Immunoglobulin G4-related disease (IgG4-RD) is a chronic inflammatory disorder associated with fibrosis and abundant tissue lymphoplasmacytic infiltrations. It typically affects the pancreas, the salivary glands, and the retroperitoneal space. However, it might also involve multiple other organs, including the orbit and the thyroid. Recent studies have suggested that IgG4 plays a role in the pathophysiology of autoimmune thyroid diseases. This ultimately led to the establishment of new clinical entities called IgG4-related thyroid disease and thyroid disease with an elevation of IgG4. The aim of this paper is to describe the pathophysiological, histopathological, and clinical features of Graves' Disease (GD) and Graves' Orbitopathy (GO) with elevated IgG4 levels. Multiple studies have demonstrated higher IgG4 serum concentrations in GD patients than in healthy euthyroid controls. Depending on the studied population, elevated serum IgG4 levels occur in 6.4-23% (average: 10.3%) of all patients with GD, 8.3-37.5% (average: 17.6%) of patients with GO, and 0-9.8% (average: 5.4%) of patients with GD without GO, while GO patients comprise 37.5-100% (average: 65.8%) of all GD patients with elevated IgG4 levels. Characteristic features of GD with elevated IgG4 levels include lower echogenicity of the thyroid gland on ultrasound examination, peripheral blood eosinophilia, higher prevalence of orbitopathy, and better response to antithyroid drugs with a tendency to develop hypothyroidism when compared to patients with GD and normal levels of IgG4. Typical signs of GO accompanied by increased concentration of IgG4 include younger age at diagnosis, and more severe course of the disease with a higher Clinical Activity Score (CAS).. We strongly recommend considering the diagnosis of GO with elevated IgG4 in patients with an established diagnosis of GD, elevated serum IgG4 levels, and clinical features of ophthalmic disease overlapping with those of IgG4-related orbital disease. Human immunoglobulin G (IgG) subclasses were numbered in an order reflecting the time of their discovery, which also corresponds to their prevalence in plasma . Thus, IImmunoglobulin G4-related disease (IgG4-RD) is a chronic inflammatory disorder associated with fibrosis and abundant lymphoplasmacytic infiltrations, often with obliterative phlebitis and storiform fibrosis within the affected tissues. IgG4-RD appears to underlie several fibroinflammatory disorders earlier regarded as idiopathic or isolated –7. TypicThe symptoms depend on the affected organ. On histopathology, this fibroinflammatory disease is characterized with specific findings such as lymphoplasmacytic infiltration of IgG4-positive plasma cells, obliterative phlebitis, and storiform fibrosis, in most cases accompanied by increased levels of serum IgG4 .To date, an association between IgG4 and various other diseases like allergies, cancer, or rheumatoid arthritis has been revealed . Until nThe aim of this paper is to summarize current knowledge on the role of IgG4 as a biomarker of Graves' Disease and Graves' Orbitopathy.Increased immunoglobulin G4 (IgG4) concentration is a common, though nonspecific, finding observed in most IgG4-RD patients . High IgIgG4 is the least abundant class of IgG. Due to the specific structural features, IgG4 exerts a unique ability of Fab arm exchange . ExchangThe direct cause of IgG4-RD remains unknown. Growing evidence suggests an autoimmune basis for the disease. Potential best-studied autoantigens include annexin A11, laminin 511, and galectin-3; however, their role requires clarification –21. The Exposure to foreign antigens activates circulating monocytes and basophils. In consequence, they produce the B cell-activating factor (BAFF) that induces B cell proliferation and differentiation towards IgG4-producing plasmablasts and plasma cells , 23. In β, interferon-gamma (IFN-γ), and transforming growth factor-β1 (TGF-β1) inducing local cytotoxicity, tissue damage, and fibrosis [T cells predominate in IgG4-RD tissue infiltrates and play an important role in its pathogenesis . The latfibrosis , 33. Higfibrosis , 32. Thiβ produced by Treg are considered to drive tissue damage. The IL-13 and TGF-β promote myofibroblast differentiation and extracellular matrix protein synthesis [Other T cell subsets are also involved in IgG-RD pathogenesis. Both T helper type 2 cells and regulatory T cells (Treg) were reported to increase in IgG4-RD –36. The ynthesis , whereasynthesis . The folynthesis , seem toynthesis . Increasynthesis –44. Tfh ynthesis , 45. Tfhynthesis . Tfh celynthesis .+20−27+38hi plasmablasts, producing IgG4, were found to increase in circulation and tissue infiltrates, compared to healthy individuals, and to correlate with the IgG4-RD activity and extension (number of affected organs) irrespective of IgG4 concentration. Plasmablasts declined after B cell depletion treatment with rituximab and glucocorticoid treatment, which resulted in clinical improvement, while in relapse, they reemerged [β (PDGF-β), enzymes modifying the extracellular matrix such as lysyl oxidases, or chemokines such as CCL4, CCL5, and CCL11  [In IgG4-RD, the B cell subpopulations and their regulatory cytokines are altered. The aforementioned mechanisms result in the development of IgG4-producing plasma cells and their short-lived precursors—plasmablasts. Clonally expanded CD19eemerged –50. B ce and 11) .In several disorders, including myasthenia or pemphigus, the pathogenic role of IgG4 is well defined. Due to the complexity of IgG4-RD pathogenesis, the potential contribution of IgG4 in this disease remains unclear.Graves' Disease (GD) is an autoimmune thyroid disease, where the most important clinical feature is hyperthyroidism . It can The pathophysiology of GO is very complex and still not fully understood. It involves complex interactions between a variety of immune cells, cytokines, interleukins, adhesion molecules, and growth factors and increased oxidative stress. A key role is being assigned to abnormally activated orbital fibroblasts, overexpression of the human leukocyte antigen-DR (HLA-DR), and autoimmunity against the thyrotropin receptor (TSH-R) and insulin growth factor 1 receptor (IGF-1R) –66.The orbital tissue in GO is infiltrated by a variety of mononuclear inflammatory cells like CD4+ T cells, CD8+ cells, B cells, and macrophages . Cell-meThe TSH receptor antibodies (TRAb) play a key role in GD and GO pathogenesis. Elevated TRAb serum concentrations are associated with the development of GO as well as the activity and severity of the disease –71. The in vitro studies suggest that the reactivation of Epstein-Barr virus (EBV) might play a role in the development of GD and IgG4 might be one of the contributing factors in this mechanism. Nagata et al. induced EBV reactivation on peripheral blood mononuclear cells containing TRAb(+) EBV(+) cells sourced from GD patients and healthy controls. EBV reactivation resulted in TRAb production, which was significantly higher in GD patients than in controls [in situ hybridization and immunohistochemistry on thyroid glands revealed that EBV-encoded small RNA1- (EBER1-) positive cells and IgG4-positive plasma cell infiltration were detected in the same areas along with lymphoid cell infiltration [Recent controls . The samcontrols . Finallyltration .Studies evaluating the presence of histological features of IgG4-RD in GD patients are very scarce and deliver contradictory results. According to a study by Nishihara et al., only 11 out of 1484 thyroid glands resected from GD patients had a diffuse lymphoplasmacytic infiltration in the stroma, which is a characteristic feature of IgG4-RD and IgG4 thyroiditis. Only 5 of those 11 had IgG4-positive plasma cells in the thyroid, only one showed fibrosis, and none of the 11 specimens had obliterative phlebitis . In contWhile thyroid biopsies cannot be used to diagnose IgG4-RTD, the biopsy procedure is the gold standard of IgG4-ROD. It is usually obtained from the extraocular muscle, lacrimal gland, orbital mass lesion, and infraorbital nerve or a combination of these . The diaA study by Wong et al. reported no significant IgG4 staining in 26 orbital biopsies of GO patients. This needs to be interpreted with caution as the study analyzed mostly specimens obtained from orbital fat, while only four biopsies were obtained from the lacrimal gland and none from extraorbital muscles . This isThere are currently a handful of case reports describing patients with GO, and the biopsy procedure confirmed IgG4(+) plasma cell infiltration of the orbital tissue –88. In tThere are only a few studies, often with ambiguous outcomes, assessing the role of IgG4 in Graves' Disease and even less in Graves' Orbitopathy. We summarized data from those studies in Multiple studies showed higher serum IgG4 concentrations in GD patients than in healthy euthyroid controls , 89, 92.Elevated serum IgG4 levels occur in 6.4-23% (average: 10.3%) of all patients with GD, 8.3-37.5% (average: 17.6%) of patients with GO, and 0-9.8% (average: 5.4%) of patients with GD without GO, while GO patients comprise 37.5-100% (average: 65.8%) of all GD patients with elevated IgG4 levels . The frep < 0.001) [According to the first and largest (109 patients) study on this subject published in 2014 by Takeshima et al., patients with GD and elevated IgG4 were older than those with normal IgG4 levels (54.7 ± 6.2 vs. 43.4 ± 15.4 years), respectively.  