105 °C, 500 °C and 1000 °C. After heating, the sample was taken out and cooled to room temperature. After the sample was weighed, it was then heated in a high temperature furnace to the next target temperature. The difference between the mass of the sample at 500 °C and the mass at 1000 °C was due to the decomposition of CaCOTwo methods were used to test the pH of the concrete section after splitting. The first method used ethanol phenolphthalein solution to spray the surface of the cracked concrete. When the pH was lower than 9.5, the concrete surface did not change color, and when the pH value was greater than 9.5, the concrete surface turned purple. The second method used the PH110  with a flat head sensor to test the pH. After the cube test piece was split, a piece of 10 mm × 10 mm absorbent paper was put on the point where the pH was to be measured, and then 100 μL of distilled water was dripped onto the absorbent paper. The absorbent paper was closely attached to the concrete section. After 15 min, the distilled water and the concrete pore solution were fully ion exchanged to reach equilibrium. The pH value of the absorbent paper was measured as the pH of the measuring point of the concrete ,28.Electric flux is an indicator for evaluating the corrosion resistance of concrete against chloride ions and can reflect the compactness and durability of concrete to some extent ,30. In oThe abrasion resistance of concrete samples was tested in a self-designed experimental device ,23. The In order to compare the difference in abrasion resistance between carbonation-cured concrete and moisture-cured concrete, samples at 28 days were selected for abrasion resistance testing. The specific parameters of the test are shown in A sample of the pulverized pulp with a volume of about 10 mm × 10 mm × 10 mm was taken and dried in a vacuum drying oven at a temperature of 65 °C for more than 48 h for the pore structure test. The pore structure was tested using a Autopore 9620 mercury intrusion meter . The mercury intrusion analyzer had a maximum pressure of 25 psia for low pressure analysis with a low pressure pore size analysis range of 360–3.6 μm. The mercury intrusion analyzer also had a maximum pressure of 60000 psia for high pressure analysis with a high-pressure pore size analysis range of ≥ 3 nm, and a volumetric accuracy of 0.1 μL for entering mercury and withdrawing mercury.A 10 mm × 10 mm ×10 mm sample was cut from the inside of the paste block and polished stepwise using a EcoMet 300 polisher . After polishing, it was placed in absolute ethanol to terminate hydration for more than 48 h. Then the sample was put in a vacuum oven and kept at 65 °C for more than 48 h. The micro-hardness of the sample was measured using a HXS-1000A digital intelligent micro-hardness tester . The test load can be set from 10 to 1000 gram of force, the force retention time range is 1–99 s, the objective magnification for measurement is 40 times, and the magnification of the eyepiece is 10 times. The load and load time were set to be 100 gram of force and 10 s, respectively. 2 uptake of carbonation-cured concrete is shown in 2 uptake is 14–15%. The data measured by the mass change method differs from the data measured by the thermal analysis. Evaporation of moisture in the mass change method may not be fully collected, resulting in data that may be less than the actual value. According to the above pH data, the degree of carbonization in different parts of the carbonation-cured concrete is different. Therefore, when the CO2 uptake of the concrete is calculated by the thermal analysis method, samples from the surface to the internal five layers are taken to ensure the accuracy of the calculation. The amount of CO2 uptake calculated by the thermal analysis method is slightly larger than that calculated by the mass change method. This is because the CO2 decomposed by the carbonate mineral in the cement is also included in the CO2 uptake amount, resulting in a value that is higher than the actual value. The CO2 uptake of the carbonation-cured concrete at 28 days obtained by thermal analysis was 15.8%, which is slightly higher than the 15.1% at 1 day. This is because the sample also undergoes a carbonization reaction in the process of conventional curing, hence, the amount of CO2 uptake increases.The CO2 diffusion, so the carbonization reaction takes place within this range. However, due to the hindrance of moisture in the pores, CO2 failed to reach the range of 30–50 mm, so a carbonization reaction did not occur in the core region of the concrete test block. It can also be seen from the data in 2 with a high pH value is produced, so the pH value of the sample increases. The pH value of each layer of the carbonation sample was greater than 12.0.The variation in the mass loss of concrete with the number of abrasion cycle under different angles is shown in The total mass loss after six cycles of abrading of the sample is shown in Since the data for micro-hardness is highly discrete, the data was processed using a box plot, and the processed data is shown in 2 uptake, chloride ion permeability and abrasion resistance of the carbonation-cured concrete were studied. By comparing these characteristics in carbonation-cured concrete and moisture-cured concrete, and analyzing the pore structure and micro-hardness of the concrete paste, we were able to make the following conclusions:In this study, carbonation-cured concrete was prepared by pre-curing, carbonation curing and then moisture curing. The strength, pH value, CO(1) The strength of carbonation-cured concrete is more than 10% higher than that of moisture-cured concrete at the same age.(2) The steel bar in carbonation-cured concrete can exist stably. However, considering the low pH value in the surface layer at the initial stage of carbonation curing, the thickness of the steel protective layer of carbonation-cured concrete should be deeper than that of moisture-cured concrete.(3) Carbonation-cured concrete exhibited better abrasion resistance than that of moisture-cured concrete.(4) Carbonation-cured concrete had better chloride ion permeability than moisture-cured concrete.(5) The pore structure was optimized after carbonation curing, thereby improving the strength and impermeability of the cement paste to some extent.(6) The abrasion resistance of carbonation-cured concrete was better than that of the moisture-cured concrete because it has greater micro-hardness and compressive strength.


