CRYβB2 gene expression has drawn particular interest because of its association with overall survival and African-American ethnicity in multiple cancers. Several reports indicate that overexpression of the CRYβB2 pseudogene, CRYβB2P1, and not CRYβB2 is linked with race and poor outcome. It remains unclear whether either or both genes are linked to breast cancer outcomes. This study investigates CRYβB2 and CRYβB2P1 expression in human breast cancers and breast cancer cell line models, with the goal of elucidating the mechanistic contribution of CRYβB2 and CRYβB2P1 to racial disparities.In the USA, the breast cancer mortality rate is 41% higher for African-American women than non-Hispanic White women. While numerous gene expression studies have classified biological features that vary by race and may contribute to poorer outcomes, few studies have experimentally tested these associations. CRYβB2 or CRYβB2P1 were generated and used to identify reads that uniquely aligned to either gene. Gene expression according to race and tumor subtype were assessed using all available TCGA breast cancer RNA sequencing alignment samples (n = 1221). In addition, triple-negative breast cancer models engineered to have each gene overexpressed or knocked out were developed and evaluated by in vitro, biochemical, and in vivo assays to identify biological functions.Custom scripts for CRYβB2P1 is expressed at higher levels in breast tumors compared to CRYβB2, but only CRYβB2P1 is significantly increased in African-American tumors relative to White American tumors. We show that independent of CRYβB2, CRYβB2P1 enhances tumorigenesis in vivo via promoting cell proliferation. Our data also reveal that CRYβB2P1 may function as a non-coding RNA to regulate CRYβB2 expression. A key observation is that the combined overexpression of both genes was found to suppress cell growth. CRYβB2 overexpression in triple-negative breast cancers increases invasive cellular behaviors, tumor growth, IL6 production, immune cell chemoattraction, and the expression of metastasis-associated genes. These data underscore that both CRYβB2 and CRYβB2P1 promote tumor growth, but their mechanisms for tumor promotion are likely distinct.We provide evidence that . Our data demonstrate novel molecular mechanisms of two understudied, disparity-linked molecules.Our findings provide novel data emphasizing the need to distinguish and study the biological effects of both CRYβB2 and CRYβB2P1 as both genes independently promote tumor progressionThe online version of this article (10.1186/s13058-019-1191-3) contains supplementary material, which is available to authorized users. While breast cancer is the most common cancer among women, a survival gap exists among African-American and Caucasian/White women . This din = 33) and 94% of all White patients (n = 36) were correctly classified according to race using CRYβB2 as one of the two-gene signatures in prostate tumors, PSPHL being the other gene classifier [CRYβB2 to be differentially expressed in non-malignant African-American breast tissue [CRYβB2 expression has been correlated to poorer outcome in cancer, regardless of race [CRYβB2 in cancers.Crystallin β B2 (CRYβB2) has recently drawn particular interest because of its genetic association with overall survival and African-American ethnicity in multiple cancers, including breast, colorectal, renal cell carcinoma, glioblastoma, and prostate tumors , 14–17. assifier . Similarassifier , 18. Add of race , 14–16. −/− mice have reduced fertility compared with wild-type mice via reduced expression of cell cycle and survival genes [CRYβB2 pseudogene, CRYβB2P1, and not the parental gene was linked with the observed poor outcome in African-American breast cancers and congenital cataracts in particular ethnicities/populations [CRYβB2 and CRYβB2P1, due to their high sequence similarity. Therefore, the potential exists that CRYβB2P1 expression has confounded prior results. It remains unclear whether either gene, or both genes, is linked to breast cancer outcomes. This study investigates racial expression differences and regulatory relationship between CRYβB2 and CRYβB2P1.The CRYβB2 protein is an abundant ocular lens protein, and mutations have been associated with congenital cataracts and macular degeneration , 20. Moual genes , 22. Criulations –27. Pseuulations . A critin = 1221) were retrieved from GDC Data Portal . Sequence data aligned to chromosome 22: 25,216,000–25,525,000, which span the CRYβB2 and CRYβB2P1 genes were extracted for further study. Custom scripts were used to search through the alignment files and identify reads that aligned to either CRYβB2 or CRYβB2P1, or both genes. The genomic coordinates for each position of each read were marked as either uniquely-mapped to either gene, or as part of a multi-mapped region. Unique and multi-mapped positions from each alignment file were combined, and those that were consistently unique across all samples were merged to form composite regions of unambiguous alignments. The algorithm was tested and validated by adding synthetic reads that mapped to unique regions for each gene and in the multi-mapped regions to several alignment files. Only after validating each stage of the process was the algorithm applied, and the results were used for downstream analyses.All available Cancer Genome Atlas (TCGA) breast cancer RNA sequencing alignment files  transformed and used for analysis unless specified otherwise.PySam 0.15.0, a python wrapper for Samtools, was used to quantify the reads that mapped to any of the composite unique regions in either 5 cells per insert; each model plated in duplicate). Cells migrated through the membrane for 4 h towards the indicated cell model in 24-h conditioned media. After this time, non-migratory cells were wiped from the top surface of the membrane; migratory cells were then fixed in methanol and stained with 1.0% crystal violet. Cell numbers were determined from microphotographs taken over five (non-overlapping) areas of the membrane.The SUM159 cell line was obtained from Asterand  and all remaining cell lines from American Type Culture Collection . Immunofluorescence was performed with appropriate controls as previously described [Cells were lysed in mPer Lysis Buffer  supplemented with protease and phosphatase inhibitors , then subjected to western analyses as previously described –31. Antiescribed .