However,< 0.001) .p = 0.046, odds ratio (OR) = 1.351, and 95% CI: 1.006–1.815). The same study found that IgG4 levels and the IgG4/IgG ratio are elevated in the moderate-to-severe orbitopathy group compared with the group with mild orbitopathy and in the active GO group compared with the inactive GO group. IgG4 levels were also associated with the Clinical Activity Score (CAS), which is an often employed and renowned classification used to grade the severity of GO. Average IgG4 levels were 46.5 ± 15.9 vs. 74.1 ± 9.4 vs. 93.9 ± 19.9 mg/dl (p = 0.001) for groups of patients with a CAS of 1, 2, and 3, respectively. IgG4/IgG ratios were 3.6 ± 1.6 vs. 5.3 ± 2.0 vs. 6.0 ± 1.5% (p = 0.033) for groups with a CAS of 1, 2, and 3, respectively [In a study by Yu et al., female sex, smoking history, and levels of IgG, IgG4, triiodothyronine (T3), free thyroxine (FT4), TRAb, and antithyroglobulin antibodies (TgAb) were associated with GO development in univariate analysis. However, the serum IgG4 concentration was found to be the only independent factor associated with GO development in multivariate analysis (ectively .p = 0.024) [In another study, IgG4 levels were observed to increase in concordance with CAS and IgG4 levels and were also higher in the GO group compared with GD patients without GO  . Convers= 0.024) , 90. Thide novo diagnosed with GD (225.69 ± 93.19 vs. 140.82 ± 93.97 mg/dl) [GD affects mostly women, but thyroid eye disease in general tends to be more severe in older patients and men. In a study by Perros et al., the female to male ratio was 9.3 in patients with mild orbitopathy, 3.2 in those with moderate orbitopathy, and only 1.4 in those with severe orbitopathy. This was often related to the fact that men tend to be heavy smokers , 95, and7 mg/dl) . However2 and 136 ± 122/mm2, respectively [Another thing indirectly linking GD to IgG4-RD is the peripheral blood eosinophilia. Approximately 40% of patients with IgG4-RD have eosinophilia , 99, whiectively .p = 0.01) and TgAb levels (p = 0.006) [In one study, patients with immunoglobulin G4 levels above the 75th percentile were reported to have higher concentrations of antithyroid peroxidase antibodies (TPOAb) (= 0.006) . TPOAb cp = 0.001) and significantly higher FT4/FT3 ratios  than patients with IgG below the 90th percentile [Regarding GD treatment with antithyroid drugs, patients with IgG4 levels above the 75th percentile require a shorter duration of the first methimazole treatment cycle than patients with immunoglobulin G4 below the 75th percentile. At diagnosis, patients with immunoglobulin G4 levels above the 90th percentile tend to have lower total T3  for a mean of 10.4 ± 8.0 months. It described a return of IgG4 levels elevated above 135 mg/dl to normal values after antithyroid treatment; however, one of the patients with normal baseline IgG4 levels had an increased level after antithyroid treatment .According to a study by Luo et al., patients with GO with an elevation of IgG4 present most commonly with bilateral muscle thickening (in 7/8 cases), which affects the medial rectus muscle, the inferior rectus muscle, and relatively often the lateral rectus muscle . Infraorbital nerve thickening and lacrimal gland involvement, which are key features of IgG4-ROD, were less prevalent  . All thoThe diagnostic dilemma of distinguishing between GO and IgG4-ROD was described in a very detailed and informative case series by Tooley et al. . In this+ B cells. CD20+ B cells are progenitors to plasmablasts. Plasma cells that produce IgG4 are usually short-lived, and they undergo naturally programmed cell death within a couple of weeks. After this process is completed, the plasma cell pool cannot be repeated because the B cells, which are their precursors, are already depleted. This explains also why it usually takes rituximab a couple of weeks to work [To date, no trials assessing response to standard or potentially different treatment regimens in GO with elevated IgG4 have been conducted. However, based on studies on IgG4-RD, IgG4-ROD, GO in general, and some case reports, treatment with glucocorticoids is the first line of therapy. However, the disease has a relatively high risk of recurrence when the dose is tapered, and in these cases, other immunosuppressive regimens might be helpful. Currently, also, rituximab is considered a primary therapeutic strategy, which allows steroid discontinuation and relieves signs and symptoms , 104–107 to work . FibrotiSymptoms of IgG4-ROD may overlap those seen in other diseases, though in most patients, both eyes are affected. Likewise, CT or MRI scans of the orbital region also present unspecific features . The pren = 42), GPA (n = 6), sarcoidosis (n = 12), and thyroid eye disease (n = 26). With the IgG4 plasma cell threshold set at >10/HPF, positive staining was present in patients with GPA (83%), NSOI (38%), and sarcoidosis (42%). Only a few cases had more than 30 IgG4-PC in HPF. In contrast to IgG4-RD, in examined biopsies, storiform fibrosis or obliterative phlebitis was not detected [In the study by Wong et al., they collected orbital biopsies from patients diagnosed with nonspecific orbital inflammation  and resected thyroid samples are also needed.We strongly recommend considering the diagnosis of Graves' Orbitopathy with elevated IgG4 in patients with an established diagnosis of Graves' Disease, elevated serum IgG4 levels, and clinical features of ophthalmic disease overlapping with those of IgG4-related orbital disease.


In pediatric inpatient settings, unconfirmed penicillin allergy labels (PALs) are associated with increased broad-spectrum antibiotic use, costs, and adverse events. However, 90% of antibiotics are prescribed in the outpatient setting and 70% of these antibiotics are given for upper respiratory tract infections (URTI.) Little is known about the effect of PALs on antibiotic prescribing in the pediatric outpatient population.st 2010 and June 30th 2020 and seen at one of 91 Texas Children’s Pediatrics or Children’s Hospital of Philadelphia primary care clinics. Children with an ICD10 code for an URTI and an antibiotic prescription were stratified into those with or without a penicillin allergy label at the time of the infection. Rates of second-line and broad-spectrum antibiotic use were compared.A retrospective birth cohort was created of children born between January 1The birth cohort included 334,465 children followed for 1.2 million person-years. An antibiotic was prescribed for 696,782 URTIs and the most common diagnosis was acute otitis media. Children with PALs were significantly more likely to receive second-line antibiotics  and broad-spectrum antibiotics  Children with PALs received more third generation cephalosporins (60% vs. 15%) and more macrolide antibiotics (25% vs. 3%) than those without a PAL. Overall, 18,015 children (5.4%) acquired a PAL during the study period, which accounted for 23% of all second-line antibiotic prescriptions and 17% of all broad-spectrum antibiotic use for URTIs.Multivariable logistic regression for receipt of second-line antibiotics for upper respiratory tract infectionsPALs are common and account for a substantial proportion of second-line and broad-spectrum antibiotic use in pediatric outpatients treated for URTIs. Efforts to de-label children with PALs are likely to increase first-line antibiotic use and decrease broad-spectrum antibiotic use for URTIs, the most common indication for antibiotic prescribing to children.Debra Palazzi, MD, MEd, AAP AHRQ (Research Grant or Support)Elsevier JAMA Pediatrics (Board Member)


Ceramic-based wastes generated from different industrial activities have increasingly been reused as construction material incorporated into concrete. In general, these wastes just replace common concrete aggregates such as sand and gravel. In the present work, waste from clay brick industries composted of kaolinite minerals were for the first time evaluated for their potential to be reused as the pozzolan constituent of a cement for structural concrete. Initial standard testes revealed that the clay ceramic waste (CCW) displays high pozzolanicity. Concrete was then produced with 10 and 20 wt.