Streptomyces. The produced substances include all important drug classes used in clinics today, such as β-lactams, tetracyclines, macrolides, aminoglycosides, or glycopeptides. However, in the past years the effectiveness of these impressive weapons have become endangered by the rise of resistances of live-threatening pathogenic bacteria. In addition to that, the golden era of antibiotic discovery has been over for quite a while and the antibiotic pipeline was feared to run dry. Next-generation sequencing techniques in combination with genome mining approaches revolutionized the field of antibiotic research and might reboot the pipeline in the near future. In 2002, the first Streptomyces genome sequence was published [Streptomyces coelicolor. Mining this sequence revealed that S. coelicolor harbors 22 secondary metabolite gene clusters but indeed produces only four of the encoded metabolites under standard lab conditions. Currently, more than 625 genome sequences are available only of the genus Streptomyces [Streptomyces and, with actinomycin being the first antibiotic that was isolated from an actinomycete, these bacteria still represent a treasure chest for the identification of novel antibiotics. Actinomycetes are well known as an inexhaustible source for antibiotics. Most of the known antimicrobials today were originally isolated from actinomycetes, especially from the genus ublished . This waptomyces , which iNovel cultivation strategies, elaborated screening techniques, new genetic manipulation tools, further insights into physiological aspects of actinobacterial live style, and also knowledge on new secondary metabolite biosynthetic pathways may open up a new era of antibiotic drug discovery. In the Special Issue of "Actinomycetes: The Antibiotics Producers" we will highlight the latest research findings in the field. The current issue brings together 19 publications (9 research articles and 10 reviews) from expert groups, which address scientific topics associated with the biology of actinomycetes as well as their antibiotic biosynthesis capacity.Macrotermes natalensis and analyzed them in terms of their antibiotic production properties. Thereby, the authors isolated several new actinobacterial species and identified a dichlorinated diketopiperazine derivative and two tetracyclic lanthipeptides (rubrominins A and B) as new secondary metabolites [Streptomyces actinomycetes represent good sources for novel natural compounds. This is also underpinned by the study of Iorio et al., which specifically focusses on the underexplored genus Actinoallumurus, for which two novel polyether substances (α-770 and α-823) were described [Two research articles deal with the isolation and characterization of actinobacteria from unique habitats. Using a high-throughput sequencing approach, Maciejewska et al. were able to show that there is a quite complex actinobacterial community present in cave moonmilk deposits obtained from the cave “Grotte des Collemboles”, Belgium . By applabolites . Howeverabolites . Indeed,escribed ,10. Thesescribed . Finallyescribed . Streptomyces albus. Viana Marques et al. provide an update on β-lactam inhibitors and their applications [Besides the studies that aim to identify novel antibiotics, the Special Issue also contains four papers dealing with specific antibiotics and their biosyntheses: Schwarzer et al. report on the biosynthesis of the polyketide rishirilide B , whereasications , whereasications . Streptomyces tsukubaensis [Nonomuraea sp. [Especially when it comes to engineering and production attempts of the respective compounds, regulation and self-resistance of the producer organisms are important factors to be targeted in order to increase yields. Thus, aspects of regulation and self-resistance are also addressed in the Special Issue: Ordóñez-Robles et al. summarize the current knowledge on regulation of tacrolimus biosynthesis in ubaensis . Alduinaraea sp. , whereasraea sp. .Streptomyces differentiation in liquid cultures with the aim to elicit antibiotic production [Streptomyces lividans [Streptomyces, which exerts a dual function as 3′-5′-exoribonuclease and RNA 3′-polyribonucleotide polymerase [Besides their overwhelming importance as antibiotic producers, actinomycetes undoubtedly have further imposing characteristics, and some of them are handled in the Special Issue. For example, actinomycetes undergo a complex life cycle, whereby morphological development and secondary metabolism often coincide. Manteca and Yagüe review strategies to improve oduction . Furtherlividans . Finallylymerase . Antibiotics.We are happy to bring together so many valuable works from respected experts in the field. It was our great pleasure to be Guest Editors of this special issue of


Evidence-based medicine relies on current best evidence from the medical literature, the patient’s history, and the clinician’s own experience to provide the best care for patients. Systematic reviews and meta-analysis are considered the highest levels of evidence for informing clinical decisions. Recently, reports have shown an increase in the number but a decrease in quality of meta-analysis publications. We reviewed publication trends and determined the countries with the most journal articles and types of publications in PubMed from 1995 to 2015.We examined journal entries in PubMed from 1995 to 2015 from top publishing countries for total number of publications and citations in core clinical journals and in specific publication types .Yearly, only 30 countries generated 94.6% of all publications and 98.1% of core clinical journals worldwide. All publication types increased but with a significant increase in meta-analysis publications from China. Collaborative and