ΔΔCT method and/or the standard curve analysis when indicated. Primer sequences are listed in Additional file Total RNA was isolated using the RNeasy kit . Subcellular RNA fractionation was performed using the Active Motif kit #25501  following the manufacturer’s instructions with the modification of lysis shortened to 2 min. RNA was reverse transcribed using the High Capacity cDNA RT Kit (Life Technologies), and qPCR analysis conducted using the Absolute Blue qPCR Mix, Low Rox (ThermoFisher) with the Applied Biosystems QuantStudio™ 6 Flex Real-Time PCR system each according to the manufacturer’s instructions. Relative fold changes in gene expression were determined via the Foxn1nu mice  were injected orthotopically into the right abdominal mammary gland with 5 × 105 of the indicated cell model suspended in 30.0 μl of 50/50 PBS:Matrigel. Weekly tumor growth was measured via calipers and tumors excised when volume neared 400 mm3. Whole tumors were homogenized and RNA extracted using the Qiagen RNeasy kit as instructed. Three independent experiments were performed to ensure repeatability. Mice that did not form tumors were euthanized 5 months post-injection. Detection of metastatic xenograft cells in liver and lung was conducted as previously described [Animal experiments were conducted in accordance with accepted standards of humane animal care and approved by the Animal Care and Use Committee at North Carolina Central University. Xenografts were generated as we previously described . Brieflyescribed .3D cultures were performed as previously described –36. BrieIL6 signaling was inhibited using a 24-h dose of 75.0 ng/ml in vitro and 100.0 μg/kg in vivo intratumoral injection of a recombinant human IL6 receptor blocking antibody or isotype control  . InhibitT tests were also performed using GraphPad Prism Software 6.0 unless otherwise noted. Data was considered significant at P < 0.05.For all assays, a minimum of three independent experiments were performed using a minimum of duplicate samples in each experiment. For correlation determination, the Spearman correlation was used. Significance was determined via one-way analysis of variance (ANOVA) with Tukey Honest Significant Difference (HDS) or Bonferroni post hoc analyses. CRYBB2 and CRYBB2P1 to the promotion of breast cancer and breast cancer disparities, custom scripts were used to search through all available TCGA breast cancer RNA sequencing alignment files (n = 1221) to identify reads that aligned to either CRYβB2 or CRYβB2P1, or both genes. The genomic coordinates for each position of each read were marked either as uniquely mapped to either gene or as part of a multi-mapped region . Overall, there were consistently higher counts for CRYβB2P1 compared to CRYβB2 in all breast tumor subtypes and ethnicities evaluated , while CRYβB2P1 expression was significantly higher in the basal tumors compared to luminal A and B tumors . Divergent from previous reports, the levels of CRYβB2 were not significantly different between Asian, Black, and White tumors. However, CRYβB2P1 had significantly higher expression in Black tumors compared to Asian and White . These data correspond with a subset of studies that suggest CRYβB2P1, and not CRYβB2, is the gene associated with health disparities and poor outcome in breast cancers [CRYβB2 expression was no longer significantly higher in Her2 compared to luminal A tumors . However, CRYβB2P1 was significantly differentially expressed by race as well as by subtype after conditioning for race when evaluating age/menopause status . To test if similar patterns of expression were observed in breast cancer cell lines, a panel of 24 luminal and basal breast cancer cell lines were evaluated via qPCR using primers specific to either CRYβB2 or CRYβB2P1  cell lines were established  compared to parental control cells, gene knockouts, and dual gene overexpression models . Cells overexpressing CRYβB2P1 also demonstrated increased tumorigenicity, with tumors readily detected in all animals at 3 weeks post-injection compared to other models that exhibited 100% mice bearing tumors after 8 weeks or later  compared to the control parental cell lines . The most striking observation was the significant increase in invasive structures in cells with high levels of CRYβB2 (CRYβB2 overexpression and CRYβB2P1 knockouts). Total RNA was extracted from 3D cultures grown for 10 days and analyzed via tumor metastasis and epithelial-mesenchymal transition (EMT) pathway-focused qPCR arrays. Table To directly observe cellular morphology and behaviors, cultures of all the SUM159 and Hs578t cell models were grown in 3D culture and monitored over 10 days via live cell imaging. Consistent with in vivo study results, the size of spheroids was significantly larger in the models overexpressing CRYβB2, p < 0.01, t test; Fig. p = 0.052, t test).Invasive structures similar to those observed in the CRYβB2-overexpresing models were previously observed using 3D cultures of MCF10AI human breast epithelial cells . These ip < 0.01, t test; Fig. CRYβB2P1 modified models tested  protein was inhibited by expression of the NOS pseudogene . We hypotionship .CRYβB2 and CRYβB2P1. First, our data highlight that expression of the pseudogene, CRYβB2P1, is associated with Black/African-American breast cancer patients compared to White and Asian patient samples, and CRYβB2P1expression levels are higher compared to CRYβB2 in all TCGA breast cancer samples. Other studies have highlighted CRYβB2 as a health disparity gene in breast cancer, but our data suggest technical difficulties in distinguishing between the expression levels of the two genes and that the more likely disparity target is CRYβB2P1 [CRYβB2P1 functions as a ncRNA in triple-negative breast cancers to alter transcription.Another major conclusion that can be drawn from our investigation is the distinction of the race-related expression of CRYβB2P1 , 14–17. CRYβB2P1 is currently under investigation in our laboratory. Of note, a review of seven cancer lines in the publicly available University of California, Santa Cruz, genome browser database indicates CRYβB2P1 is active  and rich in transcription factor and chromatin regulatory marks, while not many regulatory marks are present for CRYβB2. These data strongly suggest CRYβB2P1 ncRNA may act in cis at the CRYβB2 locus and/or function in trans genome-wide.Pseudogenes are copies of protein-coding genes that no longer produce the same functional product as their parental gene, but still share a high sequence similarity, and can thus regulate or mediate the function of their parental genes through mechanisms such as the generation of ncRNA. Pseudogenes can be transcribed in parallel with their parental genes, or with their own tissue or temporal specific patterns . EvidencCRYβB2 as one of four genes significantly associated with African-American race and survival in luminal A breast cancers [CRYβB2P1 in the promotion of breast cancer, including increased proliferation, tumorigenesis, and invasive behaviors. Whether CRYβB2P1 alters CRYβB2 expression, or has tumor-promoting effects independent of CRYβB2, specifically in luminal cells was not investigated. Another constraint of our study is that although triple-negative breast cancer is a heterogeneous disease, we restricted