Myocardial infarction (MI) leading to heart failure displays an important cause of death worldwide. Adequate restoration of blood flow to prevent this transition is a crucial factor to improve long-term morbidity and mortality. Novel regenerative therapies have been thoroughly investigated within the past decades.Increased angiogenesis in infarcted myocardium has shown beneficial effects on the prognosis of MI; therefore, the proangiogenic capacity of currently tested treatments is of specific interest. Molecular imaging to visualize formation of new blood vessels in vivo displays a promising option to monitor proangiogenic effects of regenerative substances.Based on encouraging results in preclinical models, molecular angiogenesis imaging has recently been applied in a small set of patients. This article reviews recent literature on noninvasive in vivo molecular imaging of angiogenesis after MI as an integral part of cardiac regeneration. Heart failure following myocardial infarction (MI) still displays a major cause of death and disability worldwide . Even thAngiogenesis represents the growth of new capillaries from preexisting vessels . It is aInsufficient vessel maintenance can lead to MI . Intact Molecular imaging describes in vivo targeted, noninvasive visualization and quantification of various molecular pathways without interfering with them –18. Thro18F (in form of 18-fluorodeoxyglucose), 18F-FDG, is the most frequently employed PET tracer ATV-1 was assessed in a rat model of MI. Standard uptake values of [11C]ATV-1 correlated with immunohistochemical staining of VEGFR-2, Tie-2, and PDGFα [Several tyrosine kinases are upregulated in heart tissue undergoing angiogenesis and remodeling after MI. Immunohistochemical analyses of MI samples revealed increased levels of VEGFR-2, Tie-2, and PDGFα suggesting its use as an angiogenesis marker in non-invasive molecular imaging. ATV-1 can act as an inhibitor of those kinases. PET imaging with [nd PDGFα .Even though proangiogenic therapies have so far largely failed as an effective treatment of MI, targeting angiogenesis after MI to mitigate heart failure is still considered a promising strategy. In order to assess the success of such therapeutic interventions in the clinic or in preclinical development, reliable and sensitive noninvasive imaging modalities are needed. Molecular imaging of angiogenesis via PET, SPECT, MRI, and CEU has been investigated intensively within the past decade. A variety of tracers have been translated from tumor angiogenesis models to MI models, and promising results were achieved. These methods offer the unique opportunity to study in vivo molecular mechanisms characterizing myocardial healing after infarction and to evaluate angiogenic effects of regenerative treatments. Combination of high sensitivity PET and SPECT with high-resolution X-ray CT images allows better identification and quantification of tracer uptake within the region of interest. Multimodal imaging with highly specific tracers yields reliable and detailed data of cardiac angiogenesis in small and large animal models and also in humans. Because PET and SPECT imaging use ionizing radiation, these imaging modalities might also expose patients to a risk of growing neoplastic lesions. For clinical applications, further research is warranted to develop radiotracers with a reasonable level of ionizing radiation or even replacing PET and SPECT with MRI or CEU while still featuring high affinity for visualizing growing blood vessels. Continuing development of noninvasive imaging modalities for future clinical applications may enable improved patient risk stratification and pave the way for personalized therapy.


A set of computational methods that contains a brute-force algorithmic generation of chemical isomers, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations is reported and applied to investigate nearly 3000 probable molecular structures of polydopamine (PDA) and eumelanin. via π–π interactions to form graphite-like layered aggregates. We also show that there is a group of tetramers notably more stable than the others, implying that even though there is an inherent chemical diversity in PDA and eumelanin, the molecular structures of the majority of the species are quite repetitive. Our results also suggest that larger oligomers are less likely to form. This observation is also consistent with experimental measurements, supporting the existence of small oligomers instead of large polymers as main components of PDA and eumelanin. In summary, this work brings an insight into the controversial structure of PDA and eumelanin, explaining some of the most important structural features, and providing a set of molecular models for more accurate modeling of eumelanin-like materials.A set of computational methods that contains a brute-force algorithmic generation of chemical isomers, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations is reported and applied to investigate nearly 3000 probable molecular structures of polydopamine (PDA) and eumelanin. All probable early-polymerized 5,6-dihydroxyindole (DHI) oligomers, ranging from dimers to tetramers, have been systematically analyzed to find the most stable geometry connections as well as to propose a set of molecular models that represents the chemically diverse nature of PDA and eumelanin. Our results indicate that more planar oligomers have a tendency to be more stable. This finding is in good agreement with recent experimental observations, which suggested that PDA and eumelanin are composed of nearly planar oligomers that appear to be stacked together  PDA shares chemical functionalities  with mussel adhesive proteins such as Mytilus edulis foot protein-5 (Mefp-5).2 Mussels can attach to various types of surfaces with high bonding strength, even under harsh marine conditions. Such extraordinarily robust adhesion is due to the presence of significant amounts of catechol and amine-rich amino acids in their threads.5 Accordingly, synthetic materials containing these functional groups are the object of growing interest, not only for adhesives but also for many other applications. PDA is a mussel-inspired material since its fundamental building blocks contain both catechol and amine groups. As with mussel threads, PDA can spontaneously deposit on the surface of almost any material regardless of its chemical nature. Therefore, PDA has drawn extensive interest as a versatile surface functionalization and coating material for a broad range of applications.7 Surface coating provides protection of the underlying material against external erosion by agents such as strong oxidants and acids. Furthermore, surface modification can change the surface properties and create additional functionalities of the underlying material. Due to the advantages mentioned above, PDA has entered the scene of materials science in recent years, with applications that go beyond surface coatings,9 into the fields of solar energy,10 water purification,11 shape memory polymer,12 microrobots,13 biomedicine,16 and nanotechnology.17Polydopamine (PDA), first reported by Messersmith, Lee, and co-workers in 2007,19 scanning tunneling microscopy (STM) measurements,22 atomic force microscopy (AFM) and transmission electron microscopy (TEM) images24 suggested the existence of small nearly planar oligomers that appear to be stacked together via π–π interactions to form graphite-like layered aggregates. However, proper explanations for the small molecular size of these oligomers and their detailed molecular structures have not been provided yet. As a result, unlike the vast majority of natural pigments , eumelanin cannot be described in terms of a well-defined structure, but rather as a polydisperse mixture of oligomers. The structural investigations of eumelanin are challenging due to its amorphous characteristic as well as insolubility in water and most organic solvents. Therefore, it is currently impossible to provide an accurate picture of eumelanin structure beyond a description of its fundamental building blocks, which are 5,6-dihydroxyindolequinone (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA).26 In addition, very little information is available regarding the further evolution of these building blocks to eumelanin structure, and its polymerization mechanism is also ambiguous.On the other hand, eumelanin, the most common type of melanin, is a natural pigment in the human skin, hair, and eyes. Eumelanin shares many physiochemical properties with PDA due to their similarities in structures and chemical functionalities. Eumelanin is an essential pigment in most organisms and has been studied for decades. X-ray diffraction studies,27 However, unlike eumelanin, DHICA-derived units are absent as building blocks in PDA.28 As a result, even though PDA is sometimes called synthetic eumelanin in literature,29 these two materials are not synonymous. In this work, to simplify the problem, we ignore the differences between PDA and eumelanin and consider them as similar materials composed of DHI and its redox forms. As with eumelanin,30 despite the significant effort on structural investigations of PDA since it was first reported in 2007, the structure of PDA has yet to be determined. Recently, an experimental study29 using solid state spectroscopic techniques suggested that PDA is a supramolecular aggregate of DHI monomers, which are held together by a combination of charge transfer, π-stacking, and hydrogen bonding interactions. Another experimental study31 using high-performance liquid chromatography-mass spectrometry (HPLC-MS) reported that a physical, self-assembled trimer of (dopamine)2/DHI exists in PDA. However, it is generally accepted in this field that the polymerization of DHI generates a group of covalent oligomers including dimers, trimers, tetramers, and even larger oligomers. From the chemical analysis, it is clear that the existence of various redox forms of DHI monomers, but the way such monomers connect together to form oligomers is still unclear due to the formation of numerous intermediates during the polymerization. This variety of oligomers and redox forms that constitute PDA and eumelanin is referred to as chemical disorder model.32The lack of precise knowledge on the molecular structures and polymerization mechanism of eumelanin does not stop the applicability of synthetic eumelanin-like materials, among which PDA is the most popular one. Dopamine, the main constituent of PDA, stands as a natural choice for synthesizing universal coating materials since it is the simplest building block containing the required catechol and amine groups. DHI, the intermediate oxidized product of dopamine, is the precursor of PDA and has been successfully synthesized and characterized in experiments. Consequently, the fundamental building blocks of PDA are DHI and its redox forms, as well as a portion of uncyclized amine-containing units. A recent study suggested that increasing the dopamine concentration while synthesizing PDA leads to higher proportions of uncyclized amine-containing units. On the other hand, lower