co-authored papers among the 30 countries also showed an increasing trend.The USA leads in all publication citations and specific publication types, except for meta-analysis where China publishes more. Collaborative publishing among international collaborators is also increasing. Recent best evidence from the literature and health care providers experience form the foundation of evidence-based medicine (EBM) . In the The publication type category in the MeSH translation table represents the article’s type of material  . The pubThis paper is an attempt to review recent trends of some publication types in PubMed. It is not an assessment of the quality of journal articles themselves but rather a quantitative look at recent publication trends from top research countries worldwide.n = 235) based on IPv4 Internet address allocations . The top publishing countries was generated from searches of all publications in PubMed for the year 2015. An example of a country search query for Germany is in PubMed searches were performed in December 2016 using the following limits: all publications sorted from top publishing countries from 1995 to 2015 and the following search strategies and publication types: meta-analysis, systematic reviews, clinical trials, and randomized controlled trials. A list of countries was created n = 25 based ohttps://www.nlm.nih.gov/pubs/techbull/so13/brief/so13_author_affiliations.html). For example, both “UK” and “United Kingdom” and “Russia” and “Russian Federation” were included in the affiliation filter to search for publications from the UK and Russia, respectively. Changes in indexing of affiliation information were instituted by the NLM in late December 2013 that required a modification of the search algorithm (https://www.nlm.nih.gov/pubs/techbull/so13/brief/so13_author_affiliations.html). For the USA, the search algorithm was modified by adding all the names of the 50 states in the USA to compensate for this change in practice. Filter algorithms were continuously revised when “unexpected” results were encountered, such as, when entries from New Mexico state were included in results from Mexico, the country , which was second highest (Table The Abridged Index Medicus (AIM) or “Core Clinical,” a subset of MEDLINE,  which cuA review of meta-analysis and systematic review publications from the top 10 publishing countries are shown in Fig. Since “Systematic Review” is not a publication type filter in PubMed, but rather, a subset filter, the search algorithm was modified by including the title and “text word” combination to find articles of that type. The modified filter for systematic reviews was “(systematic review [tw] OR systematic review [tiab] OR meta synthesis [ti] OR cochrane database syst rev [ta] OR systematic literature review [ti] OR pooling project [tw] OR umbrella review [tw])” . Figure Co-authored publications with the US collaborators account for the highest percentage among the top 30 countries . Figure Recent reports of the rapid increase in meta-analysis publications from China prompted this study –7. Our rSome reports cite evidence-based medicine as the stimulus for the rapid rise in the number of meta-analysis publications in China  and to tInternational collaboration improves the quality of research for all partners although it has some challenges as well . The grohttps://www.nlm.nih.gov/pubs/techbull/so13/brief/so13_author_affiliations.html). For example, many publications from the USA only list the states or even only the academic institution or the organization where they originated. After 2013, PubMed discontinued adding “USA” in the affiliation field. In our search algorithm, we added all the states in the USA to compensate for this. Some publications from China, Russia, or the Russian Federation that may have listed only their academic affiliations or cities would have missed their country affiliation. Unlike the authors in the USA, we cannot compensate by adding the names of all the states for China and Russia. However, a random search of over 200 articles from Russia and China all showed their countries in the affiliation field. It is unlikely, but this is still a possible limitation of the study.Although we attempted to include all publications from the 235 countries with citations in PubMed, we may have missed some publications because some co-authors’ affiliations may not have been indexed in PubMed before 2013. Other reasons may include NLM indexing affiliations of authors only if provided by publishers in their XML submissions; association of authors with multiple institutions and not listing all affiliations; and discontinued editing of author affiliation field in MEDLINE/PubMed citations. In the past, NLM policy was to indicate the affiliation for the first author only and edited the field by adding “USA”  are responsible for an average of 93.9% of all publications in PubMed. What might explain the few numbers of countries responsible for a majority of the world’s literature? It is likely a consequence of country priorities, lower levels of funding, and the high cost of doing research, although labor costs may be lower . BiomediWe had also considered whether the rapid increase of publications might be explained by so-called predatory journals—low-quality journals with little or no peer review, which promise rapid publication but exist solely for profit . HoweverThis study showed the continuing increase in research publications globally. The USA still leads the world in all publications and specific publication types except in meta-analysis where recently China now publishes more. Increasing collaborations and authorships among countries with the USA, China, and other top publishing countries is also increasing.