our TCGA and cell line analyses to basal-like subtypes [CRYβB2P1 on tumor cell behaviors is tissue dependent. Pilot studies in our laboratory show CRYβB2 but not CRYβB2P1 is expressed significantly higher in pancreatic cancer cell lines compared to primary pancreatic cells Additional file 2: Table S2. qPCR Primer Sequences (PDF 37 kb)Additional file 3:CRYβB2 *adjusted p = 0.00427; CRYβB2P1 *adjusted p=7.7E-05, **adjusted p =1.3E-02. e CRYβB2P1 *adjusted p = 0.0043, **adjusted p = 0.0008. Distribution of CRYβB2 and CRYβB2P1 among race within subtype (c) and CRYβB2P1 among age/menopausal status (d). Significant results for CRYβB2 *adjusted p = 0.0304, and for CRYβB2P1 *adjusted p = 2.4E-06, #adjusted p = 5.3E-03, and **adjusted p = 2.3E-04. d CRYβB2P1 distribution among age before conditioning for race: *adjusted p = 0.0334, **adjusted p = 0.0231, #adjusted p = 0.0052, ##adjusted p = 0.0269, and (e) subtype after conditioning for race: * adjusted p = 0.0071, ** adjusted p = 0.0369, and #adjusted p = 0.0257. Young <40 yrs, Pre = pre-menopausal 40-46, Peri = peri-menopausal 46-55, and Post = post-menopausal >55 yrs. Figure S2. Distribution of transcript localization for each gene following subcellular fractionalization of RNAs. RNA was isolated and separated into cytosolic and nuclear subcellular fractions from proliferating cells. U6 and ACTB expression show successful separation of the nuclear and cytosolic subcellular compartments, respectively. 159 = SUM159, P-/- = CRYβB2P1 knockout, C-/- = CRYβB2 knockout, cyto = cytosolic fraction, nuc = nuclear fraction. Figure S3. CRYβB2 alters breast cancer cell growth behaviors in 3D cell culture. a Cells were grown in Matrigel and imaged on day 8. Data is one representative assay of a minimum of four independent experiments from Hs578t models. Figure S4. Cells were grown to 80% confluence, washed, then incubated in serum-free media 24 h. Images are representative immunoblots from the indicated models of control parental or CRYβB2-overexoressing cells. All data represent a minimum of three independent experiments. +C = CRYβB2 overexpression. Figure S5. CRYβB2 and CRYβB2P1 expression patterns of pancreatic cancer cell models. qRT-PCR analysis of the indicated cell lines. HPNE = hTERT-HPNE non-cancerous pancreatic ducal cells. Remaining cell models are pancreatic cancer cell lines. (PDF 5920 kb) Figure S1. Distribution of each gene among subtype and race using linear regression: conditioning on race (a) or subtype (b).


The primary cause for many of these changes is the development of sensitization at dopamine receptors located on striatal projection neurons (SPN). This initial priming, which is particularly evident at the level of dopamine D1 receptors (D1R), can be viewed as a homeostatic response to dopamine depletion and is further exacerbated by chronic administration of l-DOPA, through a variety of mechanisms affecting various components of the G-protein-coupled receptor machinery. Sensitization of dopamine receptors in combination with pulsatile administration of l-DOPA leads to intermittent and coordinated hyperactivation of signal transduction cascades, ultimately resulting in long-term modifications of gene expression and protein synthesis. A detailed mapping of these pathological changes and of their involvement in LID has been produced during the last decade. According to this emerging picture, activation of sensitized D1R results in the stimulation of cAMP-dependent protein kinase and of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa. This, in turn, activates the extracellular signal-regulated kinases 1 and 2 (ERK), leading to chromatin remodeling and aberrant gene transcription. Dysregulated ERK results also in the stimulation of the mammalian target of rapamycin complex 1, which promotes protein synthesis. Enhanced levels of multiple effector targets, including several transcription factors have been implicated in LID and associated changes in synaptic plasticity and morphology. This article provides an overview of the intracellular modifications occurring in SPN and associated with LID.A large number of signaling abnormalities have been implicated in the emergence and expression of  A number of molecular changes produced in response to administration of l-DOPA have been identified and many of them have been tested for their involvement in the development and expression of dyskinesia. Most of this information has been obtained from studies centered on the striatum, which is the main component of the basal ganglia and the principal target of anti-parkinsonian medications. This article provides an overview of the signaling abnormalities implicated in l-DOPA-induced dyskinesia (LID), with special emphasis on postsynaptic mechanisms occurring in striatal neurons.The last two decades have witnessed a remarkable progress in the understanding of the mechanisms mediating the effects of dopamine replacement therapies in Parkinson’s disease (PD). This is particularly evident with regard to l-DOPA are particularly evident at the level of the GABAergic projection neurons that connect the striatum to the efferent nuclei of the basal ganglia . Striatal projection neurons (SPN) express high levels of two major classes of dopamine receptors: the dopamine D1 receptors (D1R), whose activation increases the production of cAMP, and the dopamine D2 receptors (D2R), which inhibit cAMP synthesis. The opposite actions of these receptors on cAMP depend on their coupling to distinct G-proteins. Thus, D1R activate a Gαolf protein that stimulates the activity of adenylyl cyclase (AC), whereas D2R are coupled to Gαi/o-mediated inhibition of AC  the ability to promote motor activity, whereas the SPN of the indirect pathway (iSPN) suppresses motor activity. In addition, the selective expression of D1R in dSPN, and of D2R in iSPN, confers to dopamine the ability to enhance excitability of dSPN and to reduce excitability of iSPN, thereby promoting motor activity , a member of the membrane-associated guanylate kinase proteins   6. GRK6 is highly expressed in the striatum and involved in the desensitization of D1R -treated monkeys and that this effect is reversed by Similarly to GRK6, also GRK3 is highly expressed in the striatum and down-regulated in response to dopamine depletion  has been proposed as a potential mechanism , which is highly expressed in SPN . This effect is implicated in LID, which is counteracted by administration of the PKA inhibitor, Rp-cAMPS  at glutamatergic corticostriatal terminals  pathway, plays a particularly relevant role and has been the subject of intense investigation  and the calcium- and diacylglycerol-regulated guanine nucleotide exchange factor II , which is particularly abundant in SPN. Calcium-dependent activation of Ras-GRF1 and CalDAG-GEFII induces the exchange of GDP for GTP on the