% of CCW mixed with ordinary Portland cement (OPC) as its pozzolan constituent. Compression strength of these concretes and of pure OPC as a control sample were determined in standard tests after 14 and 28 days of curing. In addition, the corresponding density, water absorption, capillarity and percentage of voids were measured together with the evaluation of microstructural indices by scanning electron microscopy. The initial tests confirmed that the CCW is indeed an effective pozzolanic potential due to a chemical effect by reacting with CH to generate C–S–H. Moreover, the technological results proved that CCW might effectively replace the pozzolan cement constituent for structural concrete. The study of materials with pozzolanic potential is a major focus in the application of cementitious materials, within the materials of science ,2. This Pozzolans, naturally found or as waste, are defined as materials that do not have agglomerating power on their own but finely ground in the presence of cement and water and have the potential to undergo hydration reaction for consolidating as a solid material . For thiThe pozzolanic reaction that occurs in the materials with this behavior are considerably complex , mainly C–S–H is hydrated calcium silicates and CH is portlandite.The release of heat indicates that Equation (1) is exothermic. C-S-H is the main component responsible for the strength of the concrete. CH (portlandite) has a secondary role in the strength of concrete, when compared to the C–S–H, also presenting contributions with regard to the durability of the material . With thIt should be noticed that with the use of pozzolanic materials, or simply pozzolans, there is a readjustment in the amounts of C–S–H obtained in the final product, which will naturally contribute in some way to the strength of the concrete ,21. ThisIn Campos dos Goytacazes, in the north of the state of Rio de Janeiro, Brazil, the red ceramic manufacturing process, which involves manufacturing, transporting and storage, generates approximately 19 thousand tons of waste per month . HoweverSome important works have already been carried out with this type of waste in construction materials ,31,32,33Regarding the specific application of ceramic waste as a pozzolan material to act as OPC for structural concrete, 2 and 18.2% Al2O3. Irassar et al. [2, 20% Al2O3 and 11.5% CaO and feldspar, anorthite and hematite. Pokorný et al. [2, 14.3% Al2O3, 15.2% CaO and 17.9% SO3, in addition to microcline, albite and muscovite. With similar work as in [2, 16.04% Al2O3, 4.08% MgO and 3.15% Na2O. Pacheco-Torgal and Jalali [Kannan et al.  studied r et al.  investigý et al.  evaluaterk as in , Vejmelkrk as in  used therk as in  and Chenrk as in  applied d Jalali  considerd Jalali  and Wangd Jalali  the respBased on these aforementioned ceramic wastes, kaolinite clayey waste has so far been considered as pozzolanic material. Another relevant point highlighted in Another important point regarding the results present in In this context, it is highlighted that the main objective of this work is to evaluate the application of ceramic waste as a substitute for Portland cement in concrete, evaluating the pozzolanic potential of the material through results of compressive strength, microscopy and Luxán method. The main research gap is the characterization of the ceramic residue using methods that evaluate the pozzolanic activity of the material by a coherent procedure, such as the Luxán method, different from other authors, such as those highlighted in To carry out this study, clay ceramic waste (CCW) was used, see in The waste (CCW) was characterized chemically by X-ray Fluorescence (XRF), as seen in 2 and Al2O3, besides the presence of other minor oxides such as Fe2O3, Na2O, K2O, P2O5, TiO2 and CaO. In Can be observed in The CCW had its pozzolanic activity evaluated through the Luxán method of the conductivity measurement . In thisThree concrete compositions were produced using CP III-40 RS cement, a commercial Brazilian OPC . Owing to the reduced presence of pozzolans, favoring the evaluation of the activity, coarse aggregate of the type gravel 1 and fine aggregate of the type natural sand washed from the Paraíba do Sul river with grains of 0.01–2.4 mm in diameter were also included into the concrete. The concrete compositions were selected as 0%, 10% and 20 wt.% of CCW in substitution for OPC, as indicated in For the execution of the technological tests, the specimens were removed from the submerged cure 30 min before, with a cloth the excess surface water was removed, to allow the execution of the tests. After, compressive strength tests were carried out after 14 and 28 days of curing, using a Solotest hydraulic press with a speed of 50 N/s, for 3 specimens in each mixture . NeoprenMass density tests in the hardened state, capillary water absorption tests and immersion and boiling water absorption tests were also performed, in all of these tests 3 specimens were made for each mixture . The purFinally, a microstructural analysis was performed using scanning electron microscopy (SEM) in a Jeol microscope equipment model JSM 6460 LV  for complementary characterization of the material. For microstructural analysis the reference mixture (without any addition) and the optimal mixture were selected, which had the best technological properties, according to the results found.2 + Al2O3 + Fe2O3), a 92.80% ratio was obtained. This was above the limit of 70% established to classify the material as class N pozzolan, which is the most noble class of pozzolans, calcined clays and materials of volcanic origin [c origin . To undec origin , to be aIn relation to the pozzolanic activity index, according to the Brazilian norms , 75% of Another important characteristic observed in fck strength of concrete is the value that presents a 95% probability that individual strength values of test specimens are higher, that is, only 5% of the values are less than or equal [fck). This rule is applied through the Gauss curve, using the following equation:Regarding the characteristic compression strength, it is noteworthy that this is the main parameter used in structural engineering projects. This parameter was obtained from the experimental results of compressive strength. As defined by the Brazilian standard, this characteristic or equal . Assuminfcm represents the average strength obtained in the compression test at 28 days and the S represents the standard deviation related to that test.fck, which leads to the conclusion that all compositions can be used as structural concrete [fck [After applying the values obtained for strength to 28 days for the compression test in the CCW compositions of 0, 10 and 20%, 28.98, 27.06 and 25.86 MPa were obtained, respectively. According to Brazilian standards, concrete is considered structural when it has more than 20 MPa of concrete . Additioete [fck , which iThe density results in It is also verified by the water absorption values and voids index, -Evaluation of the use of other percentages of CCW in the properties of the studied concrete. For example, using 5 or 15% of the waste as a substitute for cement;-Use of the plasticizer additive to reduce the water/cement (w/c) ratio, making it possible to obtain a concrete with superior strength, aiming to study how the CCW affects the properties of concrete with a lower w/c ratio;-Evaluation of the compactness of concrete using ceramic residue using techniques of porosometry, electrical resistivity or ultrasound;-Evaluation of the rheological properties of concrete containing CCW;-Evaluation of the durability of concretes containing CCW through high temperature tests, salt spray, ice–thaw cycles, immersion and drying tests and/or chloride attack;-Evaluation of the durability of concretes containing CCW in a situation of water and gas permeability;-Life cycle analysis of concretes containing CCW comparing them to concretes without incorporating waste;-Economic analysis, using the principles of circular economy, of the application of CCW as a substitute for cement in concrete.The results obtained in this research can be increased with the realization of future works presenting continuity of the theme. It is suggested:It can be concluded that the CCW presented a variation in electrical conductivity of 1.21 mS/cm, which made possible its classification in high pozzolanicity, corroborating with the chemical and mineralogical characterization, which showed the presence of metakaolinite with amorphous regions in CCW. Through the index of pozzolanic activity and the characteristic compression strength, we observed that the concrete even with 20% of CCW presents potential structural applications in urban environments, where the pollution aggressiveness was intermediate and concrete with compression strength higher than 25 MPa was required. It is observed, for example, that the compressive strength for the C10 composition was 27.2 MPa at 14 days and 31.42 MPa at 28 days, while the C20 composition showed compressive strength of 24.5 MPa at 14 days and 27.82 MPa at 28 days. The reduction in compressive strength, comparing the compositions with the reference, was attributed to the exhaustion of portlandite in the hydration of the cement, due to the pozzolanic reaction. However, even with the reduction, the observed values were compatible with concretes with structural applications.In addition to the chemical and pozzolanic effect, illustrated by the result of resistance to compression, it was observed that the ceramic residue had an effect on the physical properties. This was verified through the results of density, capillarity, water absorption and voids index. From a physical point of view, it is observed that the CCW had a physical filling effect, because even using smaller amounts of cement, the ceramic residue maintained the density and voids of the concretes with statistical equivalence.In the SEM microstructure analysis, it was confirmed that the CCW had a significant effect as a pozzolanic constituent, since the amount of portlandite detected in the reference composition, 0% CCW, was substantially reduced in the composition with 20% CCW (optimum mixture), proving the effectiveness of the pozzolanic activity. Finally, it can be concluded that the CCW has a pozzolanic potential and is feasible to be used as a partial substitute for OPC in 20% for the production of structural concrete.