Carbon fiber-reinforced polymer (CFRP)-confined foam concrete can be applied in structure protection, e.g., as an impact barrier of bridge piers, in which it is used as the core of the composite impact barrier. Applying CFRP to the foam concrete exterior enhances both the CFRP and the foam concrete, leading to improved compressive performance due to their interaction. In the present study, the carbon-fiber reinforced polymer (CFRP) confining effect on the response and energy absorption of foam concrete subjected to quasi-static and medium-strain-rate dynamic compression was experimentally investigated. The confinement by CFRP changed the response and failure mode of foam concrete specimens from shear in quasi-static load and splitting in dynamic load to crushing, resulting in a significant increase in the load bearing and energy absorption capacity. The composite consisting of CFRP and foam concrete was sensitive to strain rate. In particular, the CFRP–foam concrete interaction led to the remarkably improved resistance and energy absorption capacity of CFRP-confined specimens, which were significantly higher than the sum of those of standalone CFRP and foam concrete. The application of carbon fiber-reinforced polymer (CFRP) on foam concrete exterior remarkably improves its crushing resistance due to the confinement effect, leading to significantly increased energy absorption capacity. In engineering practice, CFRP-confined foam concrete can be applied in structure protection, e.g., as an impact barrier of bridge piers, in which it is used as the core of the composite impact barrier. The crushing absorbs a considerable amount of energy, and, at the same time, it reduces the load transferred to both the bridge and the vehicles. Moreover, the energy absorption capacity and crushing resistance can be altered by changing the foam concrete density.Cellular solids are a group of materials with a large portion of air pores inside the base materials, usually categorized as open-cell and close-cell types due to macrostructure. While some cellular solids are ductile , some cellular solids are brittle (foam concrete and foam ceramic). In addition to their superior properties such as being lightweight and low thermal conductivity, they experience a relatively long stress plateau of nearly constant stress level with increasing strain when subjected to compression, implying excellent capacity in energy absorption ,2,3,4. TFoam concrete is traditionally used as a thermal insulation material  and non-3 and polypropylene fibers can reach up to 50 MPa [The preparation and quasi-static performance of foam concrete was extensively studied ,18, whico 50 MPa , even hio 50 MPa . At the o 50 MPa .The increasing applications of foam concrete in load bearing-related applications require a thorough understanding of its response under compression load, both static and dynamic. While the performance of foam concrete subjected to quasi-static load was extensively investigated , its beh3 /s. While the specimens can be loaded with almost constant strain rate if properly designed, due to the mechanism of SHPB, it is difficult to obtain the complete stress–strain relationship incorporating the long stress plateau [The dynamic characteristics of materials including foam concrete can be investigated with testing facilities of varying loading rate. In addition to the servo-hydraulic universal machine for quasi-static and very-low-speed loading, SHPB is capable of compressively loading specimens with a relatively high strain rate, up to 10 plateau . In the In the present study, the response, failure mode, and energy absorption of foam concrete with and without CFRP confinement subjected to quasi-static and dynamic loading were experimentally investigated and compared. Under different loading rates, the crushing strengths of foam concrete and CFRP wrap were tested separately. Then, the CFRP-confined foam concrete was tested quasi-statically and dynamically with corresponding loading rates to investigate the strengthening effect due to the CFRP–foam concrete interaction in terms of crushing strength. Subsequently, the energy absorption of foam concrete with and without CFRP reinforcement subjected to various loading rates was examined, and the factors determining the strengthening effect were identified and discussed.3, 500 kg/m3, and 600 kg/m3. The size of the specimens was determined based on two factors. On one hand, the specimens should not be big, to make sure the loading capacity of the high-speed testing machine is not exceeded, especially under relatively high loading speeds such as 10 m/s. On the other hand, the specimens should have an adequate height-to-diameter ratio, to eliminate the friction end effect from the crosshead and the supporting anvil, according to St. Venant’s principle. In fact, it is difficult to accurately control the density of the foam concrete during the manufacturing process. Therefore, in the analysis, the density of each specimen is provided to facilitate comparisons of crushing strength and energy absorption.In the present study, the foam concrete was made by mixing C30 cement with a plant protein foaming agent , without any other ingredients. Foam concrete was produced under controlled conditions from cement, water, and a liquid chemical that was diluted with water in a volume ratio of 1:40 and aerated to form the foaming agent. The cement used in the tests was common Portland cement of compressive strength 30 MPa. A pre-foaming method was used to produce foam concrete comprising the produced base mix and stable pre-formed foam, which were blended thoroughly. The 100-mm cube standard blocks were cured for 28 days. All specimens were of the same geometry and size: a cylinder of diameter 40 mm and height 80 mm, cut from 100-mm cubes, as shown in The CFRP of uni-axial tensile strength 3800 MPa and elastic modulus 230 GPa was manufactured by Nanjing Haituo Company, one of the major CFRP-related product providers in China. The CFRP-reinforced foam concrete specimens were made by wrapping and gluing the CFRP around the cylindrical foam concrete specimens, whose overlapping area was half the cylinder circumference.The foam concrete with and without CFRP reinforcement, as well as the CFRP wrap alone, was subjected to both quasi-static - and dynamic compression. MTS Exceed E45  with a maximum loading capacity of 300 kN in the structure lab of Beijing University of Technology, shown in The plateau stress and densification strain are two important parameters for cellular solids such as foam concrete, determined as shown below. The energy absorption efficiency is widely accepted to be defined asThe maximum of the efficiency with respect to strain is determined asThe strain corresponding to the maximum energy absorption efficiency is defined as densification strain. The specific energy absorption is calculated as the area below the stress–strain relationship from zero to densification strain. In the present study, the discrete stress–strain data in pairs were used to calculate the specific energy absorption. Specifically, summing up all the products of stress readings and