small G-protein Ras  signaling cascade, which is involved in the regulation of protein synthesis. mTORC1 is activated by the small G proteins, Ras homolog enriched in brain (Rheb) and Ras homolog enriched in striatum (Rhes) , which promote the expression of late response genes by binding to their activator protein-1 (AP-1) consensus sites . DREAM is activated by calcium and cAMP, and functions as a transcriptional repressor by binding to a regulatory element located downstream of the transcription initiation site of several genes, including members of the zif268 expression produced by cocaine in the striatum   affected by both treatments , 3 h (long-term depression-related genes) and 6 h (genes regulating protein phosphatase activity) , which is involved in synaptic plasticity , which were most likely activated to counteract abnormal D1R transmission. A noteworthy finding observed exclusively in dSPNs was the correlation between expression of genes such as Nurr1, Itch, Fra-1 and Dusp and the severity of the dyskinetic response to l-DOPA , a major downstream target of ERK. Chromatin immunoprecipitation (ChIP) analysis showed that ERK-mediated activation of MSK1 leads to the accumulation of histone H3 phosphorylated on Ser10, at the promoter of l-DOPA in experimental parkinsonism is also leading to hyper-phosphorylation of histone H3 at Ser28 , or genes (e.g. ∆FosB and Narp) controlled by ERK, whose dysregulation is ultimately implicated in dyskinesia. By the same reasoning, it will be important to distinguish specific constituents of the chromatin remodeling machinery, particularly enzymes catalyzing covalent modifications of both histones and DNA, which may represent viable druggable targets.l-DOPA, which significantly reduces the risk of developing LID, is not associated with increased phosphorylation of DARPP-32 and ERK, or with accumulation of ∆FosB in striatal neurons (Lebel et al. Progress in the understanding of the mechanisms of LID has also led to the identification of several molecular events, such as activation of key signaling components and overexpression of specific genes, which can be utilized to evaluate the potential efficacy of various types of interventions. For instance, continuous, as opposed to pulsatile, administration of


Bovine tuberculosis (bTB) remains a prominent zoonotic pathogen on the world stage, with significant impacts on animal and human health, and economic well-being. Eradication is hampered by a complex epidemiology, which in many countries involves wildlife hosts. Indeed, despite advances in understanding gleaned from national programs of bTB eradication, much of our understanding of transmission mechanisms, diagnostics, control, and multi-host infection systems remains opaque.Frontiers in Veterinary Science, as editors, we felt these limitations could best be addressed by adopting an international perspective. Localism understandably focuses on the fine details of problems at hand, but can perhaps overlook issues that only become apparent when compared to the experiences of others.In this collection of Below we summarize the papers published in this truly international collection, and highlight some themes. We trust readers will find these articles as stimulating to read as they were to edit.Modeling approaches were used to gain insights and make inferences on a number of different problems in bTB management.Ladreyt et al. in their paper “In silico Comparison of Test-and-Cull Protocols for Bovine Tuberculosis Control in France,” developed a stochastic simulation model to explore the potential impacts of differing test-and-cull options relative to whole herd depopulations in terms of epidemiological effectiveness, cost and acceptability to stakeholders. The authors suggest the model will be of utility for decision support for comparing alternative control protocols.Brunton et al. used statistical survival models to track the future risk of herds which retained inconclusive reactor (IR) animals. The authors reported significant increased future risk in herds with IR animals detected, relative to negative herds, and suggested that careful decision-making around the management of IR reactors needs to be employed to help mitigate such risk.In “Exploring the Fate of Cattle Herds With Inconclusive Reactors to the Tuberculin Skin Test,” Frankena et al. in their paper “A New Model to Calibrate a Reference Standard for Bovine Tuberculin Purified Protein Derivative in the Target Species” to determine the potency of an in-house developed, reference standard of M. bovis purified protein derivative (PPDb). Secondarily, the model determined the precision and accuracy of the test relative to a standard (guinea pig) potency test. Such work will be important for ensuring uniformity of standards of PPD for bTB test diagnostics, as the Bovine International Standard (BIS) supply is limited.Statistical models were used by Smith and Delahay concentrate on the badger-TB episystem to highlight methodological approaches used to model disease dynamics and control interventions. The paper also highlights how future data collection could be integrated into modeling endeavors, and how such models could be optimally utilized.Simulation and mathematical modeling has been a fundamental tool for bovine tuberculosis control, especially where wildlife reservoir species are involved. In “Modeling as a Decision Support Tool for Bovine TB Control Programs in Wildlife,” The use of genetic selection to improve animal health has emerged from recent advances in genomics and their application to epidemiological data. Resistance to bTB is a heritable trait in cattle, and provides an additional tool by which the disease can be controlled.Tsairidou et al. pose a counterpoint to existing efforts to use genomic selection in cattle to improve resistance to bTB. Specifically, they raise the possibility that infectivity may be a separate genetic trait in cattle—indicating that some animals could be predisposed to being bTB “super-spreaders.” They also show by simulation modeling, how including selection for an infectivity phenotype alongside resistance could deliver reduced bTB prevalence for cattle industries.In their Perspective article, “Can We Breed Cattle for Lower Bovine TB Infectivity?,” Raphaka et al. describe their development and evaluation of a simulation model which details the outcomes of varying intensities of genetic selection for bTB resistance in UK dairy cattle. Their findings show that adding genetic selection to bTB control strategies can aid in the reduction of bTB prevalence and severity of breakdowns. The authors suggest the use of genetic selection tools, alongside more traditional test and slaughter methods, could enhance eradication schemes.In their Original Research article, “Impact of Genetic Selection for Increased Cattle Resistance