dopamine concentrations lead to higher levels of DHI.22 A recent study based on mass spectroscopy results of DHI-melanin also suggested that the majority constituents of the material are tetramers and pentamers of DHI.32 Consequently, we narrow down our investigation to DHI oligomers up to tetramers. Within the framework of this bottom-up approach and considering the location of the most reactive positions of DHI, we systematically generate and analyze all probable early-polymerized DHI oligomers, ranging from dimers to tetramers, to provide a fundamental explanation of some important structural features of PDA and eumelanin, as well as to propose a set of molecular models that represents the chemically diverse nature of these materials.The absent of proper molecular models for PDA and eumelanin implies a fundamental drawback that hampers a better understating, fast development, and property optimization of these materials. Although the science and applications of PDA-based materials have rapidly advanced in recent years, most studies in this field were based on empirical approaches instead of a solid framework of structure–function relationships due to this structural controversy. In this work, a set of computational methods, which includes a brute-force algorithmic generation of chemical isomers, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations, is implemented to investigate molecular structures of PDA and eumelanin. The investigation begins with analyzing the reactivity of DHI and its related radical species that lead to the formation of PDA and eumelanin. The most reactive positions of DHI are identified, and a possible polymerization mechanism of DHI in different redox forms based on spin density calculations, is also provided. In addition, early studies showed that the size of eumelanin protomolecules is around 15–20 Å, suggesting that tetramers and pentamers formed by covalently bonded DHI monomers are the most probable molecular sizes of eumelanin.35 In this mechanism, o-quinone groups react via aryloxy radical coupling with the unoxidized catechol to form crosslinks. This reaction scheme can be extrapolated to the case of PDA and eumelanin formation, for which the catechol functional group is thought to be the main responsible of the polymerization. A detailed investigation of the reaction mechanisms of DHI that lead to the formation of DHI oligomers, including a complete description of potential energy surfaces, activation energies, and reaction pathways, goes beyond the scope of this work. Despite such complexity, we still can analyze the reactivity of DHI to form radical species within the context of some accepted mechanisms of catechol polymerization, as well as describe the spin density and radical resonant structures that lead to the expected connections between DHI monomers. Therefore, to support the prevalence of certain atomic connections in the formation of DHI oligomers, and to provide some insight into the polymerization process that leads to the molecular structures of eumelanin-like materials, DFT calculations are performed for different redox forms of DHI. 37 functional is adopted for optimizing the molecular structures of these 18 dimers, together with the def2-QZVP38 basis set . After discarding higher energy conformations, the most stable dimer is made through 2,2′-position, followed by 2,4′-position , 4,4′-position , 4,7′-position , 2,7′-position , and 7,7′-position .Based on the results discussed in the previous section, the unpaired electron in the radical species, a key player in the polymerization mechanism of DHI, is mainly localized in 2-, 4-, and 7-position. The result implies that the covalent bonds connecting DHI monomers to generate DHI oligomers are more likely to form at these positions. As a result, there are 6 structural unique dimers , which are made through 2,2′-, 2,4′-, 2,7′-, 4,4′-, 4,7′-, and 7,7′-position. Nevertheless, to evaluate the stability of all possible connectivity, a total of 18 dimers are generated using a brute-force algorithmic generator . Fig. S140 In addition, the dimer made through 4,7′-position . After discarding those structures with higher energy conformations, the top 12 stable trimers are identified, which are made through 2,4′- & 2,2′-position, 7,4′- & 2,2′-position , 2,2′- & 4,4′-position , 2,2′- & 7,4′-position , 2,2′- & 7,2′-position , 7,7′- & 2,2′-position , 2,4′- & 7,4′-position , 4,2′- & 4,4′-position , 4,2′- & 7,4′-position , 7,2′- & 4,2′-position , 2,4′- & 7,2′-position , and 4,2′- & 4,7′-position . 40Following the same strategy applied for dimers, a total of 216 trimers are generated using the brute-force algorithmic generator. Fig. S244 functional is adopted for optimizing the molecular structures of the tetramers, together with the def2-SVP38 basis . After removing the results of higher energy conformations, the systematic search for the most stable molecular structures of the tetramers is completed. Following the same strategy, the top 16 stable tetramers are identified, which are made through 4,2′- & 7,4′- & 2,2′-position, 2,2′- & 4,4′- & 2,2′-position , 2,2′- & 4,2′- & 4,4′-position , 2,2′- & 4,7′- & 2,2′-position , 2,2′- & 7,2′- & 4,4′-position , 2,2′- & 4,2′- & 7,4′-position , 2,4′- & 2,4′- & 2,2′-position , 2,4′- & 2,2′- & 4,2′-position , 2,2′- & 7,7′- & 2,4′-position , 2,2′- & 7,4′- & 2,7′-position , 2,2′- & 4,2′- & 4,7′-position , 7,2′- & 7,4′- & 2,2′-position , 2,2′- & 4,2′- & 7,7′-position , 2,2′- & 4,7′- & 4,2′-position , 2,4′- & 2,7′- & 2,2′-position , and 2,4′- & 2,2′- & 4,7′-position . The number of DHI oligomers that need to be investigated increases exponentially as the size increases. A total of 2592 tetramers are generated using the brute-force algorithmic generator. This group of tetramers covers all 162 structural unique tetramers as well as 2430 redundant tetramers that account for different conformations to find the most stable conformation of each structural unique tetramer. As with the calculations for the trimers, the BLYP45 These tetramers are made through 2,7′- & 2,7′- & 2,7′-position, creating an interior ring where all the nitrogen atoms are in the center, in an arrangement similar to porphyrin. Such arrangement was first proposed by Kaxiras26 and have been adopted to study the structural, mechanical, and optical properties of PDA and eumelanin in our previous computational work.47 However, this particular kind of molecular structures, in which an additional covalent bond is formed between the first and fourth monomers, is not considered in this work. The reason is that two hydrogen atoms need to be removed when an additional covalent bond. Therefore, the number of atoms in porphyrin-like tetramers is different from that in the tetramers considered in this work. Consequently, the energy comparison between tetramers with different numbers of atoms will be problematic. Despite PDA and eumelanin might contain porphyrin-like tetramers in certain concentrations, it is impractical to assume all tetramers are in the porphyrin-like arrangement. Therefore, considering other species of probable molecular structures that represent the chemically diverse nature of these materials, is important to perform more accurate simulations.We are aware of the evidence of porphyrin-like tetramers that has been found in natural eumelanin pigment as well as in PDA by using electrochemical fingerprinting.pn is the probability of finding the molecular structures that are made of the top n  stable molecules including their higher energy conformations, m is the total number of molecules , εi is the energy of molecule-i (here we use its relative energy), k is the Boltzmann constant, and T is the temperature (here we use 300 K). via non-covalent interactions such as the van der Waals and charge interactions. As a result, the new monomer has to compromise on a higher energy configuration when it attaches to an oligomer. In other words, the degree of freedom for adding a new monomer to an oligomer decreases as the number of DHI units in the oligomer increases due to overlapping electron clouds (steric effects). This result explains the existence of relatively small degrees of polymerization in PDA and eumelanin.via π–π interactions can be observed in experiments.46 Furthermore, only if the oligomers are able to stack together closely to form so-called secondary structures, the excitonic effects among the oligomers can be strong enough to produce the broadband absorption spectrum of PDA and eumelanin.47Using nearly planar molecular models to simulate the self-assembly mechanism of PDA and eumelanin is important. e.g., pentamers and hexamers), to consider different redox forms , and to include DHICA, in future studies when more computational resources are available.In this work, we discuss possible reaction mechanisms of DHI and identify its most reactive positions. For the first time, a first-principle approach is applied to provide a quantum chemical explanation for the existence of certain preferential connections between DHI monomers. Using reactivity descriptors such as the Fukui function and spin densities calculations, we support some of the assumed mechanisms of the DHI polymerization. These results are then adopted as an input for the brute-force algorithmic generator that generates all probable chemical isomers for small DHI oligomers. From a total number of nearly 3000 isomers, the most stable dimers, trimers, and tetramers of DHI are selected to propose molecular models that will help more accurate modeling of PDA and eumelanin. The relative energies between molecular models reported in this work can be applied to estimate a proper ratio of these molecules in simulations. We also provide fundamental explanations for some important structural features of PDA and eumelanin, such as the structural planarity and small size of oligomers. It is important to remark that we have evaluated DHI oligomers only up to tetramers and have not considered different redox forms due to computational limitations. Note that PDA and eumelanin contain a portion of oligomers larger than tetramers, and oxidation has a profound impact on the molecular structures. Consequently, this work cannot completely elucidate the controversial molecular structures of PDA and eumelanin. However, this work provides some new possible findings and explanations to the structural properties of eumelanin-like materials. Most importantly, the theoretical framework and brute-force algorithmic generator in this work can be easily applied to more realistic situations such as to explore larger oligomers  can form a covalent bond with orientation 1 at the 7-position. The second reactive position considered is the 2-position. There are also six orientations of a DHI monomer  can form a covalent bond with orientation 1 at the 2-position. The third reaction position considered is the 4-position. Similarly, there are also six orientations of a DHI monomer  can form a covalent bond with orientation 1 at the 4-position. Consequently, a total of 18 combinations of positions and orientations can be generated on the second layer of checkerboard