The incorporation of recycled aggregates in concrete not only reduces the extraction of natural resources, but also decreases landfill disposal of construction and demolition waste. Hence, environmental impacts and costs are reduced, promoting the use of recycled aggregates and circular economy. However, the impacts of transport depend on the distance between facilities and longer distances may result in recycled aggregates being more costly and having larger environmental impact than natural aggregates. This paper discusses this topic, presents a review on the use of life cycle assessment methodology on natural and recycled aggregates for concrete, and applies this methodology in a real context pertaining the procurement of coarse aggregates to ready-mix concrete plants. A case study of two Portuguese regions, Coimbra and Lisbon, is presented. For each region, a quarry, a construction and demolition waste plant, and a ready-mix concrete plant are chosen and a comparative life cycle assessment is made. Different scenarios for the supply of natural and recycled aggregates are studied and the scenarios for recycled aggregates procurement include different hypotheses for the installation (construction and demolition waste plant or quarry) processing the construction and demolition waste into recycled aggregates. For this case study and both regions, it was found that the supply of recycled aggregates produced at the construction and demolition waste plant has lower environmental impact and cost than all other scenarios, including the provision of natural aggregates, except when it is assumed that the quarry is licensed and equipped for receiving unsorted construction and demolition waste and processing it into recycled aggregates. The paper shows that transport distance is a determining factor in the comparison of the impacts of the procurement of natural and recycled aggregates. Moreover, in the Portuguese context, the environmental impacts of the procurement of recycled aggregates may be smaller than those of natural aggregates, but cost may be larger for recycled aggregates, preventing that the most sustainable option is chosen. The incorporation of recycled aggregates (RAs) in concrete contributes to the European Union’s (EU) goal of 70% of non-hazardous construction and demolition waste (CDW) reuse/recycling, defined in EU Directive 2008/98/CE .The technical feasibility of recycled aggregate concrete (RAC) has been demonstrated in several previous studies ,3,4 in wHowever, this is not always the case and the EI (and cost) of RAC may be larger than those of natural aggregate concrete (NAC) ,13,14. SIn this context, this paper presents a comparative environmental and economic life cycle assessment (LCA) of the procurement of NAs and RAs to ready-mix plants, including their production. This LCA is made for two Portuguese regions, Lisbon and Coimbra. For each region, a quarry, a CDW plant, and a ready-mix concrete plant are selected. Three main scenarios are defined for the LCA: the production of NAs in quarries; the production of RAs in CDW plants with additional processing in quarries; and the production of RAs in quarries. The impact categories considered in the environmental LCA are global warming potential (GWP) and consumption of non-renewable energy resources (PE-NRe) due to their impact on carbon footprint and embodied energy.NAs and RAs have different characteristics, so the incorporation of RAs results in concrete with different properties than those of NAC. RAs are less dense and more porous and deformable  than NAsIn what concerns structural behavior, most resistance mechanisms rely strongly on the properties and layout of reinforcement. Therefore, the load-bearing capacity of reinforced RAC elements is not as affected by RAs as the properties of (plain) concrete ,3. Long-In order to ensure that RAC has equivalent properties to analogue NAC mixes, cement content may be increased . This imSeveral studies have been carried out regarding the comparison of environmental and economic impacts of NAs and RAs and NAC and RAC. In most cases, these studies are made with specific data from producers. In the case of the comparison between NAC and RAC, some authors ,19 resorMarinković et al.  comparedTošić et al.  studied Hossain et al.  applied Braga et al.  presenteFraj and Idir  comparedEstanqueiro et al.  presentePark et al.  analyzed2 value  very close to 1 (0.97) when the outliers circled in orange, away from the trend line, are neglected. When these results are included, the R2 value decreases to 0.83.Other authors have studied the environmental and economic impacts of aggregates, considering other types of data. Some of their results are presented below.Jullien et al.  analyzedSimion et al.  quantifiFaleschini et al.  also stuRosado et al.  comparedThe LCA of the production of NAs and RAs includes all stages regarding extraction and processing of the aggregates to be used in concrete, while the LCA of concrete with NAs and RAs appraises the production and transport to the ready-mixed plant of the various constituents of the concrete, namely cement, admixtures, water, and aggregates , and the production of concrete itself.Kurda et al.  comparedGöswein et al.  focused Colangelo et al.  analyzedPradhan et al.  comparedAs understood from this appraisal, in general RA and RAC have smaller environmental impacts than NAC. However, differences related to the shape of aggregates , to the mix design , to processing , and traIn this study, an LCA is carried out to compare the environmental and economic impacts of NAs and RAs. This requires the definition of functional unit, system boundaries, and life cycle inventory. The functional unit was chosen as 1 tonne of aggregates and implies that the impacts of NAs and RAs will be compared for the same mass of aggregate. The system boundaries define which processes are included  and the life cycle inventory gives information about the data collection.A quarry for the extraction of NAs;A demolition site for the CDW collection;A CDW plant for the reception of these wastes and production of RAs;A ready-mix concrete plant, where concrete is produced and raw materials (including coarse aggregates) received.For each of the two regions analyzed (Lisbon and Coimbra), different industrial units were selected:The ready-mixed plants selected for this study are those of an ongoing research project that aims at the industrial production of recycled aggregate concrete at those plants. The quarry selected for each region is the one that usually supplies NAs to each ready-mix plant. The CDW plants were selected based on preferences of the owners of the ready-mix plants.Transport distances between the main operating locations of Coimbra are presented in In Scenario 1—NAs are produced at the quarry, where they are crushed, washed, and sieved prior to their delivery to the ready-mix concrete plant;Scenario 2a—CDW is transported from the demolition site to the CDW plant, where the CDW is sorted, crushed into RAs, and sieved, and then the RAs are delivered to the ready-mix concrete plant;Scenario 2b—CDW is transported from the demolition site to the CDW plant, where the CDW is sorted, crushed into RAs, and sieved, and then the RAs are transported to the quarry, where they are washed and sieved, and finally RAs are delivered to the ready-mix concrete plant;Scenario 2c—CDW is transported from the demolition site to the CDW plant, where the CDW is sorted , then the CDW is transported to the quarry, where it is crushed, washed, and sieved, and finally RAs are delivered to the ready-mix concrete plant;Scenario 3—CDW is transported from the demolition site to the quarry, where it is received in conformity with a suitable code of the European List of Wastes; then, the CDW is sorted and the content of contaminants is reduced. Afterwards, CDW is crushed into RAs, which are washed and sieved. Finally, RAs are delivered to the ready-mix concrete plant.LCA was based on different scenarios shown in The life cycle inventory data, collected from previous studies from Portuguese companies, are shown in QC is the quarry of Coimbra, while QL is the quarry of Lisbon;CPC and CPL are the ready-mix concrete plants of Coimbra and Lisbon;DSC and DSL are the demolition sites of Coimbra and Lisbon;CDWpC is the CDW plant of Coimbra and CDWpL is the CDW plant of Lisbon;Places marked with * represent the locations where crushing takes place, differentiating crushing at CDW plant (scenario 2b) from crushing at the quarry (scenario 3).The results in terms of EI of the production and transport are presented below. The impacts are the lowest for the region of Coimbra in Scenario 3 and for the region of Lisbon in Scenario 2a, and the highest for the first scenario in both regions;Regarding production impacts, the best results are related to the production of RA directly at the quarry (Scenarios 2c and 3);Regarding the impacts of transport, the best results are those of Scenario 1, because of the reduced transport distances between facilities. However, differences between Scenario 1 and 2a are small in both regions;Concerning RAs, the best solution for immediate implementation is Scenario 2a (procurement of RAs produced and delivered by the CDW plant) for both regions. Moreover, this solution results in much smaller impacts than the procurement of NAs.In In this preliminary study, the functional unit was defined in terms of mass. Since RAs are less dense than NAs, the required mass of RAs to replace NAs in the production of concrete is lower and the functional unit is not exactly the same. However, as seen in −2 €/t·km [Production costs were collected from a previous study related to Portuguese companies . For NAs2 €/t·km .Scenario 2a is the least costly and the one with the most feasible and immediate implementation because there is no need to further investment on equipment or in licenses to receive CDW at the quarry;Scenarios 2b and 2c present the highest costs;In terms of production cost, the results are better for the scenarios with RAs;On the other hand, transport costs are lower for the first scenario, concerning NAs, due to the shorter transport distance.The economic impacts of the production and transport are shown in The same cost was considered for RAs produced either at the CDW plant or at the quarry;The investment needed for the quarry to be able to produce RAs with CE marking was not accounted for (Scenarios 2c and 3);The licensing cost for the quarries to be able to receive CDW was also not considered (Scenarios 2c and 3).Regarding costs, it is highlighted that:As understood in Scenario 2a is only less costly than Scenario 1 when the transport distance of RAs is 53 km shorter than the transport distance of NA.In terms of GWP and PE-NRe, Scenario 2a is better than Scenario 1 when RAs are less than 500 km more distant from the ready-mixed plant than NAs, which will occur virtually always in the Portuguese context.Based on the impacts per km of transport, the distance for which environmental and economic impacts of Scenario 1 (production and delivery of NAs from the quarry to the ready-mix plant) are the same as those of Scenario 2a (production and delivery of RAs from the CDW plant to the ready-mix plant) was calculated. It was found that:In order to better understand the differences between production and transport for both scenarios, Since the differences between the impacts of NAs and RAs could be larger if the CDW plant were closer to the ready-mix plant, an analysis considering an alternative CDW plant in Lisbon was carried out. The alternative CDW plant was selected so that it would be simultaneously closer to the demolition site, to the quarry, and to the ready-mix concrete plant. The differences of the distances between the first selected CDW plant and the alternative one are shown in The replacement of NAs with RAs is seen as an environmental-friendly option that reduces the EI associated to the extraction of coarse natural aggregates. Therefore, the environmental impact of ready-mix concrete production also decreases. However, since EI of high bulk materials (such as aggregates for construction) are strongly dependent on transport distance, the procurement of RAs may actually result in larger EI than the supply of NAs. Therefore, this analysis should be made on a case-to-case basis. Economic impacts should also be assessed, since RAs must be cost-competitive in order to be a sustainable solution.In this study a review of the state of the art of the use of NAs and RAs in concrete is made, comparing the environmental and economic impacts of both types of aggregates. Additionally, an environmental and economic LCA was carried out considering two Portuguese regions, Coimbra and Lisbon. For each region, a quarry, a CDW plant, and a ready-mix concrete plant were selected. Three main scenarios were defined, in order to evaluate the impacts of the several solutions.Regarding the EI, the findings are very similar for the two environmental impact categories analyzed . The lowest EI are related to Scenario 3, for the region of Coimbra, in which the CDW is directly sent from the construction site to the quarry, sorted, and processed into RAs, which are then delivered to the ready-mix concrete plant. However, the immediate implementation of this scenario is impaired due to the need to invest in equipment and personnel at the quarry and, foremost, legal restrictions for the reception and processing of waste at quarries. For the region of Lisbon, the best Scenario is 2a, in which the CDW is transported from the demolition site to the CDW plant and, after processing, RAs are delivered to the ready-mix concrete plant.Regarding economic impacts, the best option and the one that could be immediately implemented is Scenario 2a, for both regions.The worst EI are related to the first scenario, representing the current situation, in which NAs are produced at the quarry and then are delivered to the ready-mix concrete plant. The impacts of production are always lower for the scenarios with RAs but the impacts of transport are higher due to increased total travel distances between facilities and demolition site (NAs only travel from quarry to ready-mix plant).The worst economic impacts are related to the scenarios in which CDW is processed both at CDW plant and at the quarry. This is a consequence of the impacts of transport, due to the increase of travel distance.In order to study the influence of transport distance, an alternative CDW plant for the region of Lisbon was studied in a sensitivity analysis. This plant is closer to the demolition site, quarry, and ready-mix concrete plant. This solution resulted in a reduction of about 28% in terms of environmental and economic impacts.Future studies will focus on the comparison of the EI and cost associated with the production of natural and recycled aggregate concrete. Some aspects related to concrete mix design that were not considered in this paper will be taken into account, such as the fact that the density of recycled aggregates is lower than that of natural ones  and the consideration of probable changes in mix design, needed to offset the influence of RAs on concrete properties, in the LCA.