the corresponding small strain increment yielded the specific energy absorption. Subsequently, the plateau stress was calculated as the ratio of the specific energy absorption to densification strain.The test results of foam concrete with and without CFRP reinforcement subjected to quasi-static compression are listed in The foam concrete cylinders were compressed, and the quasi-static stress–strain relationships are shown in The typical response and failure mode of foam concrete is shown in As the confinement or constraint induced by the CFRP enhances the plateau stress, it is interesting to check the further enhancement if the single-layer CFRP is changed to a double layer. According to the test result shown in The standalone CFRP wrap, without any filling, was compressed quasi-statically, whose load-bearing capacity is indicated by the red dashed line in There were two major mechanisms for the remarkably improved plateau stress. On one hand, due to the confinement from the CFRP, the splitting along the loading direction and shear cracking was suppressed, and the failure was dominated by the crushing mode, resulting in higher resistance. On the other hand, with respect to the energy absorption, the interaction between the foam concrete and fiber wrap expanded the CFRP circumferentially, in which a considerable amount of strain energy was stored.When it comes to the foam concrete with and without CFRP subjected to dynamic compression, the response becomes more complicated due to varying loading rate. Based on the fact that the stress of foam concrete increases sharply with increasing strain when the densification strain is reached, while taking into account that the strain rate effect induced higher resistance, the nominal strain of dynamic compression on foam concrete specimen with and without CFRP reinforcement was determined as 0.5, to avoid overloading the testing machine.−1), the INSTRON VHS 160/100-20 can load specimens with intermediate strain rate in between. One of the advanced features of this machine is that, with a specially designed fixture, the specimens can be compressed with  constant speed from the initial loading. In the present study, the loading speeds were chosen as 1 m/s, 5 m/s, and 10 m/s, for all foam concrete specimens with and without CFRP reinforcement. The test results are in Unlike the universal testing machine with quasi-static loading and split Hopkinson pressure bar with relatively high loading rate  constant loading rate, i.e., from t0 to t1 for History 1 or from t0 to t2 for History 2. After that, the crosshead decelerates and finally comes to a stop at ts, during which the loading speed is not constant. The compression stroke for all specimens is designed the same regardless of the constant loading velocity, i.e., the area from t0 to ts remains fixed in Figure 13, which implies that the time of constant loading for higher velocity should be shorter than that for lower velocity. This mechanism accounts for the observation that, in the initial loading stage, the plateau stress under 10 m/s was significantly higher than that under 5 m/s, while, in the advanced loading stage, the plateau stress of 10 m/s was similar to or even lower than that under 5 m/s. In fact, the initial loading stage corresponded to the constant rate loading, while the later stage corresponded to the decelerated rate loading.Limitations do exist in the present experimental study. As stated previously, since the loading capacity of the dynamic testing machine 100 kN cannot be exceeded and a certain safety margin must be applied in the test, the compression distance of all specimens was determined as 40 mm, half the original specimen height. Further insight into the loading velocity control of the testing machine indicated the limitation of the “constant rate loading” in practice, as shown in The stress–strain relationship subjected to 1-m/s compression is shown in 3 foam concrete with and without CFRP reinforcement subjected to 5 m/s crushing was taken as an example. With respect to the response and failure mode, the 600 kg/mSubjected to dynamic load, the interaction between the foam concrete specimen and CFRP is indicated in With increasing loading rate, the response and plateau stress of foam concrete with and without CFRP reinforcement was compared, as shown in In the analysis of the foam concrete behavior subjected to quasi-static and dynamic loading, the energy absorption with and without CFRP reinforcement was calculated with a pre-defined nominal strain of 0.5, rather than the actual densification strain corresponding to the maximum energy absorption efficiency. Therefore, for those with a densification strain greater than 0.5, the energy absorption had the same characteristics with the plateau stress, as the concerned specific energy absorption (defined as the energy absorption divided by the undeformed volume of specimen) was simply the integral of the stress over the strain from zero to 0.5. For those with densification strain smaller than 0.5, generally foam concrete of relatively high density or with CFRP reinforcement, the actual densification strain should be used for calculating the specific energy absorption. In fact, in addition to absorbing a considerable amount of energy, the foam concrete should control the transferred load to a designed level regardless of strain in the application range, to facilitate protective structure design in engineering practice.While foam concrete is relatively cheap, CFRP is relatively expensive in structural engineering applications. However, applying CFRP significantly improves the protection performance. To achieve the same load bearing and energy absorption capacity, the amount of foam concrete usage can be remarkably reduced if CFRP is applied to the exterior of the foam concrete, which has multiple merits. On one hand, the core size of foam concrete protective structures is reduced, facilitating the design of such structures. On the other hand, it is more environmentally friendly as the usage of concrete is reduced.Foam concrete with and without CFRP reinforcement subjected to quasi-static and dynamic compression was experimentally investigated, in which the resistance and energy absorption capacity were examined. From the test results, the following findings can be reached:1. The response and failure modes of foam concrete varied from shear and splitting without CFRP to crushing by applying CFRP confinement;2. The resistance and energy absorption capacity improved with increasing loading rate, regardless of whether the foam concrete was confined with CFRP or not;3. The confining effect induced by the CFRP–foam concrete interaction significantly improved the compression resistance and energy absorption capacity; the compression resistance increased by approximately 10-fold, and the specific energy absorption within a strain of 0.5 increased by approximately 10-fold.4. The compression resistance and energy absorption capacity of CFRP-confined foam concrete were approximately 300% higher in quasi-static loading and 150% higher in dynamic loading than those of standalone CFRP and foam concrete combined.