to Bovine Tuberculosis on Disease Transmission Dynamics,” Genomic methods are revolutionizing traditional molecular epidemiological approaches to disease source attribution, principally due to their much superior resolution. Application to bTB infectious systems promises to improve our knowledge of transmission dynamics.Orloski et al. provide a current description of the diversity of M. bovis isolates in cattle and farmed cervids in “Whole Genome Sequencing of Mycobacterium bovis Isolated from Livestock in the United States, 1989-2018.” The authors conclude that the use of whole genome sequencing (WGS) can reduce time and costs associated with epidemiological investigations, provides a powerful tool for advancing our understanding of transmission, and may improve eradication programs.Price-Carter et al. demonstrated the increased resolution of WGS for identifying sources of infection in outbreaks, especially in areas complicated by wildlife reservoirs. WGS was also able to provide information on the evolution of M. bovis within New Zealand animal populations, becoming a key component in their eradication strategy.Similarly, in “Whole Genome Sequencing for Determining the Source of Mycobacterium bovis Infections in Livestock Herds and Wildlife in New Zealand,” In multi-host epidemics, control or eradication of bTB in domestic hosts is often unachievable if disease control in wildlife reservoir populations is not simultaneously implemented.Buddle et al. and Gormley and Corner there are few options available to control TB in wild populations that are both effective and socially acceptable. Buddle et al. in “Efficacy and Safety of BCG Vaccine for Control of Tuberculosis in Domestic Livestock and Wildlife,” highlight Bacillus Calmette Guerin (BCG) vaccination of wildlife as one such approach. The authors highlight the evidence to suggest BCG can induce protection in European badgers, white-tailed deer, wild boar, and brushtail possums. Gormley and Corner explored the palatability of wildlife interventions to different stakeholders and value systems in their review “Wild Animal Tuberculosis: Stakeholder Value Systems and Management of Disease.” The authors suggest that factors influencing consensus on management approach can depend on the species in question, the economic cost-benefit and ethical considerations. The review identified that interventions that are acceptable in one region, may not be agreed upon in another region, even amongst broadly similar stakeholder groups.As highlighted by both M. tuberculosis, and Palmer and Thacker have also recently shown its potential for wildlife in “Use of the human vaccine, Mycobacterium bovis Bacillus Calmette Guerin in deer.” Decreased disease severity in vaccinated deer would likely be accompanied by decreased disease transmission. Progress on the development of oral baits for vaccines will facilitate the effort to implement this potentially valuable tool for addressing M. bovis infection in deer, and subsequent spread to livestock.Vaccination with BCG has been shown to reduce disease in humans caused by Bouchez-Zacria et al. in “The Distribution of Bovine Tuberculosis in Cattle Farms Is Linked to Cattle Trade and Badger-Mediated Contact Networks in South-Western France, 2007–2015,” used a network modeling approach to investigate cattle farms' bTB risk in France, utilizing both cattle trade data in concert with badger contact networks based on inferred badger home ranges, and M. bovis molecular typing. This work highlighted how both spatial relationships and trade relationships between farms, along with linkages associated with badger territorial behavior can be attributed to bTB risk, highlighting the complexity of multi-host epidemics.Cosgrove et al. extend previous modeling work to investigate how spatial and temporal persistence, density and attractiveness of feeding sites for wild deer affect both prevalence in deer and subsequent interspecies transmission. They show why feeding of deer is a relevant issue not only for hunters and wildlife managers, but for cattle producers and agriculture agencies as well.Human-caused aggregations of animals are a challenging tuberculosis management problem. In “Baiting and Feeding Revisited: Modeling Factors Influencing Transmission of Tuberculosis Among Deer and to Cattle,” VerCauteren et al's. “Persistent Spillback of Bovine Tuberculosis From White-Tailed Deer to Cattle in Michigan, USA: Status, Strategies, and Needs.” The authors provide a much-needed summary of biosecurity research undertaken in Michigan to date, and lessons learned. In addition, they make a case for management tools as yet un-, or at least under-utilized: vaccination of wild deer, precision culling of deer on farms, and, notably, strategic habitat manipulations to spatially redistribute deer.The essential role of on-farm biosecurity in managing bovine tuberculosis is one of the focal points of Cross et al. “Bovine Tuberculosis Management in Northwest Minnesota and Implications of the Risk Information Seeking and Processing (RISP) Model for Wildlife Disease Management” focuses on how deer hunters sought and acquired information on potential human health risks stemming from M. bovis exposure via hunting. Understanding how stakeholders obtain knowledge and form perceptions is crucial to implementing disease management in which they are necessary participants.Also focusing on white tailed deer in the USA, Mycobacteria in European wildlife. In “Mycobacterium caprae Infection of Red Deer in Western Austria–Optimized Use of Pathology Data to Infer Infection Dynamics,”Nigsch et al. provide for the first time in English a detailed description of the M. caprae outbreak spilling over to sympatric pastured cattle. They advocate for the use of a summary measure of detailed pathology findings—the Patho Score—as a quick and easy means of drawing broader inferences about the epidemiology of the disease in red deer, with implications for control in cattle.A pair of studies in this Research Topic addresses reservoirs of Réveillaud et al. recount the design and implementation of a nationwide system for M. bovis testing. “Infection of Wildlife by Mycobacterium bovis in France Assessment Through a National Surveillance System, Sylvatub” details the structure and integration of a surveillance system that acquires samples from diverse governmental and non-governmental partners, via both active and passive means. Thus, far, in addition to cattle, infection has been detected mainly in badgers and wild boar, although not to the extent noted elsewhere in Europe.In France, M. bovis cycles between cattle and free-ranging wildlife declare the necessity of eliminating the disease, but such goals are typically easier proclaimed than achieved. Exactly how can eradication be methodically pursued in the face of real-world logistical, cost