plates. Combining the first and second layers of checkerboard plates, a total of 18 dimers can be generated. Fig. S3DHI oligomers are composed of various numbers of DHI units with different configurations. In this work, we use multi-layers of checkerboard plates to represent different polymerization degrees of DHI oligomers. For example, a tetramer can be generated with four layers of checkerboard plates . We cons49 with the consistent valence force field (CVFF) are adopted in this work. The CVFF has been widely applied in modeling organic molecules with aromatic rings such as benzene, polydopamine,50 and caffeine co-crystals.51 Energy minimization using the conjugate gradient (CG) algorithm is performed to relax the initial molecular structures. After energy minimization, MD simulations are performed to further equilibrate the molecular structures. The integration time step is 1.0 fs and the nonbonding interactions (12–6 Lennard-Jones and coulombic interactions) are computed with a cutoff of 12 Å. The MD equilibration includes 10 iterations of equilibrations in order to find the most stable geometry of each molecule. Each iteration contains four steps of the NVT ensembles. In the first step, a molecule is equilibrated with the NVT ensemble at a temperature starting from 1.0 K and increasing linearly to 300 K in 1.0 ps. In the second step, the molecule is equilibrated with the NVT ensemble at 300 K for 1.0 ps. In the third step, the molecule is equilibrated with the NVT ensemble at a temperature starting from 300 K and decreasing linearly to 1.0 K in 1.0 ps. In the final step, the molecule is equilibrated with the NVT ensemble at 1.0 K for 1.0 ps. A Langevin thermostat is performed to control the temperature. When the fourth step of the NVT ensembles is finished, the molecule is then relaxed with energy minimization again to calculate its energy. After that, its geometry and the corresponding energy are recorded. This set of the NVT ensembles is repeated 10 times. As a result, a total of 10 geometries and the corresponding energies are recorded for each molecule. The geometry with the lowest energy is then selected for further geometry optimizations with DFT. The MD equilibration scheme is shown in Fig. S4.Full atomistic MD simulations are performed to relax and equilibrate the initial molecular structures of DHI oligomers generated using the brute-force algorithmic generator. The large-scale atomic/molecular massively parallel simulator (LAMMPS)52 The dispersion correction DFT-D3 with Becke–Johnson damping (D3BJ)53 is implemented. In addition, PDA and eumelanin are both synthesized in water solution. Therefore, the SMD model,54 a continuum solvation model based on the quantum mechanical charge density of a solute molecule interacting with a continuum description of the solvent, is adopted to take water effects into account. The B3LYP37 functional is adopted for optimizing the molecular structures of the dimers, together with the def2-QZVP38 basis set. However, the B3LYP/def2-QZVP is too expensive for optimizing the molecular structures of the trimers and tetramers. As a result, the BLYP44 functional together with the def2-SVP38 basis set is adopted for optimizing the trimers and tetramers. A benchmark study is conducted to evaluate the accuracy of using the BLYP/def2-SVP for optimizing the molecular structures of DHI oligomers. The 18 dimers 56 to circumvent the BSSE. The improvement of using the BSSE-correction gCP is significant. The results (green bars) calculated using the BLYP/def2-SVP/gCP are quite close to the reference values (blue bars). The energy differences between using the B3LYP/def2-QZVP and BLYP/def2-SVP/gCP are within 1 kcal mol–1. Since the BLYP/def2-SVP/gCP can provide acceptable results and it is at least 500 times faster than the more accurate B3LYP/def2-QZVP, the BLYP/def2-SVP/gCP is adopted for the geometry optimizations of the trimers and tetramers. The energies obtained in the DFT calculations are used to benchmark the molecular structures of DHI oligomers.The geometries obtained from the MD equilibration are further optimized with DFT using ORCA quantum chemistry package.s Fig. S1 are usedTwo tetrameric models are used to study the self-assembly mechanism. For each model, eight tetramers are separated by a distance larger than 25 Å in the initial configuration, to ensure that there are no intermolecular interactions. After energy minimization, the system is equilibrated with the NVT ensemble at 300 K for 1.0 ns. The snapshots in


This Editorial refers to:Int. J. Mol. Sci. 2016, 17, 1916.Merlot, A.M.; Sahni, S.; Lane, D.J.R.; Richardson, V.; Huang, M.L.H.; Kalinowski, D.S.; Richardson, D.R. Letter to the Editor: Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques. Int. J. Mol. Sci. 2016, 17, 1917.Xu, Z.; Liu, Y.; Zhou, S.; Fu, Y.; Li, C. Response to Letter to the Editor by D. Richardson: Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques. Int. J. Mol. Sci. 2016, 17, 1915.Xu, Z.; Liu, Y.; Zhou, S.; Fu, Y.; Li, C. Correction: Analysis of the interaction of Dp44mT with human serum albumin and calf thymus DNA using molecular docking and spectroscopic techniques. Int. J. Mol. Sci. 2016, 17, 1042.Xu, Z.; Liu, Y.; Zhou, S.; Fu, Y.; Li, C. Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques.  Internal Journal of the Molecular Sciences (IJMS) publication, Xu and co-workers presented evidence on the pharmacological interaction of the anti-tumor agent di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) with human serum albumin (HSA) and DNA [In a recent  and DNA . The phaIJMS takes any complaint that is brought before us serious, IJMS started an investigation and asked Xu et al. to reply to the letter in detail [However, upon publication, this paper became the focus of controversy and was alleged to contain numerous important flaws and factually incorrect statements . Furthern detail  and assiThe overall consensus was that the alleged controversial nature of the paper was not significant enough to warrant a retraction or extensive correction. Xu et al. could certainly have used additional methods or more control experiments, but this is an issue that is not unique for this paper. We feel that the paper by Xu et al. provides new insights into the pharmacological action of Dp44mT and fits well within a climate that allows and stimulates scientific discussion. Nonetheless, two issues raised in the letter to the editor  were ideIJMS, its Editorial Board, the handling editors and MDPI uphold the highest publication standards. IJMS is a member of the Committee on Publication Ethics (COPE). We fully adhere to its Code of Conduct and to its Best Practice Guidelines . The current thorough investigation in which the opinion of external experts was solicited exactly demonstrates the adherence to such high standards.Finally,


Telmatospirillum siberiense 26-4b1 was isolated from a Siberian peatland. We report here a 6.20-Mbp near-complete high-quality draft genome sequence of T. siberiense that reveals expected and novel metabolic potential for the genus Telmatospirillum, including genes for sulfur oxidation.The facultative anaerobic chemoorganoheterotrophic alphaproteobacterium  Telmatospirillum siberiense were isolated from a mesotrophic Siberian peatland , the type strain of the only validly described species of this genus (n = 4) reassembled with SPAdes (version 3.11.1) using contigs >1 kb from the previous assembly as the “trusted contigs” input. The draft genome sequence consists of 81 scaffolds, with a total size of 6,202,994 bp, a G+C content of 62.3%, and an N50 value of 131,736 bp. Based on CheckM  were also observed (T. siberiense encodes the Embden-Meyerhof-Parnas (glycolysis) pathway, pentose phosphate pathway, Entner-Doudoroff pathway, and oxidative tricarboxylic acid cycle. It possesses genes of lactate dehydrogenases, for mixed acid fermentation, and several pathways for monosaccharide degradation. Three nitrogenase operons (two [FeMo]nitrogenases and one [FeFe]nitrogenase) and three [NiFe]hydrogenases  (bd and cbb3 types) that enable the aerobic growth of T. siberiense at low oxygen concentrations. The motility of T. siberiense is explained by flagellar genes and autotrophic growth by genes encoding the complete Calvin-Benson-Bassham cycle. Acidotolerance (kdpABCD) and a potassium uptake system (ktrAB) (observed . As expe and 2b)  were ide (ktrAB) .T. siberiense for sulfur oxidation. A partial thiosulfate-oxidizing machinery (soxEFXYZA) and the sulfur-shuttling system DsrEFH were identified. The partial sox operon is syntenic with the operon structure of the alphaproteobacterial thiosulfate oxidizer Starkeya novella (soxBCD is located separately. No other sox or dsr genes are present in the draft genome. Experimental evidence is needed to confirm a potential role of T. siberiense in sulfur cycling.Surprisingly, the genome analysis revealed a possible metabolic potential of  novella , in whicTelmatospirillum siberiense 26-4b1 was deposited in GenBank under the accession number PIUM00000000.The draft genome sequence of


Cerebral Oxygenation and the Patent Ductus ArteriosusThe hemodynamically significant patent ductus arteriosus (PDA) remains a controversial topic. Clinicians and researchers are still debating whether or not it should be treated, what the best treatment strategy is and when would be the best time to intervene (48–51).48.Pediatrics (2000) 106(4):625–32. doi:10.1542/peds.106.4.625Tsuji M, Saul JP, du Plessis A, Eichenwald E, Sobh J, Crocker R, et al. Cerebral intravascular oxygenation correlates with mean arterial pressure in critically ill premature infants. 49.Stroke (2010) 41(9):1957–62. doi:10.1161/STROKEAHA.109.575167 PMID:20651273Brady KM, Mytar JO, Lee JK, Cameron DE, Vricella LA, Thompson WR, et al. Monitoring cerebral blood flow pressure autoregulation in pediatric patients during cardiac surgery. 50.Pediatrics (2008) 121(3):e604–11. doi:10.1542/peds.2007-1487 PMID:18250118Wong FY, Leung TS, Austin T, Wilkinson M, Meek JH, Wyatt JS, et al. Impaired autoregulation in preterm infants identified by using spatially resolved spectroscopy. 51.Adv Exp Med Biol (2016) 876:501–9. doi:10.1007/978-1-4939-3023-4_63 PMID:26782251Caicedo A, Alderliesten T, Naulaers G, Lemmers P, van Bel F, Van Huffel S. A new framework for the assessment of cerebral hemodynamics regulation in neonates using NIRS. Reference 48–51 are:–Curr Opin Pediatr (2015) 27(2):158–64. doi:10.1097/MOP.0000000000000200Perez KM, Laughon MM. What is new for patent ductus arteriosus management in premature infants in 2015? –Semin Fetal Neonatal Med (2015) 20(4):272–7. doi:10.1016/j.siny.2015.03.004 PMID:25818393Evans N. Preterm patent ductus arteriosus: a continuing conundrum for the neonatologist? –Indian Pediatr (2014) 51(4):289–94. doi:10.1007/s13312-014-0403-2 PMID:24825266Ibrahim TK, Haium AA, Chandran S, Rajadurai VS. Current controversies in the management of patent ductus arteriosus in preterm infants. However, these references are supposed to be:The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.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.