However, athletes sprinting at (sub)maximal effort already surpass 5 m·s−1 after a few strides. The present study aimed to develop and validate IMU-derived SLs during maximal linear overground sprints. Recreational athletes (n = 21) completed two sets of three 35 m sprints executed at 60, 80, and 100% of subjective effort, with an IMU on the instep of each shoe. Reference SLs from start to ~30 m were obtained with a series of video cameras. SLs from IMUs were obtained by double integration of horizontal acceleration with a zero-velocity update, corrected for acceleration artefacts at touch-down of the feet. Peak sprint speeds (mean ± SD) reached at the three levels of effort were 7.02 ± 0.80, 7.65 ± 0.77, and 8.42 ± 0.85 m·s−1, respectively. Biases (±Limits of Agreement) of SLs obtained from all participants during sprints at 60, 80, and 100% effort were 0.01% (±6.33%), −0.75% (±6.39%), and −2.51% (±8.54%), respectively. In conclusion, in recreational athletes wearing IMUs tightly fixed to their shoes, stride length can be estimated with reasonable accuracy during maximal linear sprint acceleration.Inertial measurement units (IMUs) fixed to the lower limbs have been reported to provide accurate estimates of stride lengths (SLs) during walking. Due to technical challenges, validation of such estimates in running is generally limited to speeds (well) below 5 m·s Stride length can be estimated with inertial measurement units (IMUs) attached to the feet. IMUs are small sensors that measure triaxial accelerations and angular rates and often also include a magnetometer to determine orientation. Most research has focused on walking and reports (highly) accurate estimates of SL with only a few percent error compared to a golden standard ,2,3,4.In most sports, running is more important than walking. Together with stride frequency, SL determines acceleration and maximal speed, which are both important performance determining factors not only in athletics , but als−1, which, to the best of our knowledge, is the highest speed interval for which IMU-derived SLs have been reported [SL determination from sensor signals during running may be far more challenging than during walking. During running, the participants and, consequently, any sensors attached to them, move much more vigorously, which introduces large accelerations and fast changes in sensor orientation . In partreported .−1) of investigated speeds for which SL has been accurately derived from IMUs is rather low. During sprint acceleration, 5 m·s−1 is reached after the first few strides [−1 [During maximal linear sprint acceleration, there are several factors that will complicate SL determination compared to running at constant submaximal speeds. There will be (participant-dependent) changes in running technique during the transition from the acceleration phase to full speed sprinting ,8,21,22. strides ,23 and rides [−1 .Recently, we demonstrated that fairly accurate estimates of SL could be obtained during maximal sprint acceleration , but theIn walking and running, zero velocity updates are a common procedure when SL is determined from double integration of the acceleration signals ,2,17,19.The objective of the present study was to develop an algorithm that accurately estimates SLs during  acceleration across a range of running speeds that are much faster than those investigated thus far. We hypothesised that accurate determination of SL during sprints executed at different percentages of maximal effort (60–100%) is possible when touch-down-related artefacts in the IMU signals are corrected for.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the ethics committee of the Faculty of Behavioural and Movement Sciences of the Vrije Universiteit (VCWE 2016181R1). After signing informed consent and after being informed about the goals and procedures, twenty-one recreational track and field athletes from two different local clubs participated . They weThe participants did their own pre-competition warm-up, which included acceleration runs to maximal speed. Subsequently, a custom built  9-DOF IMU  was attached to each shoe. Prior to that, IMUs were switched on and tapped together on a hard surface for time-synchronization . The IMUIMUs were secured on the instep of each shoe using a tight elastic band placed under the shoelaces and hooked over the clips of the IMU . The IMUFollowing warm-up and IMU-attachment, the participants ran two sets of three sprints starting every 3 min. In each set, the sprints were done in order of increasing intensity and executed at about 60%, 80%, and 100% of maximal effort.www.kinovea.org; (accessed on 8 June 2021). Stride lengths were divided by IMU-derived stride durations (see below) to obtain stride speeds [The participants started at their own initiative. They ran on a straight white line of the track in between two parallel lines (1 m distance in between) of small cones. The cones in each line were placed at every 3 m and used for video calibration. The touchdowns of the runners’ feet were video recorded (30 Hz) from the side with 5–6 cameras (Fujifilm XP60 Full-HD) mounted on tripods (1.2 m height) and placed 8 m to the side of the sprint lane, from 3 to 27–33 m at 6 m intervals. To determine SL from video, the tips of the shoes were manually digitized for all recorded steps  using fre speeds . The higThe instances of touch-down and toe-off of both feet were derived from the IMU-signals. Specifically, the gyroscope of the sensors’ x-axis  was used for automized detection of the exact instances of touch-down and toe-off, as described in detail elsewhere . SubsequThe participants started from standstill and, hence, the first toe-off of each foot lacked a preceding touch-down event; therefore, for both feet, we had to decide how many samples before the first toe-off we would start the double integration procedure to derive length of the first stride. We established that using a 280 ms interval before toe-off included all visible  movements of both feet before toe-off for all participants, regardless the differences in split stance position and start execution among the athletes.All subsequent toe-off events were preceded by a touch-down that caused large peaks in the acceleration signals, which, upon integration, led to an offset in velocities in the horizontal  plane during the first half of the ground contact phase . These vIn the examples of This procedure resulted in (unfiltered) offset-corrected velocity signals (respectively red and blue for x and z direction in 2). The similarity between SLs obtained from video and IMUs was also investigated with Bland–Altman (BA) analyses (bias ± limits of agreement (LOA)). The BA procedure that was used accounted for the repeated measures within participants [Statistical analysis was done using Matlab  and R . Normal icipants .−1, 7.65 ± 0.77 m·s−1, and 8.42 ± 0.85 m·s−1 (p < 0.05). The respective mean values of the reference SLs obtained from video were: 3.49 ± 0.76 m, 3.46 ± 0.79 m, and 3.33 ± 0.75 m. Stride length depended on all investigated factors. There were significant main effects on SL of execution effort , method , and stride number . In addition, the following significant interaction effects were present: execution level x method , execution level x stride number , and method x stride number .Maximal stride speeds (mean ± SD) reached near the 30 m mark during sprints executed at 60, 80, and 100% effort, respectively, were: 7.02 ± 0.80 m·sp = 0.75). However, at 80% (p = 0.006) and 100% effort (p < 0.001), IMU-derived SLs differed significantly from video-derived SLs, but the respective effect sizes were small (ƞ2 = 0.005 and 0.05).Bland–Altman analysis C revealsp < 0.001), with large effects on sizes at all three levels of execution effort (ƞ2 > 0.91). This illustrates the well-known increase in SL during sprint acceleration. At 60% and 80% effort, the interaction effect between stride number and method (video or IMU) was not significant (p > 0.77). However, at 100% effort, this interaction was significant (p = 0.0005) and, although the effect size was small (ƞ2 = 0.02), this signifies that within sprints executed at 100% effort, the error of IMU-derived SL increased with stride number. There were four participants in particular  and SLs were considerably smaller, and their LOA as a percentage of average SL (0.15 m/2.59 m = 5.8%) were comparable to the 6.6% found in the present study. Moreover, in the present study, average peak speed near the 30 m mark was much higher (7.02 ± 0.80 m·s−1) and 649 of the 735 strides during sprints executed at 60% effort had stride speeds beyond the range investigated by Brahms et al. Together with the inclusion of the acceleration phase, this increased the challenge for accurate SL estimation in the present study. Zrenner et al. also didn’t include the acceleration phase in their runs, but they investigated SL estimations from foot-mounted IMUs over a wider speed range (2–6 m·s−1) compared to Brahms et al. [−1 of Zrenner et al. Nevertheless, our bias (−0.46 cm) and LOA (0.23 m) are very similar to theirs  between SLs estimated with a foot-mounted IMU and a gold standard motion capture reference during runs at constant speed in a laboratory setting . For sprs et al. . During igure 8e ). They ae 8e [−1 , which uParticipant number 1  signals are subject to inherent noise leading to errors that amplify during integration, causing errors in estimated velocity and, consequently, SL. To correct for integration drift, usually some kind of (linear) dedrifting technique is used that assumes that deviations of velocity from zero following integration of acceleration over one complete stride must be due to integration drift and should be corrected for . IgnorinDespite the critical notes made in the foregoing paragraphs, it is important to note that the errors in estimated SL are comparable to those reported in the literature for (much) slower runs executed at constant speed. We believe that the accuracy of estimated SL is good enough to be of practical relevance. Moreover, measurements with IMUs can easily be done without interfering with the athletes’ training session and the analysis is completed within a few minutes. In addition, many measurements can be made per individual athlete, which may further reduce random error and will provide athletes and coaches with the changes in SL and stride frequency  in response to training or during rehabilitation from injury. The use of wireless transmission would even make it possible to provide immediate feedback during training sessions.We conclude that reasonably accurate estimation of SL with foot-mounted IMUs during maximal linear sprinting is possible in recreational athletes. However, it remains to be established if, with the use of sensors with larger bandwidths, further improvement of sensor fixation and/or adaptation of the algorithm will suffice for accurate estimations of SL in more proficient sprinters.