and policy constraints? In “Roll-Back Eradication of Bovine Tuberculosis (TB) From Wildlife in New Zealand: Concepts, Evolving Approaches, and Progress,” Nugent et al. characterize a Bayesian “Proof of Freedom” framework currently being implemented on an area-specific basis, as well as how, incorporating decision theory, the approach is being scaled up to the national level.Many countries where Frankena et al.), host physiological and immunological status can affect the performance of diagnostic tests. Kelly et al. in “Association of Fasciola gigantica Co-infection with Bovine Tuberculosis Infection and Diagnosis in a Naturally Infected Cattle Population in Africa” present evidence to suggest an association between a liver fluke species' infection and bTB risk in different cattle breeds in Cameroon. Parasite co-infection is believed to affect the balance between Th1 and Th2 arms of the immunological response to infection, potentially impacting either diagnosis or disease progression. Kelly et al. report a complex interaction between the presence of bTB lesions and the performance of the interferon-gamma ante-mortem test in mixed breed animals, but not Fulani cattle.As well as the diagnostic regent variation  organisms in bovine tissues. Their assay exhibits good sensitivity and specificity relative to culture, and whilst the latter remains the gold standard for disease confirmation, the speed of the PCR assay could rapidly increase turnaround times, which is potentially of substantial epidemiological importance.In “Validation of a Real-Time PCR for the Detection of Mycobacterium tuberculosis Complex Members in Bovine Tissue Samples,” Hadi et al. describe in “Development of a Multidimensional Proteomic Approach to Detect Circulating Immune Complexes in Cattle Experimentally Infected with Mycobacterium bovis,” the preliminary findings of a novel proteomic method for the diagnosis of bTB infection in cattle. They identify immune complex proteins from the MTBC in experimentally infected cattle suggesting potential for this type of diagnostic approach. They also identify ways in which the method can be improved before validation on a larger dataset.Infantes-Lorenzo et al. describe the development of an ELISA based on the antigen P22 that had high specificity and sensitivity. This provides improved methods of detecting M. bovis and M. microti in New World camelids.Effective management of bTB requires development of accurate diagnostic tests in the wide spectrum of susceptible hosts. However, the conventional tuberculin skin test has low sensitivity in camelid species. In “Development and Evaluation of a Serological Assay for the Diagnosis of Tuberculosis in Alpacas and Llamas,” The “human component” of bTB epidemiology and control was highlighted in a number of papers relating to societal values and ethics of bTB control, as well as human zoonotic risk.Fekadu et al. describe the findings of their modeling study elucidating the risk factors associated with consumption of raw meat, and contraction of bTB by abattoir workers in Ethiopia. The data showed that despite workers' knowledge of the dangers involved in eating raw meat, and awareness of bTB risk, there was little uptake of preventative behavior, particularly in male workers and older demographics. The authors recommend tailoring public health interventions toward not only increasing awareness of zoonotic risk, but also influencing behavioral change.In “Risk Perceptions and Protective Behaviors Toward Bovine Tuberculosis Among Abattoir and Butcher Workers in Ethiopia,” Meiring et al. draw from the lessons of human TB control in South Africa and provide a comparison of these steps to bTB. Reduction in human TB incidence in this high burden country has been achieved through antimicrobial treatment and increasing awareness, both of which are lacking for bTB. Lack of effective movement controls, mandatory testing program, veterinary resources including state diagnostic laboratories, point-of-care tests for cattle, and presence of bTB in wildlife populations contributes to persistence of disease. A multi-sector approach, as seen for human TB, is needed to address bTB.In “TB Control in Humans and Animals in South Africa: A Perspective on Problems and Successes,” Borja et al. describe findings of bTB in Fiji in “A Retrospective Study on Bovine Tuberculosis in Cattle on Fiji: Study Findings and Stakeholder Responses.” Despite a bTB control program, goals were not being achieved due to lack of training of staff conducting TB testing, absence of standard data collection and unregulated movement of cattle. Revision of the program resulted in increased detection but also farmers' concerns. This study highlights the technical and social challenges to effective disease control.Although bTB is a global disease, it can be neglected in smaller nations. Little compares experiences with cattle producers in the two countries facing the need for heightened biosecurity on their farms. Rather than relying solely upon voluntary compliance, she demonstrates the value of negotiated outcomes in obtaining producer support for more stringent regulations necessitated by the presence of M. bovis in wildlife.Perceptions of cattle farmers are critical to implementing tuberculosis management strategies that ultimately rely upon their proactive cooperation and compliance. In “Negotiated Management Strategies for Bovine Tuberculosis: Enhancing Risk Mitigation in Michigan and the UK,” Allen et al. focus on the ongoing problems of eradicating bTB in Britain and Ireland—asking, why are these territories so different from their continental European neighbors? Is there a cocktail of unique epidemiological and ecological factors that are hindering efforts? If so, what could they be, and how can they be addressed? Suggested factors included the presence of a wildlife reservoir, differing diagnostic approaches, variation in pathogen genetics, co-infection, the structure and density of animal movement trade networks, and the potential for environmental persistence and a benign climate.Finally, in their Hypothesis and Theory article, “Bovine Tuberculosis in Britain and Ireland – A Perfect Storm? The Confluence of Potential Ecological and Epidemiological Impediments to Controlling a Chronic Infectious Disease,” M. bovis infection as a zoonotic pathogen. These themes reflect the reality that bTB control across countries is a multi-factorial problem requiring significant specialist input from various disciplines, in conjunction with “buy-in” from stakeholders across broader society, in order to move efforts from control to eradication.This Research Topic was contributed to by 150 authors, representing over 15 countries. As highlighted, some common themes emerged from these contributions . The valAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Upon activation with dMAO , complex 9 was found to promote highly isospecific styrene polymerizations ([mm] = 97.5%–99%) with high molecular weights Mw up to 181,000 g·mmol−1. When the Dmp-substituted pre-catalyst 10/dMAO system was used, the highest activity, over 7700 g·mmol(10)−1·h−1, was recorded involving the formation of precisely isospecific polystyrenes of [mm] more than 99%.