Antibiotic resistance is a global health concern and a current threat to modern medicine and society. New strategies for antibiotic drug design and delivery offer a glimmer of hope in a currently limited pipeline of new antibiotics. One strategy involves conjugating iron-chelating microbial siderophores to an antibiotic or antimicrobial agent to enhance uptake and antibacterial potency. Cefiderocol (S-649266) is a promising cephalosporin–catechol conjugate currently in phase III clinical trials that utilizes iron-mediated active transport and demonstrates enhanced potency against multi-drug resistant (MDR) Gram-negative pathogens. Such molecules demonstrate that siderophore–antibiotic conjugates could be important future medicines to add to our antibiotic arsenal. This review is written in the context of the chemical design of siderophore–antibiotic conjugates focusing on the differing siderophore, linker, and antibiotic components that make up conjugates. We selected chemically distinct siderophore–antibiotic conjugates as exemplary conjugates, rather than multiple analogues, to highlight findings to date. The review should offer a general guide to the uninitiated in the molecular design of siderophore–antibiotic conjugates. Antimicrobial resistance, including antibiotic resistance, is a major threat to global human health and society at large . The incBacterial resistance to antibiotics can occur through a number of mechanisms. Such mechanisms include the overexpression of the antibiotic target enzyme/protein, mutations in the antibiotic target protein/enzyme, overexpression of antibiotic-modifying enzymes such as β-lactamases, and the utilization of multiple efflux pumps to actively export the antibiotic out of the bacterial cell, reducing the intracellular accumulation of the antibiotic . The GraOne approach to overcome the bacteria cell wall permeability barrier and reduced intracellular accumulation is to covalently link an antibiotic to a microbial siderophore or siderophore mimic . SideropSiderophores are secreted by bacteria to the extracellular environment to solubilize and import iron, and are especially important in competitive multimicrobial environmental niches and iron-restricted host organisms. More than 500 different siderophores have now been identified with high structural diversity . HoweverA considerable number of studies have shown that siderophore iron uptake pathways can be used to transport siderophore–antibiotic conjugates into the bacterial cell by active transport. This method is often called the ‘Trojan horse′ approach . Astound5, , 5, , is a prcefepime . The majathogens ; it indueriaceae . Therefo6, has been the most studied of the sideromycins to date , FhuD (periplasm), FhuB (cytoplasmic membrane), and FhuC, which is an ATPase that provides energy for transport across the inner membrane . Ga. Ga6, ha date .Wencewicz and Miller utilized an artificial mixed bis(catechol) and hydroxmate siderophore mimic in conjugate 12  25]. Th. Th12 ; these conjugates are clearly utilizing the siderophore counterpart′s native iron transport mechanisms for active uptake. Conjugate 13 demonstrates, yet again, how utilizing natural siderophores can result in effective conjugates. Other synthetic pyoverdine-based β-lactam and DNA gyrase inhibitor conjugates have met with mixed results; these compounds generally exhibit reduced antibacterial activity compared to the parent antibiotic from which they were derived [Kinzel et al. synthesized conjugates of ch as 13  35]. Th. ThPseud derived ,37.14, which contains a siderophore directly analogous to the natural siderophore desferrioxamine B attached to the quinolone antibiotic ciprofloxacin targeting DNA gyrase [S. aureus. The authors demonstrated that only the trihydroxamate functionality, mimicking natural desferrioxamine B, was responsible for this observed S. aureus species selectivity. Desferrioxamine B is already used in patients to treat iron overload; therefore, it could be envisaged that conjugate 14 could have applications in vivo in patients with S. aureus infections. Conjugate 14 is also an example of how hydroxamate siderophores can be utilized in conjugates to gain Gram-positive species selectivity. Milliner et al. sought to gain selective antibacterial activity against S. aureus by conjugating a number of analogues of staphyloferrin A, which is a natural siderophore of S. aureus, to ciprofloxacin [Wencewicz et al. synthesized conjugate A gyrase . The confloxacin . HoweverMany functional groups have been incorporated into synthetic conjugates to form the basis of the linker, including but not limited to: amino acids , succiniThere is no doubt that designing effective conjugates with cleavable linkers for intracellular targets, mimicking nature′s sideromycins, has proved much more challenging for researchers. The cleavable linker must be stable toward the extracellular environment, but cleaved upon entry into the cytoplasm. A linker that is too stable will attenuate the activity of the antibiotic, while linkers that are more susceptible to cleavage will result in premature