Our understanding of the contribution exposure to environmental toxicants has on neurological disease continues to evolve. Of these, Parkinson’s disease (PD) has been shown to have a strong environmental component to its etiopathogenesis. However, work is still needed to identify and characterize environmental chemicals that could alter the expression and function of the nigrostriatal dopamine system. Of particular interest is the neurotoxicological effect of perfluorinated compounds, such as perfluorooctane sulfonate (PFOS), which has been demonstrated to alter aspects of dopamine signaling. Using in vitro approaches, we have elaborated these initial findings to demonstrate the neurotoxicity of PFOS to the SH-SY5Y neuroblastoma cell line and dopaminergic primary cultured neurons. Using an in vivo model, we did not observe a deficit to dopaminergic terminals in the striatum of mice exposed to 10 mg/kg PFOS for 14 days. However, subsequent exposure to the selective dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly reduced the expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH), and resulted in an even greater reduction in DAT expression in animals previously exposed to PFOS. These findings suggest that PFOS is neurotoxic to the nigrostriatal dopamine circuit and this neurotoxicity could prime the dopamine terminal to more extensive damage following additional toxicological insults. As a greater number of chemicals are manufactured and used in a variety of industrial processes and consumer products, our appreciation for their impact on human health has also increased. In particular, the nervous system appears to be uniquely vulnerable to damage following exposure to environmental toxicants, especially highly halogenated compounds ,2. IndeeMore recently, attention has been directed towards another class of halogenated chemicals, perfluorinated compounds (PFCs), of which perfluorooctane sulfonate (PFOS) is a major constituent . These cPrevious laboratory work has demonstrated the neurotoxicological effects of exposure to PFOS as well as other PFCs ,26,27,28Given our current understanding of the role exposure to environmental chemicals plays in the etiopathogenesis of PD, it is critical to extend upon these findings and further characterize the neurotoxicological effects of emerging environmental chemicals on the nigrostriatal dopamine circuit. With these ideas in mind we designed this study to specifically assess the effect of PFOS exposure on the nigrostriatal dopamine pathway through the coupling of in vitro and in vivo models. Through these approaches we found PFOS to be selectively neurotoxic to dopamine neurons, in vitro. These findings were extended, in vivo, using a subacute exposure paradigm that allowed us to evaluate PFOS neurotoxicity using adult male mice. Although exposure to PFOS did not elicit overt damage to the presynaptic dopamine terminal in the striatum, damage was exacerbated following challenge with the selective dopamine neurotoxin, methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Moreover, this potentiation may be due to the ability of PFOS to inhibit the sequestration of dopamine into synaptic vesicles. These findings suggest that PFOS can preferentially damage the nigrostriatal dopamine system and, more importantly, increase the vulnerability of the dopaminergic synapse to additional toxicological insults through alteration of dopamine handling.3H-Dopamine (3H-DA) was purchased from Perkin-Elmer . Cold tetrabenazine was obtained from Sigma-Aldrich.Perfluorooctane sulfonate (PFOS) was purchased from Sigma-Aldrich . HEK293 and SH-SY5Y cells were obtained from American Type Culture Collection . Hibernate A and Hibernate A-Calcium were purchased from BrainBits . B27, DNase1, and Neurobasal A were purchased from Life Technologies . Papain was obtained from Sigma-Aldrich. Dispase II was purchased from Roche . Aphidicolin was purchased from A.G. Scientific . Whatman GF/F filter papers were obtained from Brandel, Inc. . The bicinchoninic acid (BCA) protein assay kit was obtained from Pierce . 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was purchased from Sigma-Aldrich. Monoclonal anti-rat dopamine transporter (DAT) and polyclonal anti-rabbit tyrosine hydroxylase (TH) antibodies were purchased from EMD Millipore  or Pel Freez Biologicals . Polyclonal anti-rabbit gamma-aminobutyric acid (GABA) transporter 1 (GAT1) and vesicular glutamate transporter (vGlut) antibodies were purchased from Synaptic Systems . Monoclonal anti-mouse α-tubulin antibodies were purchased from Sigma-Aldrich. Mouse anti-microtubule associated protein 2 (MAP2) antibodies were purchased from Abcam . Secondary antibodies conjugated to horseradish peroxidase were obtained from Jackson Immunoresearch Laboratories . Secondary antibodies conjugated to fluorescent tags were obtained from Life Technologies. SuperSignal West Dura extended duration substrate and stripping buffer were obtained from Pierce. 3,3′ Diaminobenzidine (DAB) was purchased from Sigma-Aldrich. The lactate dehydrogenase (LDH) Assay Kit was obtained from Cayman Chemicals . 2 and propagated according to the protocol provided by the supplier. When cells were confluent, they were trypsinized and collected for passage to working concentrations in 96-well culture plates for treatment with PFOS.SH-SY5Y cells were originally derived from the SK-N-SH neuroblastoma cell line, which was isolated from human bone marrow. These cells were chosen for these experiments given their general neuronal phenotype and dopaminergic properties, including enzymes necessary for dopamine synthesis and metabolism, as well as dopamine handling. Cells were cultured in Dulbecco’s Modified Eagle Medium F12 (DMEM/F12) supplemented with 100 units/mL penicillin and 100 units/mL streptomycin and 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 in DMEM with 10% FBS [HEK293 cells stably expressing human- vesicular monoamine transporter 2 (VMAT2) constructs were cultured at 37 °C and 5% CO 10% FBS . ConstruCell death was assessed using the LDH cell viability assay per the standard procedures provided by the supplier and as previously published . Brieflyg (Eppendorf 5424/5424R rotor) and resuspended in 0.32 M sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), at pH 7.4 with 1× Protease Inhibitor Cocktail [g and the supernatant was removed and added to VMAT2 uptake buffer , 0.05 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), pH 7.4), or uptake buffer containing 200 µM tetrabenazine (TBZ). Cell fractions were incubated with increasing amounts of PFOS dissolved in dimethyl sulfoxide (DMSO) or control solutions lacking PFOS, followed by the addition of a 10 mM dopamine stock with a 6% 3H-DA tracer and incubated for 5 min. Reactions were terminated and samples harvested with a Brandel Cell Harvester with Whatman GF/F Filter Paper. Bound activity on membranes was measured using a scintillation counter and specific activity was determined by subtracting total disintegrations per minute (DPMs) from TBZ-sensitive  DPMs and normalized to the total protein per well as determined by BCA protein assay.Cells grown to confluency were harvested, centrifuged at 1000× d-lysine pre-coated 96-well plates at 40,000 cells per well. Plating media were removed and immediately switched to Neurobasal-A-based culture media containing B27, 1% l-glutamine and 1% penicillin-streptomycin after 2 h, in vitro. The following day, culture media containing aphidicolin (1 μg/mL) was added to reduce the proliferation of glial cells in culture. Approximately one half of the culture media from each well was replaced every four days. Primary cultures were treated on day 8 in vitro with five concentrations of PFOS dissolved in cell culture media. After 24 h, cells were fixed in 4% paraformaldehyde (PFA) for 20 min and incubated overnight in rabbit anti-TH and mouse anti-MAP2 at 4 °C. The following day, cultures were incubated with fluorescent secondary antibodies, goat anti-rabbit 488 and goat anti-mouse 572 for 1 hr at room temperature. After staining with DAPI, cells were rinsed and stored in phosphate buffer saline (PBS). Images of treated cultures were obtained using an Array Scan VTI HCS . Forty-nine contiguous fields were taken per well and TH+ neurons were counted and analyzed using GraphPad analysis software .Briefly, ventral mesencephalic neuron cultures were prepared from postnatal mice  as previously published by our group ,12. Mousn = 6) or control (n = 6) for 14 days and then sacrificed 24 h after the last dose. Cohort 2 was similarly exposed to PFOS (n = 6) or control (n = 6) and then 24 h following the last dose, mice were administered the dopaminergic neurotoxin, MPTP. MPTP was injected subcutaneously at 10 mg/kg at 7:00 a.m. and 7:00 p.m. and then animals were allowed to sit for seven days before they were sacrificed and brain tissue collected for subsequent experimental assays [Eight-week-old C57BL/6J male mice were purchased from Charles River Laboratories  and allowed to acclimate to housing for seven days before initiation of treatment. Mice were orally gavaged with 25 µL of 10 mg/kg of PFOS dissolved in a corn oil vehicle daily for 14 days, similar to previously published reports . This dol assays . ExposurN-terminus of DAT. DAT antibody binding was detected using a goat anti-rat horseradish peroxidase secondary antibody  and enhanced chemiluminescence. The luminescence signal was captured on an Alpha Innotech Fluorochem imaging system and stored as a digital image. Membranes were stripped for 15 min at room temperature with Pierce Stripping Buffer and sequentially reprobed with α-tubulin (1:1000), TH (1:1000), GAT1 (1:2500), and vGlut  antibodies. α-Tubulin blots were used to ensure equal protein loading across samples.Western blots were used to quantify the amount of DAT, TH, GAT1, vGlut, and α-tubulin present in samples of striatal tissue from treated and control mice. Analysis was performed as previously described ,11,12,33Tissue staining was performed as described previously . Brieflyt-test or one-way ANOVA. Post hoc analysis was performed using Tukey’s post hoc test. Significance is reported at the p < 0.05 level.Statistical analysis of the effects of PFOS on SH-SY5Y cells and primary cultured neurons was performed on raw data for each treatment group by one-way ANOVA. Analysis of the effects of PFOS on dopaminergic and non-dopaminergic endpoints by immunoblotting was performed on raw data from each treatment group by Student’s As the major focus of this study was to evaluate the neurotoxicity of PFOS on the dopamine circuit, we initially characterized the general cytotoxicity of PFOS using the undifferentiated SH-SY5Y neuroblastoma cell line, which