Accumulating evidence suggests that the response of bacteria to antibiotics is significantly affected by the presence of other interacting microbes. These interactions are not typically accounted for when determining pathogen sensitivity to antibiotics. In this perspective, we argue that resistance and evolutionary responses to antibiotic treatments should not be considered only a trait of an individual bacteria species but also an emergent property of the microbial community in which pathogens are embedded. We outline how interspecies interactions can affect the responses of individual species and communities to antibiotic treatment, and how these responses could affect the strength of selection, potentially changing the trajectory of resistance evolution. Finally, we identify key areas of future research which will allow for a more complete understanding of antibiotic resistance in bacterial communities. We emphasise that acknowledging the ecological context, i.e. the interactions that occur between pathogens and within communities, could help the development of more efficient and effective antibiotic treatments. The global use and misuse of antibiotics has led to the evolution and spread of bacterial resistance to all routinely used antibiotics. In order to effectively tackle the spread of antimicrobial resistance (AMR) through antimicrobial stewardship or development of novel antimicrobial compounds, understanding how bacteria adapt and evolve to survive antibiotic treatments is crucial. Here we argue that to fully understand the evolution of AMR we must study the emergence of resistance within the context of microbial communities. While the composition, structure and interactions within microbial populations are known to affect the horizontal gene transfer (HGT) of antibiotic resistance –3, it isThe ability of a bacterial strain to survive antibiotic exposure is commonly assessed in monoculture measurements of growth, through broth microdilution or agar dilution assays, to quantify a minimum inhibitory concentration (MIC) . These iThe effect of ecological context, i.e., the type and dynamics of microbial interactions that occur between pathogens and the surrounding community, is often overlooked when considering antibiotic efficacy and the evolution of resistance. We currently predominantly consider antibiotic sensitivity as an independent trait of an individual strain. However, it is clear that inter- and intra-species interactions within both clinical and environmental communities, as well as the physical properties of the associated microbial community, can alter the physiological state and antibiotic susceptibility of bacteria . InterspTo discuss the effects of antimicrobial exposure on microbial communities we must first distinguish the different ways in which bacteria survive in the presence of antibiotics. Resistance is often defined as the inheritable ability of a cell to grow at high concentrations of an antibiotic, irrespective of the duration of exposure . Its mecCollective resistance, interactions within a community that elevate the ability of its members to resist the action of an antibiotic and continue to grow in the presence of antibiotics thus increasing the MIC of the community. (2) Collective tolerance, interactions within a community that alter cell state, such as slowing down metabolism, and thus slow down the rate of cell death during transient exposure to antibiotics without an increase in MIC. (3) Exposure protection, interactions within a community that protect its sensitive members during antibiotic treatment by reducing the effective concentration of antibiotic [Interspecies interactions can have a profound effect upon the outcome of antibiotic treatment. We define three main ways in which bacterial communities, or subpopulations of communities, can survive exposure to antimicrobials due to interspecies interactions: (1) tibiotic , 25. TheMany of the known mechanisms that provide collective resistance, tolerance or exposure protection are density-dependent, providing protection to large, dense populations  through Escherichia coli can protect Salmonella enterica serovar Typhimurium from concentrations of antibiotic that would usually kill them [Staphylococcus aureus or Streptococcus pneumoniae, via a plasmid borne acetyltransferase, allows the survival of sensitive S. pneumoniae through the detoxification of the local environment in liquid culture, on semi-solid surfaces and in in vivo mouse models [Resistance mechanisms that inactivate antibiotics are widespread, providing both acquired and intrinsic resistance to some of the most widely used antibiotics including aminoglycosides, chloramphenicol and β-lactams. Such resistance mechanisms can be considered as cooperative traits; the benefit of antibiotic inactiaving enzyme production is not confined to the producer, rather it is shared across the community , 32. Cooill them . The preill them ) suggeste models . Computae models . As a reCollective resistance, collective tolerance and exposure protection to antibiotics can also be provided through biofilm formation Fig. . BacteriPseudomonas aeruginosa can cause a metabolic shift in S. aureus, reducing its growth and providing S. aureus with protection to vancomycin [S. aureus can enhance tobramycin tolerance of P. aeruginosa by increasing aggregation and altering biofilm architecture in CF model systems [, i.e., direct cell contact versus diffusible molecules, as the physical properties of the community alter the probability of cell-cell contact and diffusion rates.Much work on biofilms has focused on the interspecies interactions within the lungs of Cystic Fibrosis (CF) patients, where multi-species biofilm formation is common. Within mixed biofilms ncomycin . Recipro systems . However systems . Whether systems  might deE. coli activates the expression of an indole-dependent multi-drug efflux pump in Pseudomonas putida, which cannot produce the indole itself, leading to elevated levels of resistance in P. putida [Stenotrophomonas maltophilia, a Gram-negative bacterium that often co-occurs with P. aeruginosa during polymicrobial pulmonary infection, alters P. aeruginosa biofilm structure and stimulates the synthesis of proteins that provide resistance to cationic antimicrobial peptides such as polymyxin [P. aeruginosa induces highly tolerant small colony variants of S. aureus through the secretion of 4-hydroxy-2-heptylquinoline-N-oxide, which inhibits the electron transport chain, dramatically slowing down the growth rate of S. aureus [Bacteria secrete a vast variety of compounds ranging from signalling molecules involved in quorum sensing to exotoxins involved in virulence and competition. Secreted products within communities have been shown to directly alter gene expression, metabolic processes and the growth dynamics of co-residing bacterial species, altering their ability to survive antibiotic exposure Fig. . For exa. putida . Likewisolymyxin . In addi. aureus , 56. GivCompetitive interactions, via interference or exploitation, are prevalent within mixed bacterial communities due to overlaps in metabolic requirements and limitation of space , 30, 57.How does a multi-species community alter the selection for AMR within its members? The answer to this question depends upon the members of the community in question and the specific interactions that occur between them. Multiple interactions — including competition, exploitation, commensalism and mutualism — take place in communities and each of them has the potential to alter the strength of selection acting upon resistance mechanisms of its members. Consequently, AMR evolution within communities may occur at different rates, result in different magnitudes of resistance, or be associated with different levels of cost compared to single species populations. The presence of interacting species could further affect the evolutionary trajectory of de novo resistance by changing which mutations and molecular mechanisms are selected for.E. coli strains embedded within the community. Mechanistically, it was shown that this increase in MSC was caused either by protection of the sensitive E. coli strain by the community  antibiotic susceptibility testing, where appropriate, should be conducted upon communities in addition to single-cell cultures because resistance is determined by the interactions taking place within that specific community; (2) This should be combined with analyses exploring the consequences of antibiotic treatments on community structure and functioning, which could further change community susceptibility to antibiotics during long term or repeated treatments which are common in chronic, polymicrobial infections; (3) The evolutionary responses to antibiotic treatment should not only focus on the focal pathogen but also upon the wider community in which it resides, as the community is likely to alter the selection dynamics of resistance or could act as reservoir for antibiotic genes. While the effect of ecological context upon antibiotic resistance is important in clinical environments, it should also be addressed in natural microbial communities that are regularly exposed to antibiotic residues through contaminated manure, sewage and wastewater. Moreover, it will also be necessary to move beyond bacterial interactions to also consider the role of multi-kingdom interactions in AMR in the future. Studies increasingly highlight the importance of microbial ecology in determining tolerance, resistance and evolutionary responses to antibiotic treatment, further consideration and quantification of interactions within bacterial community should be paramount.