[OSSO]-type dibenzyl zirconium(IV) complexes  High-resolution mass spectrometry (HRMS) data were recorded by using a Hitachi-Hitec NanoFrontier eLD . All melting points were determined on a Mel-Temp capillary tube apparatus  and are uncorrected. The molecular weights (Mw) and molecular weight distributions (Mw/Mn) of the polymers were evaluated by gel permeation chromatography (GPC) on a SCL-10AVP/LC-10ATVP/DGU-14A/ CTO-10ACVP/RID-10A apparatus  using a GPC KF-804L  column. The analyses were performed at room temperature using THF as the solvent and standard polystyrene as the reference. Differential scanning calorimetry (DSC) analyses were performed on a Seiko Instruments DSC 6200 apparatus  at a rate of 5 °C/min, under continuous flow of N2 (0.5 mL/min), using aluminum capsules. [OSSO]-type ancillary ligands 7 and 8 -type ligands 7 or 8 with Zr(CH2Ph)4 in toluene at room temperature gave the corresponding dibenzyl zirconium(IV) complexes 9 or 10 as air- and moisture-sensitive pale yellow crystals in 83% or 92% yields, respectively  complexes  (2.7934(7), 2.7932(6) Å)  triad  determined by DSC analysis. The GPC analysis of the resulting polymer revealed a large molecular weight  and a monomodal distribution with a very broad polydispersity (PDI = 8.6). When the polymerization temperature was raised to 25 or 40 °C, slightly decreasing isotacticity of 96.8% or 90.7% together with narrower molecular weight distributions of 3.8 or 2.9 were observed, respectively (Runs 2 and 3). At 70 °C, 9/dMAO system showed the highest activity of 618 g·mmol(9)−1·h−1, and the narrow PDI value of 2.2, while the isotacticity of the obtained polymer decreased somewhat to 87.5% and the molecular weight Mw was reduced drastically to 9500 g·mol−1, suggesting that the active species was unstable during the chain propagation step at high temperatures (Run 4). In the varying temperature experiments, the broader polydispersity and the higher molecular weight Mw were observed at lower temperatures as characteristic trends. These results would be explained by a relatively slow generation of active species and/or a slow propagation of polymer chains in the styrene polymerization process.To elucidate the potential as an isospecific polymerization catalyst, we examined the coordinate polymerization of styrene using precursors m] triad . Since nrpentier ,42, an e10. The polymerization of styrene  employing the system consisting of 10 (2.0 μmol) and dMAO (500 μmol) in toluene at 0 °C for 10 min could also afford completely isotactic polystyrene of 0.259 g ([mm] > 99%), which corresponds to the activity of 777 g·mmol(10)−1·h−1 (Run 5). Despite the more overcrowded environment at zirconium center in 10, this activity is approximately 15 times higher than that of 9 at the same temperature (50 g·mmol(9)−1·h−1). GPC analysis exhibited that the obtained polymer was monomodal and had a high molecular weight  and a slightly large polydispersity of 3.1. Similar results were observed in the polymerizations carried out at 25 and 40 °C to produce excellent isotactic polymers ([mm] > 99%) with higher molecular weights Mw up to 380,000 g·mol−1 and narrower molecular weight distributions of 1.8 and 2.1, respectively, being consistent with a single site behavior (Runs 6 and 7). The corresponding activities recorded in the range of 2200–4100 g·mmol(10)−1·h−1 increased with elevating temperatures; at 70 °C, 10/dMAO system achieved the highest polymerization activity of 7700 g·mmol(10)−1·h−1 forming a high molecular weight polystyrene . Despite the higher polymerization temperature, the resulting polymer had a complete isotacticity ([mm] > 99%) and a monomodal molecular weight distribution (Mw/Mn = 1.8). In the DSC measurement, the melting temperatures (Tm = 221.4–225.8 °C) of polymers produced by 10/dMAO system reflected their highly isotactic microstructures. Thus, our catalyst system exhibited remarkably higher activity, even at low temperatures (777–7700 g·mmol(10)−1·h−1 at 0–70 °C) than those of reported [OSSO]-type titanium complex 1a (330 g·mmol(1)−1·h−1 at 40 °C) -type bis(phenolate) ligands based on trans-cyclooctanediyl platform. Use of the Dmp-substituted pre-catalyst 10 in the presence of dMAO as an activator was critical for the formation of precisely isospecific polystyrenes with high activities. Notably, the isotactic polymers produced by 10/dMAO system had significantly larger molecular weights and narrower polydispersity indexes of nearly 2.0.We have established a controlled isospecific polymerization of styrene employing dibenzyl zirconium(IV) complexes (


In vivo PET studies confirmed that abnormal spreading of serotonergic terminals within the putamen has a pivotal role in the development of LIDs and GIDs. However, glutamatergic, adenosinergic, opioid systems, and phosphodiesterases 10A may also play a role in the development of these motor complications. An integrative multimodal imaging approach combining PET and MRI imaging techniques is needed to fully understand the mechanisms underlying the development of LIDs and GIDs.The purpose of review is to review the current status of positron emission tomography (PET) molecular imaging of serotonergic system in Parkinson’s patients who experience levodopa-induced (LIDs) and graft-induced dyskinesias (GIDs). PET imaging studies have shown that Parkinson’s disease is characterized by progressive loss of dopaminergic and serotonergic neurons. Parkinson’s patients who experienced LIDs and GIDs have an aberrant spreading of serotonergic terminals, which lead to an increased serotonergic/dopaminergic terminals ratio within the putamen. Serotonergic terminals convert exogenous levodopa into dopamine in a non-physiological manner and release an abnormal amount of dopamine without an auto-regulatory feedback. This results in higher swings in synaptic levels of dopamine, which leads to the development of LIDs and GIDs. The modulation of serotonergic terminals with 5-HT According to Braak’s staging of Parkinson’s, the pathological processes begin early in the serotonergic raphe nuclei, prior to the onset of motor symptoms, and precede the damage of substantia nigra -benzonitrile ([11C]DASB) is a second-generation PET ligand with high selectivity for the serotonin transporter (SERT)  is a molecular imaging technique that allows the quantification of biological targets and pathological processes in vivo in the brain , such as serotonergic, which do not possess the molecular