has been used extensively as an in vitro model in PD research . Cells wExtending our SH-SY5Y data, we then evaluated the dopaminergic effects of PFOS on primary cultured neurons isolated from the dopamine-rich ventral mesencephalon, which is a major target of dopaminergic neurodegeneration in PD. Neurons were exposed to DMSO or increasing concentrations of PFOS for 24 h before they were fixed and quantified for loss of dopaminergic and non-dopaminergic neurons. Using TH as a selective marker for dopamine neurons, PFOS exposure elicited significant reductions in TH-positive neurons at each concentration of PFOS tested A. In conDrawing from our in vitro data, we further elaborated our assessment of PFOS on the nigrostriatal dopamine circuit using an in vivo platform. C57BL/6J male mice were exposed via oral gavage to 10 mg/kg PFOS, daily, for 14 days and then evaluated for alterations in expression of key presynaptic dopamine proteins in the striatum, known to change in PD. Interestingly, exposure to PFOS did not elicit any measureable deficits in either DAT or TH . Previou3H-dopamine. With this approach we found PFOS effectively inhibits VMAT2 with an IC50 of 4.56 μM. To confirm that these reductions in VMAT2 function were not due to cytotoxicity, we treated HEK-hVMAT2 cells with similar concentrations of PFOS for 10 min and found not change in LDH release at any of the concentrations tested. These findings suggest that inhibition of VMAT2 function was not due to cytotoxicity of the cells  were acutely treated with increasing concentrations of PFOS and then assessed for the ability of VMAT2 to sequester he cells .The nigrostriatal dopamine system has been shown to be extensively vulnerable to damage by a variety of environmental toxicants, highlighting exposure to these chemicals as critical risk factors in PD ,29. The Our initial approach to characterizing the neurotoxicity of PFOS on dopaminergic cells was to employ an acute exposure to the SH-SY5Y neuroblastoma cell line, which resulted in significant cytotoxicity at the micromolar range. Establishing a generalized understanding of the neurotoxicity of PFOS was critical, as it served to guide our subsequent exposure paradigm in a more biologically complex model, to further assess the impact of PFOS on the nigrostriatal dopamine circuit.N-acetyl cysteine.Primary cultured neurons isolated from the ventral mesencephalon provide a powerful model to efficiently evaluate the neurotoxicity of chemicals on select neuronal populations and can assist in further refining neuronal targets for investigation, in vivo. Using this model, our findings demonstrate a preferential neurotoxicity of PFOS to dopaminergic neurons at low micromolar concentrations that was not seen in the non-dopaminergic population, predominantly composed of GABAergic cells. To our knowledge, this is the first evidence of PFOS-induced dopaminergic neurotoxicity using primary cultured neurons. The explanation for the preferential damage of the dopaminergic neurons to PFOS could be due to the ability of PFOS to generate reactive oxygen species coupled with the innate vulnerability of dopaminergic neurons to oxidative stress ,36. IndeIf PFOS selectively damaged nigrostriatal dopamine neurons, in vitro, we surmised that exposure to PFOS would have similar effects, in vivo, potentially via alteration of presynaptic machinery that is critical to proper dopamine handling. To evaluate this hypothesis we employed a subacute exposure paradigm of 10 mg/kg PFOS for 14 days. As our understanding of the neurotoxicological impacts of PFOS and how these relate to the body’s burdens in the human population is still being elaborated, we based our current treatment regimen on our previously published studies that have evaluated similar highly halogenated chemicals in adult mice ,4,6,12. In order to address the possible underlying mechanistic processes that could contribute to the unmasking of dopaminergic terminal damage following exposure to MPTP, we focused on the function of VMAT2, which functions to sequester cytosolic dopamine into synaptic vesicles and prepare them for release at the plasma membrane during neurotransmission. Using an in vitro assay that allows us to assess the function of VMAT2 to transport dopamine into vesicles, we found that PFOS significantly inhibits VMAT2 function at low micromolar concentrations. Packaging of free dopamine into vesicles is a crucial part of maintaining not only normal dopamine homeostasis, but also the health of the dopamine neuron. Indeed, the inability to sequester dopamine into vesicles through loss of VMAT2 function has been shown to be neurotoxic to dopamine neurons through the generation of reactive species ,44. Our In addition to packaging dopamine, VMAT2 is also able to package MPP+ into vesicles, in turn, preventing it from being transported to the mitochondria and disrupting ATP synthesis. Indeed, MPP+ neurotoxicity to the dopamine system is predicated on the expression and function of VMAT2 ,53,54. TIn conclusion, in this study we have coupled in vitro and in vivo approaches in an effort to evaluate the neurotoxicological effects of PFOS on the nigrostriatal dopamine system. These findings build upon previous work and serve to extend these findings by highlighting the vulnerability of the dopaminergic synapse to PFOS exposure and identifying a potential target in VMAT2, whose function may serve as a critical mediator of PFOS neurotoxicity. Moreover, these findings draw attention to PFOS as a potential risk factor for damage to the nigtostriatal dopamine circuit, which is damaged in PD, as well as other neurological diseases and disorders.


The environment is increasingly being recognized for the role it might play in the global spread of clinically relevant antibiotic resistance. Environmental regulators monitor and control many of the pathways responsible for the release of resistance-driving chemicals into the environment . Hence, environmental regulators should be contributing significantly to the development of global and national antimicrobial resistance (AMR) action plans. It is argued that the lack of environment-facing mitigation actions included in existing AMR action plans is likely a function of our poor fundamental understanding of many of the key issues. Here, we aim to present the problem with AMR in the environment through the lens of an environmental regulator, using the Environment Agency (England’s regulator) as an example from which parallels can be drawn globally. The issues that are pertinent to environmental regulators are drawn out to answer: What are the drivers and pathways of AMR? How do these relate to the normal work, powers and duties of environmental regulators? What are the knowledge gaps that hinder the delivery of environmental protection from AMR? We offer several thought experiments for how different mitigation strategies might proceed. We conclude that: (1) AMR Action Plans do not tackle all the potentially relevant pathways and drivers of AMR in the environment; and (2) AMR Action Plans are deficient partly because the science to inform policy is lacking and this needs to be addressed. Many of the hurdles to combating antibiotic-resistant infections in the clinic are well understood and, as such, have been used to inform existing antimicrobial resistance (AMR) Action Plans . It is aIn this review, we aim to present the AMR challenge through the lens of an environmental regulator, using the Environment Agency (England’s regulator) as an example from which parallels can be drawn globally. We argue that there is an evidence gap that hinders the ability of policymakers and environmental regulators from delivering environmental protection from AMR. The following five questions exemplify the approach taken by an environmental regulator when tackling the AMR challenge. The inability to answer these questions, in this point in time, highlights some of the evidence gaps that need to be prioritized to facilitate a holistic, evidence-based AMR Action Plan:(1)What are the benefits of controlling AMR in the environment over and above mitigating the potential transmission of resistance to humans?(2)What is the relative contribution from the release of antibiotics, metals, biocides, and antibiotic resistance genes (ARGs) to the emergence, maintenance and spread of AMR in the environment?(3)How well do current technologies and approaches limit AMR in the environment?(4)If reducing AMR needs to be reflected within our regulatory framework, how could this best be tackled?(5)Is there evidence to suggest that expansion of existing regulation/control measures for pollutants to AMR will translate into a decline in AMR in the environment?Here, we review the pertinent issues that lie at the root of the aforementioned questions, such as: What are the drivers and pathways of AMR? And, How do these relate to the normal work, powers and duties of environmental regulators? The discussion is then turned to: What are the knowledge gaps that hinder the delivery of environmental protection from AMR? Finally, we offer several thought experiments for how different mitigation strategies might proceed in the light of a holistic understanding of AMR drivers and pathways. It is the intention that this review would be a catalyst for future discussions among scientists, policymakers, clinicians, veterinarians, and regulators. It is also our expectation that this review will stimulate a more systematic review and meta-analysis of the literature to further examine and critique the evidence base and the changing state of our knowledge gaps.reduction of the environmental pollution by antimicrobial medicines particularly from production facilities.” The highlighting of pharmaceutical manufacturing is echoed in the O’Neill AMR Review, discussed below. Although rivers that receive eﬄuent from drug manufacturers have been shown to be a very important local issue with global implications . Moreover, it will be shown that antibiotics are only one of the many drivers of AMR in the environment.The World Health Organisation (WHO) and its Global Action Plan broadly outlines five strategic objectives to tackle AMR: (1) to improve awareness and understanding of AMR; (2) to strengthen knowledge through surveillance and research; (3) to reduce the incidence of infection; (4) to optimize the use of antimicrobial agents; and (5) to ensure sustainable investment in countering AMR . The WHOications , it willFigure 1). Moreover, it is unproven that WWTPs receiving eﬄuent from hospitals discharge significantly more resistance-driving chemicals into the environment, with measurably greater impact, than other similarly sized WWTPs. Although hospitals are an easy target and