antibiotic release; in both scenarios, the benefits of a conjugate are lost. Hence, a ‘fine balance′ of chemical reactivity is required for a cleavable linker, and a number of studies utilizing cleavable linkers have been unsuccessful.15  to Fe(II) reduction in the bacterial cell [17 in this study demonstrated moderately reduced MICs in comparison to a directly analogous non-cleavable counterpart. However, the conjugate was not as potent as ciprofloxacin itself, suggesting that more optimization of the cleavage mechanism of 17 is required. Similar results were observed by Miller et al. this time using an esterase-cleavable trimethyl lock [18 have been reported by Neumann and Nolan [18, which exhibited antibacterial activity against only a limited number of E. coli strains. The authors concluded that disulfide-containing linkers may not be a broad strategy for conjugate deign.Ji and Miller investigated linking the antibiotic to siderophores via a ‘trimethyl lock′ in conjugate 17 . The antial cell . The deshyl lock ; these cnd Nolan . Again, 19, which was designed to be susceptible to intracellular enzymatic cleavage. The conjugate 19 could perhaps be considered as a siderophore-mediated cleavable conjugate rather than a linker-mediated cleavable conjugate. The catecholate siderophores enterobactin and salmochelin are internalized naturally in Gram-negative bacteria such as E. coli and undergo cleavage to a number of siderophore subfragments; Neumann et al. used these observations as inspiration for the conjugate design of 19. Conjugate 19 was designed to have an amide bond on enterobactin in the same position as the glycodside linkage in the analogous siderophore salmochelin. Salmochelin undergoes intracellular enzyme-mediated hydrolysis by the esterase IroD. Remarkable results were observed with conjugate 19, which was not only as active as its parent antibiotic , but demonstrated selective antibacterial activity against pathogenic E. coli over non-pathogenic E. coli. This selectivity was rationalized by esterase IroD being predominantly present in pathogenic E. coli in comparison to non-pathogenic E. coli. There are other significant observations in cleavable conjugate design in this study that should not be overlooked. (1) A compound directly analogous to 19 with a longer PEG linker instead of the alky chain of 19 was not active against any of the tested E. coli strains. (2) It could be assumed a modified analogue of ciprofloxacin, such as a linker or siderophore subfragment analogue of ciprofloxacin, was released that was capable of retaining DNA gyrase inhibition and antibacterial activity. Hence, the chemical make-up of the linker is just as important as the cleavage mechanism/design, and although cleavage is necessary, an analogue of the cleaved antibiotic may be tolerated to retain antibiotic target engagement. Such a discovery offers further hope and research for cleavable linkers, and perhaps further highlights the use of natural siderophores in conjugates to mimic nature more closely for success.Despite all these challenges, a significant advancement in cleavage methodologies has recently been reported by Neumann et al. . The aut6 and 15 in this review being good examples. However, antibiotics other than β-lactams and DNA gyrase inhibitors have not been as well represented in recent decades. Considering the extensive literature on β-lactam and ciprofloxacin conjugates [Synthetic conjugates consisting of β-lactam antibiotics are the most reported in the literature to date followed secondly by DNA gyrase inhibitors such as ciprofloxacin. Exciting results have been observed with β-lactam conjugates enhancing MIC potencies and inducing multi-drug resistant (MDR) bacterial susceptibility. Ciprofloxacin is a broad spectrum antibiotic that is widely used to treat a number of bacterial infections; with a low molecular weight (331 Da) and a readily functionalized piperazine heterocycle, this antibiotic provides a robust chemical scaffold for investigating conjugate delivery to the cytoplasm. Many other antibiotics have been conjugated to siderophores, with conjugates njugates ,53,54,5520  to trihydroxamate siderophore mimics as well as to norneoenactin and an erythromycin analogue [21, which was functionalized for ester-mediated cleavage, did display enhanced antibacterial activity compared to the 5-FC active metabolite alone against Gram-positive Staphylococcus and Enterococcus spp. In a related study, Lu and Miller carried out an interesting modification to the ‘typical′ siderophore–antibiotic conjugate design; the authors functionalized the conjugate with multiple 5-FC metabolite antibacterial groups [Miller et al. conjugated the Gram-positive antibiotic vancomycin to catechol ligands and mixed catechol–hydroxmate ligands ; however20  did exhianalogue . The eryl groups  rather tMycobacterium tuberculosis [22 should not be underestimated, as the synthetic route involves the synthesis of both the mycobactin T scaffold and the endoperoxide 1,2,4-trioxane ring of artemisinin. The biological results were also impressive; the attachment