Aconitum carmichaelii Debx.) is a traditional Chinese medicine that is cultivated in more than eight provinces in China. However, it can be easily devastated by post-harvest rot, causing huge losses. Therefore, it is extremely important that the primary causal pathogens of post-harvest Fuzi rot are identified and appropriate detection methods for them are developed to prevent and control losses. In this study, two bacterial strains (X1 and X2) were isolated from rotten post-harvest Fuzi. Based on their morphological, physiological, and biochemical characteristics, housekeeping gene homologies, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) results, these isolates were identified as Pseudomonas aeruginosa and Serratia marcescens. The pathogenicities of these isolates were confirmed by fulfilling Koch’s postulates demonstrating that they were post-harvest Fuzi rot pathogens. Two loop-mediated isothermal amplification (LAMP) methods targeting the gyrase B subunit (gyrB) gene of P. aeruginosa and the phosphatidylinositol glycan C (pigC) gene of S. marcescens were successfully developed, and it was found that the target genes were highly specific to the two pathogens. These LAMP methods were used to detect P. aeruginosa and S. marcescens in 46 naturally occurring Fuzi and their associated rhizosphere soil samples of unknown etiology. The two bacterial assays were positive in some healthy and rotten samples and could be accomplished within 1 h at 65°C without the need for complicated, expensive instruments. To our knowledge, this is the first report of P. aeruginosa and S. marcescens causing post-harvest Fuzi rot. The newly developed methods are expected to have applications in point-of-care testing for the two pathogens under different Fuzi planting procedures and will significantly contribute to the control and prevention of Fuzi rot.Fuzi (the lateral root of  Each meAconitum carmichaelii Debx. is a traditional Chinese medicinal plant  and Jiangyou (Sichuan Province), A. carmichaelii is often planted in the first November and harvested the following summer (from late June to early August). Under high temperature and humid conditions, post-harvest Fuzi is quickly decays within 12 h (2) to prevent decay /mL] or distilled and deionized water (ddH2O) as a blank control (n = 15 Fuzi per treatment consisting of three replicates). The inoculated Fuzi were then placed in a plant growth chamber (30°C and 70% relative humidity) for 15 days, and any apparent symptoms were recorded. When the visible rot zone beyond the wounded area on each Fuzi surface was more than 2 mm wide, it was scored as infected. The incidence of disease was calculated and expressed as a percentage (%), using the formula:In July 2016, rotten and healthy post-harvest Fuzi materials were collected from Mianyang (Sichuan Province), Lijiang (Yunnan Province), and Hanzhong (Shaanxi Province) in China. Diseased tissue was excised from the rotten root and disinfected in 70% ethanol solution for 20 s, soaked in 0.1% NaOCl for 50 s, and rinsed in sterilized water three times. They were then cut with a sterilized blade, and a small block was taken from the inside of each sample and cultured on Luria-Bertani (LB) agar at 37°C. This resulted in 10 bacterial strains being isolated which were further cultured with LB liquid medium. Surface-sterilized healthy Fuzi were each inoculated with one of the isolate suspensions , two outer primers (forward primer F3 and backward primer B3), and two loop primers [loop forward primer (LF) and loop backward primer (LB)] , 1 × ThermoPol® Reaction Buffer , 8 mmol/L of MgSO4, 1 mol/L of betaine , 1.6 mmol/L of each deoxynucleotide (dNTP), 0.2 μmol/L of each outer primer (F3 and B3), 0.4 μmol/L of each LF and LB primer, 0.8 μmol/L of each inner primer (FIP and BIP), 8 U of Bst DNA polymerase , and 5 μL of DNA template. The LAMP reaction mixture was heated at 65°C for 60 min and the reaction was terminated by heating at 85°C for 5 min. The color of the solution, which was observed with the naked eye under blue light (470 nm), turned bright green in the presence of LAMP products and remained dark red in the absence of amplification. PCR reactions were conducted as described above. In addition, 3 μL of the products of both the LAMP reaction and PCR amplification were further detected on a standard 1% agarose gel.The specific LAMP primer sets of X1 and X2 were designed to target the housekeeping genes of er (LB)] . The PCRer (LB)] . All priThe two isolates X1 and X2, and the other closely related bacterial strains  were culA total of 46 healthy and rotten post-harvest Fuzi, and their associated rhizosphere soil samples were collected in Mianyang (Sichuan Province), Lijiang (Yunnan Province), and Hanzhong (Shaanxi Province) . Both X1All of the physiological and biochemical analyses, LAMP method establishment procedures, and sample assays were repeated at least four times. The data were analyzed through the chi-square test to determine whether the observations meet the theoretical expectations. The significance level when calculating the chi-square value is set to 0.05.2O and the 8 candidate bacterial isolates showed no rotten apparent symptoms. However, Fuzi inoculated with the two isolates X1 and X2 showed visible rotten symptoms, and the incidences of disease were 100% which were different significantly with the control groups . The Fuzi that were inoculated with isolate X1 showed symptoms that were identical to those observed in rotten post-harvest Fuzi, with the appearance of yellow lesions that were sunken and contained mucus  (gyrB of X1 (MW691198) shared 100% identity with P. aeruginosa CP030910.1 and JUVT01000188.1. The MALDI-TOF MS results indicated that X1 was P. aeruginosa (confidence > 99.0%)  and shared 100% identity with the other 33047.1) . Moreove> 99.0%) . TherefoThe Fuzi inoculated with isolate X2 also showed symptoms that were identical to those observed in rotten post-harvest Fuzi. Following inoculation with X2, red lesions appeared that were sunken, and the whole Fuzi was brown and soft . The indS. marcescens identified by an NCBI BLAST query (MT434357.1 and MN519524.1) (pigC of X2 (MW691199) showed 99.8% identity with the S. marcescens strains (CP0313161.1 and AP019009.1). The MALDI-TOF MS results indicated that X2 was S. marcescens (confidence > 99.0%)  and showed 99.8% identity with the 16S rRNA genes from several other strains of 19524.1) . Moreove> 99.0%) . TherefogyrB and a 1138-bp-long band from the housekeeping gene pigC were amplified in P. aeruginosa X1 (S. marcescens X2 (2O). Furthermore, bright green reaction products were only observed in the samples containing DNAs of P. aeruginosa X1  (Allium cepa) bulbs (Praecitrullus fistulosus) (Glycine max) and lettuce (Lactuca sativa) (S. marcescens can induce lesions and necrosis in tobacco (Nicotiana tabacum) leaves, wilt virus in carrot (Daucus carota), yellow vine disease in sunflowers (Helianthus spp.), leaf chlorosis and necrotic spots in squash (Cucurbita pepo var. styriaca), leaf spot disease in industrial hemp (Cannabis sativa), soft rot in ginger  rhizomes and many other diseases  , internaa) bulbs , fruit rtulosus) , and lea sativa) . Moreovediseases . In thisP. aeruginosa and S. marcescens strains can be PGPBs or pathogens of humans, animals, or plants, these species are not typical plant pathogens as they do not ordinarily cause visible damage to plants unless they are subjected to moderate to high temperatures and high moisture conditions. In this study, P. aeruginosa X1 and S. marcescens X2 were confirmed as post-harvest Fuzi rot pathogens. Other research has shown that the capacity of clinical isolates of P. aeruginosa to induce rot in vegetables was indistinguishable from that of agricultural isolates , lettuce, potato (Solanum tuberosum), and tomato (Solanum lycopersicum)  . Moreovean cells .P. aeruginosa and S. marcescens have mainly focused on the human pathogen candidates. A number of methods have been used as diagnostic tools in clinical settings to identify P. aeruginosa in patient samples, such as culture-based assay, the high-throughput immunochemical method, the electrochemical detection method, and qRT-qPCR . MALDI-TOF MS, qRT-PCR, repetitive element palindromic PCR, and next-generation sequencing methods have also been used to detect S. marcescens and identify potential environmental sources of infections  can be found below: MF, XZ, and BC: formal analysis and writing. GZ: investigation. ML and LC: review, editing, and project administration. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To assess the productivity and visibility in research, clinical studies, treatment, use and production of antivenoms against poisonous snakes, scorpions and spiders.® v.1.6.13 was used to construct bibliometric networks for country co-authorships and co-occurrence of terms.Bibliometric analysis of research and other activities. Articles on venoms and antivenoms published between 2000 and 2020 were retrieved from the Scopus database. The records were analyzed by bibliometric indicators including number of documents per year, journals, authors, and citation frequency. VOSviewerr® allowed visualization through joint publications of networking between countries. Visualization by co-occurrence of terms showed differences in the research carried out.Australia, Brazil, Costa Rica and India were among the six top countries with most documents and were selected for more detailed analysis. Costa Rica was the country with the largest percentage of its publications dedicated to antivenom production and venomics. Only a few papers dealt with the issues of quality, safety, and efficacy of antivenoms or the role of the national regulatory authorities. The use of VOSvieweWorking in a collaborative and coordinated manner these four countries could have a major impact on envenoming globally. Attention should be given not only to antivenom production but also to strengthening regulatory oversight of antivenom products. Almost 2.5 million people annually undergo envenoming by snakes and other venomous animals such as spiders and scorpions of which nearly 120 000 die and 300 000 remain with some type of physical or psychological sequelae. This is such a public health problem that the World Health Organization (WHO) has included these medical emergencies within the neglected diseases for many tropical and subtropical countries . It is iThe bites of venomous animals can cause acute medical emergencies that can range from severe paralysis with respiratory failure, bleeding, fatal hemorrhages, irreversible renal damage, and/or tissue necrosis with subsequent permanent disability that may lead to the amputation of limbs. These effects are caused mainly by toxins produced and secreted by the animal’s glands ,8. Childst World Health Assembly adopted resolution WHA 71.5 formally providing the World Health Organization with a strong mandate to develop a comprehensive plan to support countries in implementing measures to increase access to effective treatment for people who get bitten by venomous animals , the largest abstract and citation database of scientific peer-review literature including more than 22 000 titles from international publishers. All types of documents were included in the analysis.Search strategy. The literature search was conducted by the authors in Scopus for publications using the following terms in the title, abstract and keywords fields in the following sequence: (a) snake OR viper OR scorpion OR spider, (b) antiven* OR antiser*, (c) selection of period 2000 to 2020 and final selection of (d) Australia, (e) Brazil, (f) Costa Rica, and (g) India. The documents were exported to an Excel database where the validity of the search strategy was tested by manually reviewing retrieved articles, and for conducting further analysis. Each publication of the Excel database was assigned a country of authorship based on affiliation of authors .Data analysis. Scopus provides tools to extract some bibliometric indicators that include number of documents per year, languages, countries, journals, authors, institutions, and citation frequency. It also allows the selection and analysis of the most cited articles.For citation analysis, the top five publications per country, where the principal author is from the country in consideration, were selected. Multi-country publications  were not considered in the citation analysis.Additional analyses of Scopus and Excel databases were conducted to identify publications related to antiserum/antivenom production, by searching for specific terms such as production, manufacture, and polyvalent sera. Another search was done in relation to the terms “omic” OR “proteomic” OR “venomic” in the titles and abstracts of the publications. The list of publications selected through Scopus was further searched for the following words: “quality” AND “safety” AND “efficacy” to select those publications that could have some link to regulatory issues.Considering that “cross-reactivity” can be of great help for the development or improvement of antivenom manufacturing, the term was searched in the abstracts of all the publications from the four countries. In addition, the term “neutralization” was also searched, and the abstracts were revised manually to select those compatible with the concept of cross-reactivity.Visualization of bibliometric indicators. For the construction and visualization of bibliometric networks the software VOSviewer® v.1.6.13 for Microsoft Windows  (wer.com)  was used® thesaurus file for terms was created to merge terms that are synonyms, to correct spelling, to merge abbreviated terms with full terms, or to ignore terms that are not specific or are too general.A VOSviewerResults from the search. The first search, using the terms a) snake OR viper OR scorpion OR spider provided 101 736 documents, followed by b) antiven* OR antiser* that reduced the amount to 9 428 documents. By selecting the period 2000 to 2020 there was a further reduction to 6 375 documents. Australia, Brazil, Costa Rica and India were among the six top countries with most documents, together with the United States and the UK, and were selected for more detailed analysis. After screening the information collected , the following summary can be made: 560 articles from Australia were published in 153 journals, 1 183 articles from Brazil in 278 journals, 342 articles from Costa Rica in 83, and 719 from India in 286 journals. The five principal journals for each country analysis are shown in Supplementary material), an analysis was performed to identify multi-country publications. The principal five collaborators in multi-country publications for Australia were the United Kingdom, United States, Sri Lanka, Costa Rica and Papua New Guinea with 64, 56, 43, 22 and 18 publications, respectively. For Brazil, the collaborators were Costa Rica, United States, France, United Kingdom and Spain with 72, 61, 43, 42 and 29 publications, respectively. For Costa Rica, the five main collaborating countries were Spain, Brazil, United States, Colombia and the United Kingdom with 75, 72, 42, 38 and 37 publications, respectively. In the case of India, the collaboration was with United States, United Kingdom, Australia, Pakistan and Singapore with 48, 13, 11, 10 and 9 publications, respectively. From the assignment of country of authorship based on affiliation of authors , Brazil had 78 publications (6.6%), Costa Rica had 88 (25.7%) and India had 40 publications (5.6%).Publications related to omics, proteomics and venomics. To identify the status of use and application of these technologies and concepts by the four countries, a search for the terms “omic” OR “venomic” OR “proteomic” was conducted in titles and abstracts of the publications. Costa Rica was the country with the largest percentage of its publications containing the terms, 21.9% in titles and 31.3% in abstracts. For Australia, Brazil, and India, these values were 5.4% and 12.9%, 4.5% and 10.7%, and 3.3% and 10.6%, respectively.Cross-reactivity. Searching for studies on cross-reactivity, the keywords used were “cross-reactivity” and “neutralization”. The selected publications were analyzed manually and yielded 31 publications (5.5%) for Australia, 88 (7.4%) for Brazil, 63 (18.4%) for Costa Rica and 28 (3.9%) for India.Regulatory issues. The search for publications covering regulatory issues identified 14 publications from Australia, 13 from Brazil, 33 from Costa Rica and 10 from India that once combined in one database, represented 61 publications. Looking into the contents of the abstracts, 23 publications were confirmed as being closer to the topic searched.Collaborative network among countries. VOSviewer® v.1.6.13 extracted countries from the Scopus databases for Australia, Brazil, Costa Rica, and India. Selecting for countries with at least 5 documents, the collaboration map based on co-authorship shown in Co-occurrence of terms. For the construction of the diagram of co-occurrence of terms, from each country individual database, the software identified initially in the titles and abstracts of the publications a large set of terms.In the case of Australia, from the initial total of 12 245 terms, 164 qualified as appearing in at least 15 publications (15 co-occurrences) by binary counting (presence or absence of the term is what counts). The number was further reduced to the 125 more relevant terms. In In the case of Brazil, from the initial total of 23 921 terms, 503 qualified as appearing in at least 15 publications (15 co-occurrences) by binary counting. The number was further reduced to the 300 more relevant terms. In For Costa Rica, from the initial total of 8 523 terms, 144 appeared in at least 15 publications (15 co-occurrences) by binary counting, and the number was further reduced to the 125 more relevant terms. In Finally, for the case of India, from the initial total of 13 065 terms, 198 qualified as appearing in at least 15 publications (15 co-occurrences) by binary counting and the number was further reduced to the 125 more relevant terms. The pattern for India differs from those of the other countries and shows only two main clusters, the red cluster consolidating the terms related to the venom, composition, structure and activity and the green cluster identified with clinical symptoms and management . A few i® that provides us with the option of constructing and visualizing maps of networks of co-authorships and of the relevant terms extracted from titles and abstracts of the publications. With these tools we were able to follow the evolution and status of the field of envenoming in Australia, Brazil, Costa Rica, and India. Besides being among the top publishers of articles on snake, spider, and scorpion envenomation, these countries also have a tradition of antivenom production and use, and represent an important global resource for assisting other countries with similar problems but low research activity and access/availability to antivenoms.Bibliometric analysis is a tool that can be used to perform an objective evaluation of a scientific activity and to measure productivity ,14, as wToxicon, Toxins, Plos Neglected Tropical Diseases, Journal of Proteomics and Journal of Venomous Animals and Toxins including Tropical Diseases were the main journals involved in publishing research on venoms and antivenoms.It is important to mention that the present study only deals with envenoming by snakes, spiders and scorpions although it is recognized that other group of animals  produce Interestingly, two out of the five most cited papers for Brazil and India see  were relThe results of the analysis also show that the selected countries have made important advances in omics technologies such as proteomics, transcriptomics, and genomics –18, partIn general, there was significant work over the twenty-year period investigated on antivenom production, such as innovation and improvement of production processes, changing antigens used in immunizing animals  or usingThe visualization map of the most frequent terms found in the titles and abstracts of the publications of the four countries shows the main clusters of the research published . Except Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins (It was expected that using the three terms quality, safety, and efficacy together one would select publications linked to regulatory authorities or regulatory issues. While in several of these selected publications there is a concern that products are being used in many countries all over the world that lack the basic principles of quality and efficacy and there are serious doubts about their safety and efficacy , at the lobulins , which eThe analysis also highlights the remarkable and intensive intercountry collaboration that has occurred in this field. This is seen mainly in the work of Australia, Brazil and Costa Rica which have developed extensive cooperation with several countries in assisting antivenom production and providing other technical assistance. This is clearly visualized in Recognizing further that snakebite envenoming has been categorized by WHO as a high priority neglected tropical disease, the WHA71.5 Resolution urged Member States to promote the transfer of knowledge and technology between Member States in order to improve the global availability of antivenoms and the effective management of cases and requested the WHO Director General to foster international efforts aimed at improving the availability, accessibility and affordability of safe and effective antivenoms for all .One limitation of the analysis is that the articles considered were those that were published in journals indexed in Scopus. Although Scopus contains over 40 000 serial titles and all Med Line documents, it is possible that some national, local, and regional titles are not indexed and thus the results may not reflect the totality of research being carried out in the countries. Nevertheless, we believe most high impact publications were captured.Results from this bibliometric analysis show that there is sufficient knowledge, expertise, and capacity to organize a coordinated international effort to address these issues. Australia has been helping countries in South East Asia; Brazil has supported African and Latin American countries; and Costa Rica has gone further to produce antivenoms for Central America, Sub Saharan Africa, and Papua New Guinea and has established an important transfer of technology with different countries. India has also its share of collaboration in its subregion. During the process to select the countries for the study it was clear that, measured by the number of publications, there are many other low- and middle-income countries where envenoming is a public health issue and the scientific and the clinical communities are not as involved in conducting relevant research. Promoting and establishing strategic alliances between these communities and involving other national or international stakeholders could be the basis for the development and application of knowledge into solving envenoming problems in particular contexts.There is, however, a need for a centralized and concerted coordination of activities  with theRPSP/PAJPH and/or PAHO.The authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the