in many ways a tractable target, it does not necessarily make them the highest priority for reducing the prevalence of AMR in the environment. It is argued that the vision for these action plans is not sufficiently holistic to safeguard our natural environment. To appreciate these critiques, one must first understand what drives AMR in the environment.The final Review on Antimicrobial Resistance entitled “Tackling drug-resistant infections globally: Final report and recommendations”  highlighBroadly speaking, antibiotic use in humans and animals carries an inherent risk of selecting for ARGs. These ARGs are often, if not always, found in bacteria with other genes promoting resistance to other potentially harmful chemicals . It is mA review of the literature highlights three well-characterized classes of resistance-driving chemicals: (1) antimicrobials, of which there are four subclasses, antibiotics, antifungals, antivirals, and antiparasitics; (2) heavy metals; (3) biocides . However, many other chemicals, natural , and xeCo-selection of genes that confer resistance to chemical hazards , in line with the median across Europe in 2011 of 21.3 median DDD per 1000 inhabitants per day , where tIn 2010, India was the largest consumer of antibiotics when assessing total tonnage, however, their per capita usage (7.5 units per capita), was low by comparison to Australia and New Zealand which recorded among the highest usage rates of 87 and 70 units per capita, respectively . An antiHeterogeneity in antibiotic use is reproduced at seemingly every geographical scale. For example, nested within the larger global differences are differences between countries within smaller regions, e.g., Europe, where nearly 300% more antibiotics were used per capita in Turkey than Armenia . Within In addition, to there being large differences between countries within regions, there are significant differences in antibiotic use within countries. The highest combined general practice and hospital antibiotic consumption was in Merseyside, with similar levels reported in Southern Europe with 30.4 DDD per 1,000 inhabitants per day, over 30% higher than Thames Valley with the lowest consumption,  . Durham,According to The State of the World’s Antibiotics 2015, two-thirds of all of the antibiotics produced globally each year  are used in animal husbandry . The varIn 2013, the total antibiotic sales for therapeutic use in animals, was 420 tons, approximately 44% of the total antibiotic usage in the UK . The numAntibiotics are important for maintaining animal health and welfare. Antibiotics are often dispensed to treat or prevent infections in herds/flocks . AntibioThe use of antibiotics for growth promotion has been banned in Europe and is in decline in North America. Major food suppliers such as Tyson , McDonalA large fraction of the antibiotics consumed by humans are excreted in the urine and feces in their biologically active form . The antThe persistence of an antibiotic in a WWTP is a function of: (1) influent composition  ; (2) sal3 year-1, of which 15% was categorized as gray water  and the bedding for a month. Treatment of the calves with oxytetracycline resulted in no significant effect–a result the authors attribute to the small trial size, but similar to a previous study comparing the differential effects of antibiotic administration route on antibiotic resistance generation in the microbiome  persistence and shedding of the drug into the environment in urine and feces; (2) selection of antibiotic resistant organisms in the environment; and (3) transmission of drug-resistant microbes acquired from the environment , respectively, with mean concentrations of: 36 ± 8 and 12.6 ± 3.8 mg kg-1 (dw). The second most abundant class of chemicals was antibiotics, in order of decreasing concentration: ciprofloxacin > ofloxacin > 4-epitetracycline > tetracycline > minocycline > doxycycline > azithromycin, ranging between: 6.8 ± 2.3 and 0.8 ± 0.2 mg kg-1 dw  in clinical settings refers to the concentrations needed to inhibit the growth of, or kill, a target pathogen. The concentration at which a resistance genes offers a selective advantage to its ‘host,’ will be below the MIC, otherwise the microorganism would not survive. The MSC is a useful term that refers to the minimal concentration of chemical required to provide a selective advantage to a microorganisms carrying the resistance gene relative to the same bacterium that is sensitive to the chemical, i.e., not containing the resistance gene . The MSCBiocides are disinfectants that are commonly used in hospitals, cosmetics, household cleaning products, wipes, and furniture preservatives, farmyards for purposes such as: wheel and foot washes and a range of industrial processes, including the control of fouling and souring of pipes including oil wells  . Some coPseudomonas aeruginosa, E. coli, Staphylococcus aureus, Citrobacter sp.). Triclosan, along with other biocides, such as chlorhexidine and QACs, have been shown to select for antibiotic resistance  a better understanding of the quantity of biocides and their residues in the environment and its effects on AMR selection; (2) methodologies for assessing impact on AMR; and (3) studies characterizing AMR and co-selection .The route into the environment for biocides is similar to antibiotics, most notably WWTPs. Improper disposal of biocides through dilution and discharge to WWTPs can also increase the load of biocides entering the environment, while also increasing the likelihood of generating resistance.Major urban inputs of heavy metals to WWTPs come from household eﬄuent, drainage water, business eﬄuent , atmospheric deposition, and traffic-related emissions  . Metal nThe annual heavy metal inputs to agricultural land in England and Wales for the year 2000 was substantially higher for livestock manures than for any other soil input . SourcesMetal and antibiotic resistance can share structural and function resistance systems, such as: (1) reduced membrane permeability ; (2) drug and metal alteration ; (3) drug and metal eﬄux ; (4) alteration of cellular targets ; and (5) drug and metal sequestration  .The toxicity of metals to microorganisms directly relates to the affinity of the metal to thiol compounds such as glutathione within the cell, in order of increasing ‘toxicity’/S-affinity: Mn < Co < Zn < Ni < Cd < Pb << Cu << Ag < Hg . A log cConsiderable attention has been given to understanding the speciation of metals for predicting their mobility and toxicity in soils, sediment and aquatic system . HoweverBiological wastewater treatment processes have been shown to be disrupted as a result of the toxic effects of heavy metals in the influent , such asMetals have similar pathways to that of biocides and antimicrobials, however, the relative proportion of each source is likely to differ depending on the prevalence of industry, agriculture , or waste water/stormwater. Rivers downstream of urban areas, where stormwater runoff is acute, and regions that have experienced mining, are often impacted with elevated concentrations of metals . It remaThe chronic release of antibiotics into WWTPs is concomitant with the release of resistance genes. Resistance genes in wastewater originate from the gastrointestinal tract of, primarily, humans . The co-Antibiotics can trigger the bacterial SOS response in which bacteria undergo a transient, genome-wide hypermutation that generates genetic variability in times of stress . The SOSResistance can be intrinsic, which suggests it is universally found within the genome of the bacterial species, while acquired resistance suggests it could be acquired by means of new genetic material or through sporadic mutations of intrinsic genes . IntrinsIntrinsic resistance can be further enhanced through the modification or enhancement of intrinsic resistance, e.g., upregulation of cellular components that protect against the effects of the antibiotic  , such moTable 1). Equivalent legislation, if it exists, will differ by country, though many regulations are treated similarly across countries within the European Community with which England is aligned.There are a large number of activities or products that are currently monitored or regulated by the Environment Agency of England, which intersect with the pathways and drivers of AMR  eﬄuent, and (2) sludge (biosolids). The sludge can be composted or anaerobically digested and applied to land or incinerated. The proportion of antibiotic, metal, and biocide in the eﬄuent and sludge is highly variable depending on the STP catchment characteristics, presence of hospital wastewater, nature of STP and its operational parameters. WWTPs do not efficiently remove all ARGs that are subsequently released in treated eﬄuents . So the Upon entering the river, estuary or coastal water, sewage eﬄuent will be diluted. The resulting concentration of pollutants  will interact with the native flora and fauna and begin to change the microbial community structure and genetic makeup . Changes in the microbial community by means of any number of mechanisms, of which pollution  is but one, have been shown to have significant impacts on the aboveground diversity and functioning of terrestrial ecosystems .The introduction of these pollutants into recreational and coastal bathing water, primarily through combined sewer overflows (CSOs), will elevate exposures to humans  and by eHuman and animal use antibiotics, biocides, metals, and ARGs can all be found within products, such as: sewage sludge, anaerobic digestate, manure or slurry, which are spread upon agricultural soil for the purpose of fertilizing crops and/or improving soil properties. The dissemination of these materials on soil increases the ARG exposure risk to: (1) (wild) animals; (2) crops; (3) adjacent surface water bodies; (4) groundwater; (5) farm workers; and (6) air as dust particles from land spreading or aeolian erosion . The perintI1), has been shown to be maintained for several months after application   . Strong otection .A significant number of papers exist in the literature demonstrating the sensitivity and uptake of antibiotics from irrigation water, manure- or sludge-amended soils by crops into the plant biomass . For exaOf those antibiotics that are able to be phytoextracted, some have been shown to phytoconcentrate . Those tThe effect of chronic antibiotic exposure on aquatic plants has been poorly characterized, but there is evidence to suggest that antibiotic exposed aquatic plants demonstrate significant changes in the phyllosphere microflora –a change that has unknown ecological consequences .Where there is veterinary use of antibiotics, biocides and/or metals in fish farms there will be increases in their