of mycobactin T appeared to convert artemisinin from an exclusively antimalarial agent into a dual potent anti-tuberculosis (TB) agent, with MICs ranging from 0.125–1.25 µg/L against eight differing MDR M. tuberculosis clinical isolates. The authors hypothesized that microbial Fe(III) reduction to Fe(II) leads to the conversion of artemisinin into the reduced redox state; the reduced artemisinin induces the production of oxygen free radicals that invoke antimicrobial activity. Therefore, in this case, the siderophore not only enhances delivery and uptake, but also enhances the mode of action of the antimicrobial agent. Within our group, we recently reported the synthesis of a catechol azotochelin–phenothiazine conjugate 23 [24 utilizing synthetic biology rather than chemical synthesis [24 contains an analogue of the DNA gyrase inhibitor clorobiocin. The conjugate utilizes TonB-dependent active transport and demonstrates enhanced antibacterial activity compared to clorobiocin in TolC efflux pump mutants. However, conjugate 24 was not as active as clorobiocin against E. coli K12; the authors suggest that, although the active uptake of 24 is increased compared to clorobiocin alone, so too is the TolC-mediated active efflux of conjugate 24.In another elegant study by Miller et al., an acetal analogue of the antimalarial agent artemisinin was conjugated to an analogue of the siderophore mycobactin T, which is utilized by rculosis . The difugate 23 . The pheugate 23 . NDH-2 iugate 23 . As NDH-ynthesis . Conjuga25,  can indeed be functionalized for siderophore-mediated delivery. In another approach, Paulen et al. synthesized an analogue of linezolid, one of the newer antibiotics in the market, to catecholates with the aim of lowering the MICs of this Gram-positive antibiotic against P. aeruginosa [27 exhibited an MIC of 128 µM against P. aeruginosa, which is significantly lower than that of linzeloid alone (>1024 µM); this is an intriguing result, as the linker was not designed to be cleavable, and linzeloid′s antibiotic target is in the cytoplasm. The activity of conjugate 26 was also enhanced in iron-deficient growth medium, suggesting the iron transportation of the conjugate.One area of siderophore–antibiotic research that has readily driven new conjugates is the desire to generate new molecules to treat Gram-negative pathogens. The additional lipopolysaccharides in their outer membranes, which are not present in Gram-positive bacteria, present a formidable barrier to the penetration and diffusion of antibiotics into Gram-negative bacterial cells . Hence, 25, , is not aumannii . The actnnii [26  was deperuginosa . ConjugaP. aeruginosa siderophore pyochelin, forming conjugate 28 as well as analogues (16 prepared by Noël et al. [29 synthesized by Liu et al. consisted of linezolid conjugated to a catechol siderophore; however, the linker was a cephalosporin β-lactam antibiotic [29 was 125-fold more potent than cephalosporin alone and the conjugate lacking the β-lactam linker against A. baumannii, which indicates that the penicillin-binding protein or β-lactamase cleavage is important for the observed enhancement of antibacterial activity. The cephalosporin-linked conjugate 29 was active against all four clinical isolates of A. baumannii that it was tested against.In a different study, Paulen et al. conjugated linzeloid to the nalogues . Unfortul et al. . A rathetibiotic . The aut5, in phase III clinical trials with potent activity against MDR Gram-negative bacteria [5, progresses to the market, further drug discovery research into siderophore–antibiotic conjugates would most likely be stimulated. Indeed, β-lactam–siderophore conjugates are already in the realm of medicinal chemistry and drug discovery. However, non-β-lactam siderophore–antibiotic conjugates seem to remain, as yet, an academic pursuit. Perhaps such large molecular weight conjugates may put medicinal chemists off the drug discovery process. Despite this, importantly, caution needs to be taken in overlooking such larger conjugates, since if there is one field in medicinal chemistry that perhaps ‘tolerates′ bigger molecules and violations of Lipinski′s rules, it is natural product antibiotics [The advantages that can potentially be offered by siderophore–antibiotic conjugates are clear; enhanced antibacterial potency, induced bacterial species selectivity, and even enhanced selectivity for pathogenic strains over non-pathogenic strains, transforming Gram-positive antibiotics into potent Gram-negative antibiotics, and rendering MDR pathogenic bacteria susceptible to killing. These advantages are further exemplified by cefiderocol, bacteria . If cefiibiotics . FurtherIt would be interesting to see more conjugates utilizing antibiotics and antimicrobial agents other than the β-lactam antibiotics and DNA gyrase inhibitors that currently dominate the field. Linzeloid conjugates are becoming more common. Further to this, it would be interesting to see more conjugates utilizing more structurally diverse natural siderophores, considering that over 500 siderophores have been reported . Given t