environmental release, and concomitant selection for resistance genes. The accumulation and chronic exposure of river, estuarine and coastal environments to these AMR drivers can persist  and spreAntimicrobials are used in aquaculture to prevent and treat bacterial infections in fish and invertebrates , howeverThe use and misuse of antibiotics in aquaculture has led to an increase in antibiotic resistance in fish pathogens, in the transfer of these resistance determinants to and from the sediment microbial community . AquaculAntibiotics found in manure- or sludge-amended agricultural soils will enter groundwater as a result of rainfall, irrigation, and other human activities . A pan-EThe toxicological response of organisms to exposure to antibiotic biocides and metals goes beyond the scope of this review. However, there are intriguing reports in the literature that document ecologically relevant effects on a range of target organisms from exposure to sub-lethal concentrations of pollutants, including antibiotics. Measurable changes in the metabolites found in feces of mice have been reported after exposure to sub-lethal concentrations of antibiotics. In a study on mice, the metabolites were found to be critical for host physiology  and an inflammatory response in the gut . ChronicIn the case of plants, there is evidence to suggest that seed germination, root elongation and overall plant health can be sensitive to sub-inhibitory concentrations of antibiotics and metals in the soil . Exposurde novo , which are highly deleterious molecules that can interfere with the normal functions of oxygen-respiring organisms . ROS, sude novo .It has been noted that the wider problem of AMR has many parallels to that of climate change, as they are both, arguably, an interdisciplinary, complex, global “tragedy of the commons” . Many huThe following is a first attempt to shortlist the knowledge gaps that emerge from a review, with a focus on those which could directly inform policy.(1)Figure 1, in the environment?Are there direct or indirect implications to the health, reproduction or ecosystem services of organisms or populations resulting from chronic exposure to elevated AMR drivers, as in  Sub-question: (a) Can exposure to elevated AMR drivers increase susceptibility to infection, and how does this differ with age, gender, habitat quality, exposure time, mixture exposures?; (b) Are there implications to the environment from chronic exposure to elevated levels of ARGs and the MGEs in which they might be contained? (c) Do antibiotics, biocides or metals bioaccumulate and what are the implications for AMR selection, maintenance, and benchmarking regulatory thresholds?(2)pathways, as in Figure 1, for establishing, maintaining and disseminating ARGs in the environment?What are the relative contributions of the different AMR  Sub-question: (a) Are there temporal, spatial, or physical/geological factors that impact on the selection, maintenance, and spread of AMR in the environment? (b) Should pathways be prioritized for mitigation based solely on potential impact, or should it include tractability ?(3)drivers, as in Figure 1, for establishing, maintaining and disseminating ARGs in the environment?What are the relative contributions of the different AMR  Sub-question: (a) How does ARG co-selection operate with mixtures of antibiotics, biocides and metals ?; (b) Can understanding co-selection help define targets for mitigation–is this a tractable problem?; (c) Do bacteriophage play a meaningful role in the maintenance and transmission of ARGs in the environment?  Wha(4)What concentrations of AMR drivers are relevant for assessing the risk of AMR selection and co-selection? Sub-question: (a) What is a useful benchmark for assessing risk and the success of any mitigation/intervention measures ; (b) What is the most appropriate time period for assessing the success of mitigating measures?(5)Are there direct or indirect implications from the trophic transfer of antibiotics, biocides, metals, or ARGs found within microorganisms, animals (aquaculture), or plants? Sub-question: (a) Are some chemicals more susceptible to uptake  in plants and do these pose the greatest risk to organisms and ecosystems?; (b) What are the effects of pH on the ionization of antibiotics and their differential toxicity to different target organisms?; (c) What role does metal speciation and metal bioavailability play in AMR selection and maintenance?; (d) How does antibiotic bioavailability in soil influence exposure and risk?These knowledge gaps have been, in part, summarized in Eq. 1:ij = the j-th of n chemicals with similar modes of action from the i-th of m pathways. E is the therefore the sum total of all chemicals with similar modes of action from different pathways per site. It is the aspiration to use minimum selection concentrations (MSC) derived from complex microbial communities in different environments  to calculate a risk coefficient (R):where cR > 1 represents a high risk of ARG selection and R < 1 represents a lower risk. An evidence-based determination (estimation) of the ARG-selection risk at each location can better inform and prioritize subsequent mitigation efforts.where It has been widely shown that ARGs are a natural phenomenon, and that we have been living with ARGs in the environment (and within our microbiome), since the dawn of our species . It has The decision by the European Commission to include macrolides on the Watch List indicates that their environmental impact, based on classical toxicity studies, might be sufficiently high to justify their regulation. Will regulating macrolides contribute to a reduction in AMR? Where macrolides compose the majority of AMR drivers in a habitat, one might expect it could be impactful in reducing the prevalence of ARGs. However, there are few scenarios where this condition is met–possibly downstream a macrolide manufacturing facility. The reality is that most antibiotics enter the environment through sewage eﬄuent or from manure/biosolid-amended soil, all of which are rich in a wide range of AMR drivers . Hence, it is unrealistic to expect macrolide regulation to substantially lower ARG selection. Establishing EQS’s for macrolides should not be interpreted as a mitigation measure for AMR since there is no evidence to date to suggest these two goals are necessarily linked. Moreover, regulating macrolides will not eliminate their use, but simply lower their use and subsequent concentration the environment. The question remains whether a reduction in macrolide release into the environment to satisfy an EQS will reduce macrolide-ARG prevalence.Knowledge Gap 3)? If the release of ARGs into the river are deemed relatively unimportant, then there is more reason to suspect that the removal of metals, biocides or antibiotics could be useful. However, if metals are important relative to the other drivers, then treatment technologies will need to address this challenge. Technologies used to remove metals  . RegulatIf the solution to mitigating AMR in the environment must move ‘upstream’ from the WWTP, it likely becomes the remit of several additional government regulators. AMR Action Plans might benefit from adapting the philosophy of the recycling movement “reduce, reuse, recycle” as it offers a more holistic and cost-effective mechanism for a reduction in the load of resistance-driving chemicals entering the waste stream. Without knowing the relative role of each of the drivers of AMR , it would be necessary to identify, quantify and reduce each of the chemical drivers entering the wastewater. There are many activities currently underway on a global scale that aim to tackle some of these upstream sources of AMR drivers, e.g., reducing antibiotic misuse in human and veterinary use; reducing biocide use in personal care and household products; capture, reuse and recycle metals within the waste stream. The monumental task that this represents highlights the need to prioritize research to address these Knowledge Gaps.The relevance of the AMR pathways and drivers to the environmental regulator can be crudely summarized in three, high level, points:(1)Low levels of antibiotics, metals, and biocides can directly select (and co-select) for ARGs within the AMR pathways that are under the remit of the Environment Agency, i.e., sewage eﬄuent, rivers, sludge, irrigation system, (bio)aerosols, aquaculture, industry, and groundwater.(2)Pathways for the dissemination of antibiotics, metals, biocides, and ARGs into animals, plants, food , groundwater, rivers and bathing waters, are within the remit of the Environment Agency.(3)Sub-lethal effects of antibiotics, metals and biocides on organisms, their microbiome and ecosystem services  can impact the health, yield and safety of economically important food products and the wider biome.Department of Health , Department of Food, Agriculture and Rural Affairs (Defra) , Veterinary Medicines Directorate, Environment Agency  as well as, Water and Sewerage Companies, industry and wider society. The first step toward this vision for a holistic AMR mitigation strategy, is for each of the stakeholders to critically evaluate the role they play in contributing to the composition and dissemination of resistance-driving chemicals. This document reflects a first effort to that end in England and the wider U.K.It is clear that environmental regulators should have an important role to play in future efforts to mitigate the environmental and human health risks from AMR. However, it is also apparent, that they represent only one of many stakeholders that can contribute to the solution(s). Implementation of ‘upstream’ solutions, such as ‘reduce, reuse, recycle,’ will necessitate coordinated strategies across multiple regulatory bodies and stakeholders, including: It is important to recognize that the lack of environmental focus in AMR Action Plans and the O’Neill Review is, arguably, because: (1) our understanding of AMR in the environment is so unsatisfactory that there is very little that can be suggested for mitigation without employing the precautionary principle as the primary rationale for action, and (2) the disciplines of pollution, environmental microbiology, and the wider infectious disease fields have been chronically underfunded , leavingHowever, there is reason to be optimistic, as for the first time, all seven UK Research Councils have come together to collaborate on funding long-term cross-disciplinary, integrated research on AMR . An AntiAH, VR, and AS conceived of the review outline. AS researched and drafted the manuscript. HS provided significant input to several sections to improve clarity and accuracy.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.


EditorThe following manuscript has been retracted from our November – December, 2016 issue. It has been retracted on a request from the authors as they forgot to properly acknowledge the source of their data. The authors are grateful to those who pointed out this mistake. - Retraction in: Pak J Med Sci 2016;32(6):1500-1505. doi: https://doi.org/10.12669/pjms.326.11460Link: http://pjms.com.pk/index.php/pjms/article/view/11460/4800Effective role of lady health workers in immunization of children in Pakistan1, Azka Naeem2, Unaiza Shahid3, Wajiha Noor Syed4, Urva Khan5, Nayyar Misal Zaidi6Saira AfzalRetracted on February 7, 2017


Precise fabrication of molecular assemblies on a solid surface has long been of central interest in surface science. Their perfectly oriented growth only along a desired in-plane direction, however, remains a challenge, because of the thermodynamical equivalence of multiple axis directions on a solid-surface lattice. Here we demonstrate the successful fabrication of an in-plane, unidirectional molecular assembly on graphene. Our methodology relies on nanomechanical symmetry breaking effects under atomic force microscopy tip scanning, which has never been used in molecular alignment. Individual one-dimensional (1D) molecular assemblies were aligned along a selected symmetry axis of the graphene lattice under finely-tuned scanning conditions after removing initially-adsorbed molecules. Experimental statistics and computational simulations suggest that the anisotropic tip scanning locally breaks the directional equivalence of the graphene surface, which enables nucleation of the unidirectional 1D assemblies. Our findings will open new opportunities in the molecular alignment control on various atomically flat surfaces. Since the first direct observations of well-organized molecular assemblies on surface using scanning probe techniques6, orientation control of such assemblies has been intensively studied because of their importance in chemistry7, electronics8, biomimetics4, and optics10. There have also been a number of reports on polymerization of well-oriented mesoporous silica thin films using on-surface assembly of surfactant molecules as templates11. In addition, modulation of the electronic or optical properties of the two-dimensional (2D) material substrates, including graphene and related 2D semiconductors13, via interactions with molecular assemblies has recently emerged as a new interdisciplinary research field over surface chemistry and nanomaterials science. Thus, the development of the precise on-surface molecular assembly alignment method has become increasingly important.Precise formation of on-surface nanostructures at the molecular level has long been of central interest in surface science15, molecular concentration16, solvent properties18, ionic properties19, and substrate friction20. In particular, the orientations of in-plane molecular assemblies are strongly affected by the symmetry of the substrate lattice because individual molecules tend to be first adsorbed with orientations along the well-defined axes on the surface23. Although precise orientation control is essential for various applications, perfectly unidirectional formation remains a challenge because of the surfaces normally have multiple axis directions with thermodynamical equivalence and one of them is just randomly selected for nucleation. Consequently, a typical in-plane molecular assembly on a surface has multiple domains with various orientations25. To lower the surface symmetry, researchers have proposed applying electric/magnetic fields26, using a macroscopic shear flow27, and controlling the surface boundary shape28. However, for the single-domain molecular assembly formation along a desired direction at any demanded position, further development of a simple and effective methodology of local surface symmetry breaking has still been strongly desired.The growth mode of molecular assemblies on solid surfaces is determined by delicate balances among various factors such as surface substrate properties31. Assembly of typical surfactant molecules, sodium dodecyl sulfate (SDS), on graphene-water interface was studied as a prototypical case. Under the AFM tip scanning condition, we found that ribbon-like one-dimensional (1D) assemblies of the SDS molecules were generated. The orientation of the 1D molecular assemblies showed clear correlation with the symmetry axis of the graphene lattice and the AFM tip scan direction. The computational simulations proved that the AFM tip scanning led to the anisotropic adsorption stability of the molecules on graphene surface. On the basis of these understandings, we eventually achieved the unidirectional formation of the 1D molecular assemblies along a selected symmetry axis after removing the initially adsorbed molecules, by sequential fine tunings of the scan parameters. These findings offer new opportunities for the on-demand unidirectional molecular assembly alignment on atomically flat 2D surfaces, and shed light on the usefulness of the anisotropic nanomechanical effects in the AFM tip scanning, which has rather been considered as undesirable in conventional AFM observations.Here, we report the successful fabrication of an in-plane, unidirectional molecular assembly on graphene. We use nanomechanical symmetry breaking effects induced by scanning with the probe tip of an atomic force microscope (AFM); the inherently directional tip scanning is expected to strongly modify the surface thermodynamical conditions as well as to introduce effective anisotropy. This concept is distinctive from that previously proposed for mechanical nanomanipulation, where a quasistatic AFM tip operation was used to cut molecules or to move them to a desired positionTo demonstrate our concept, we first examined the effects of AFM tip scanning by comparing molecular adsorption morphologies at a liquid–solid interface with and without tip scanning. Figure 24  drastically modified the SDS adsorption morphology; the initially adsorbed SDS molecules were removed, and new patterned structures emerged on the surface (see Supplementary Movie θrs (0° ≤ θrs ≤ 90°), as shown in the inset of Fig. θrs < 15° were rarely observed, and those with θrs > 60° exhibited the longest total length , and only the SDS ribbons with θrs ≈ 90° emerged on the same graphene surface (regrowth). This successful erasing–regrowth operation clearly demonstrates that one specific lattice direction can be selected for the ribbon growth among the multiple thermodynamically equivalent directions on the graphene surface. This result also clearly demonstrates that the presented mechanism concerning the stability of the adsorbed molecules is valid because the ribbons with θrs ≈ 90° were more stable than those with θrs ≈ 30°. The presented method for the precise unidirectional formation of individual molecular assembly nanostructures may enable the on-demand fabrication of more complicated and functionalized molecular nanostructures.On the basis of the aforementioned understandings, we demonstrate perfectly unidirectional formation of the SDS ribbons through sequential fine-tuning of the tip–sample force. Figure In summary, we demonstrated the formation of in-plane-oriented molecular assembly nanostructures under fine-tuned AFM tip scanning conditions. The SDS molecules were assembled into ribbon-like nanostructures at the water–graphene interface, with well-defined in-plane orientations determined by both the underlying graphene lattice and the AFM tip scan direction. Moreover, the unidirectional formation of the ribbons was achieved via sequential fine-tuning of the tip–sample force. The angle-selective destabilization through angular rotation of the adsorbed molecules forced by the AFM tip scanning was proposed as the possible origin of the directional molecular assembly formation, as supported by experimental statistics and computational simulations. These results will pave the way for the on-demand formation of molecular nanostructures at a desired position and direction on various atomically flat solid surfaces.The SDS molecules were dissolved in water (0.6 wt%), and multilayer graphene flakes were transferred onto silicon substrates. We used this concentration because only the SDS aggregations were observed at higher concentration (2.0 wt%). An AFM probe tip and multilayer graphene on a silicon substrate were placed in a water droplet, into which the SDS solution could be slowly injected at a rate of 75 μL/h using a microsyringe pump. Consequently, the concentration of SDS molecules reached approximately 1.36 mM. All of the measurements were performed at room temperature.fts) of 20 pN (see Supplementary Note 6 for the detailed estimation method) and a scan velocity (vscan) of 4.3 μm/s. The intense tip scanning for generating ribbons was performed in 15 min with an fts of 30 pN and a vscan of 55.7 μm/s. In independent 291 scanning experiments, the scanning time was set as about 20 min. In the demonstration of unidirectional ribbon formation, the AFM images . In the examination of the effect of AFM tip scanning, the AFM images Fig.  were obtges Fig.  were colSupplementary InformationSupplementaryMovie_S1.movSupplementaryMovie_S2.mov


Vibrio vulnificus causes human sickness throughout the world via the consumption of undercooked seafood or exposure to contaminated water. Previous attempts at phylogenetic analyses of V. vulnificus have proven unsuccessful, mainly due to the poorly understood impact of factors on its divergence. In this study, we used advanced statistical and phylogenetic methods to strengthen the classification of V. vulnificus. This updated classification included the impact of geographical and host factors. The results demonstrate the existence of hierarchies and multidimensional effects in the classification of V. vulnificus, from the molecular level using biotypes, to the distributional level using geographical location, to the adaptational level through host immune response. These findings have implications for the classification of bacteria, bacterial evolution, and public health. There are three main disease syndromes caused by this bacterium: invasive septicemia, acute gastroenteritis, and necrotizing wound infections. Vibrio vulnificus naturally exists in coastal regions globally and can cause seafood‐borne disease and wound infections in humans and a range of fish diseases. Although V. vulnificus can cause severe and even rare fatal infections in infected individuals, the impact of climate change and geographical distribution on the ecology of V. vulnificus is little known. A series of environmental parameters contribute to the virulence and abundance of V. vulnificus, which includes ocean temperature, turbidity, conductivity, sea level height, sea ice, winds, salinity, and wave length and velocity of propagation. The ecology of V. vulnificus is also related to dissolved organic carbon and chlorophyll  to provide a better understanding of how to classify V. vulnificus. We estimated the divergence times of some specific strains of V. vulnificus and constructed a phylogeny. We also evaluated the distribution of genetic variation within and among defined groups and pairwise fixation indices (FST) were estimated. For each defined group , the presence of population clusters was inferred by Bayesian analysis. The ultimate goal was to understand the interaction between the ecology of bacteria and their global distribution.Although genomic and geographical data for 22.1V. vulnificus were utilized in this analysis. The isolate data contain information on country, host, sampling year, sequence type (ST), and ten housekeeping genes via multilocus sequence typing (MLST) (PubMLST: http://pubmlst.org/). Neighbor‐joining trees were produced on all the isolates using MEGA 7  is a general statistical method for identifying the topology that explains the evolution of observing the data  under a given substitution model of evolution with the greatest likelihood  was conducted to attribute the total variance into geographically related genetic structures or host‐associated genetic structures using Arlequin software  and used as a training set. Host sources and geographical locations were prespecified for these 100 isolates in STRUCTURE Table . Probabi2.4The choice of isolates for Bayesian evolutionary analysis by sampling trees (BEAST) analysis was based on both geographical locations and host sources Table , which mV. vulnificus and further to construct phylogenies of specific strains that represent different sources and geographical locations. The selected sequence types (STs) used are listed in Table The program BEAST   was assigned to within each geographical region and within each host source.For the AMOVAs, isolates from three geographical regions were separated into three groups and, within each group, the isolates were divided by different host sources: human, aquatic animals, and environment. Table 3.3From the BEAST analysis Table , the est3.4For the source assignment Table , the thrV. vulnificus phylogeny is the molecular divergence of biotype 3 isolates.There are 294 isolates analyzed in STRUCTURE. Among them, 100 isolates were used as training datasets. One hundred and ninety‐four isolates were assigned into predefined clusters. The number of predefined clusters ranged from 3 to 9 . One possible reason why most previous attempts focused solely on host attributions, which have proven nondefinitive, is technical sampling limitations  of For neighbor‐joining analyses Figure a, there V. vulnificus types, regardless of the sampling source. Although it has only been reported in Israel so far, this sequence type caused two severe outbreaks within a 10‐year span (Bisharat et al., V. vulnificus. The geographical variation of V. vulnificus genotypes exceeds the host association, a finding that is also supported by the AMOVA results.Both the STRUCTURE and NJ tree results Figures  and 3 deV. vulnificus should be considered as hierarchical rather than one dimensional. This is one of the first studies to successfully detect geographical effects on the ecology and evolution of V. vulnificus. The findings presented here provide strong evidence that the evolution of bacteria is affected by their micro‐ and macro‐ecology.In summary, biotype 3 defines the first divergence level, which is more important than geographical location, and the effect of geographical location exceeds that of the source attribution. In other words, the classification of V. vulnificus and related global climate data. Even with the currently limited number of isolates, we can still detect significant effects; more data would allow for more precise analyses. Future research should focus on integrating advanced tools from interdisciplinary fields, including statistics, phylogenetics, computer science, and microbiology. Although it is just a starting point, we have demonstrated the importance of integrating geographical factors into the evolutionary analysis of bacteria.Further efforts should be made to collect genomic data from https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/FFKIMS.The datasets generated during the current study have been deposited in the public available repository, The author declares no conflict of interest. Click here for additional data file.