machinery to properly control the release of dopamine  scores in preclinical models of Parkinson’s disease induced by 6-hydroxy (6-OH) dopamine  and the previous one , we suggest that the imbalance caused by a normalization of serotonin terminals in the dopamine-denervated striatum creates increased dopamine release after levodopa administration, resulting in an increased negative input to the globus pallidus neurons controlling thalamic output. Greater LIDs might be the results of increased dopamine release at pre-synaptic dopaminergic receptors located at the synapses of striato-pallidal GABAergic neurons in the globus pallidus. These neurons control the projection neurons to the thalamus and thereby the thalamic output. By over-inhibition of these neurons, the dysregulated basal ganglia output then results in LIDs. This is in line with the preclinical evidence of a profound suppression of globus pallidus output activity in monkeys experiencing LIDs .In three Parkinson’s patients with GIDs who received striatal transplantation with foetal ventral mesencephalic tissue, we evaluated the density of serotonergic terminals, using [Taking these results into account, we suggest that SERT/DAT ratios could be used as biomarker for LIDs and GIDs. Moreover, pharmacological modulation of serotonergic terminals sprouting, such as with brain growth factors, might also improve the severity of LIDs.In vivo PET studies confirmed in humans that an abnormal spreading of serotonergic terminals within the putamen has a pivotal role in the development of LIDs and GIDs. Serotonergic terminals’ release of abnormal amounts of dopamine after levodopa administration is the most widely recognized factor underlying the pathophysiology of dyskinesias but cannot be considered a necessary and sufficient condition for the development of this motor complication. The implementation of novel PET ligands is warranted for unveiling unexplored mechanisms of underlying pathophysiology of Parkinson’s disease. An integrative multimodal imaging approach combining different techniques is needed to untwist the debate around putative players on the pre-synaptic side of the cascade that lead to the development of dyskinesias in Parkinson’s disease.


The Asia Pacific region, home to two-thirds of the world’s population and ten of the least developed countries, is considered a regional hot-spot for the emergence and spread of antimicrobial resistance (AMR). Despite this, there is a dearth of high-quality regional data on the extent of AMR. Recognising the urgency to close this gap, Singapore organised a meeting to discuss the problems in the region and frame a call for action. Representatives from across the region and beyond attended the meeting on the “Antimicrobial Resistance in the Asia Pacific & its impact on Singapore” held in November 2018. This meeting report is a summary of the discussions on the challenges and progress in surveillance, drivers and levers of AMR emergence, and the promising innovations and technologies that could be used to combat the increasing threat of AMR in the region. Enhanced surveillance and research to provide improved evidence-based strategies and policies are needed. The major themes that emerged for an action plan are working towards a tailored solution for the region by harnessing the One Health approach, enhancing inter-country collaborations, and collaboratively leverage upon new emerging technologies. A regionally coordinated effort that is target-driven, sustainable and builds on a framework facilitating communication and governance will strengthen the fight against AMR in the Asia Pacific region. Mycobacterium tuberculosis (MTB), carbapenem-resistant Enterobacteriaceae (CRE), XDR Pseudomonas aeruginosa and Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA). These pathogens are part of the World Health Organisation (WHO)’s priority list published in 2017, widely known as the “dirty dozen” that pose a significant threat to human health [Antibiotics have revolutionised modern medicine and facilitated advances in transplantation, chemotherapy and surgery, while drastically reducing the mortality and morbidity from infectious diseases. The ever-increasing demand for antibiotics in healthcare is evident in the 65% increase in global consumption from 21.1 billion to 34.8 billion daily doses from 2000 to 2015 [The Asia Pacific region (APAC), home to two-thirds of the world’s population and ten of the least developed countries , 6, is hThis report summarises the information and insights shared by 26 experts who represented the academic, industry and government sectors from Papua New Guinea, Timor Leste, Indonesia, Thailand, Cambodia, Myanmar, India, Singapore, US and the United Kingdom. This report consists of three sections, namely, the challenges and progress in surveillance, drivers and levers of AMR, and potential innovations and technologies to combat the increasing threat in the region. We will present potential solutions and a roadmap as discussed during the meeting.Escherichia coli and Klebsiella pneumonia were estimated to cause 3.7 million to 6.4 million bloodstream infections, and 28.9 million to 50.1 million serious infections, and carbapenem-resistant strains caused 0.4 to 0.5 million bloodstream infections, and 2.7 to 3.1 million serious infections in 2014 [Drug-resistant pathogens have been found in every continent; however, differences between countries in the prevalence of AMR depend on multiple factors including levels of antibiotic consumption, access to clean water, adequate sanitation, vaccination coverage, the availability of quality healthcare, and access to high-quality medical products. Increasing international travel has played a key role in the spread of drug-resistant pathogens including but not limited to MRSA and extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae –9, incre in 2014 . In the  in 2014 –31.Asia Pacific is highly vulnerable to the threats of AMR. The challenges that impede the progress in controlling AMR in APAC are wide-ranging and affect both the low-to-middle-income countries (LMICs) and high-income countries. The region is projected to be home to 27 of the world’s 43 megacities by 2030 , and theThe WHO Global AMR Surveillance System (GLASS) and Organisation for Animal Health (OIE) global database on the use of antibiotics in animals were established to enable the systematic collection of data globally. By 2018, 9 of 11 WHO SEA region countries and 6 of 27 Western Pacific Region countries had enrolled into GLASS, but reporting of surveillance data is limited . Complem. The generation of high-quality data is even more challenging, particularly in settings with limited laboratory infrastructure. In these settings, clinicians frequently prescribe antibiotics without the support of microbiological results and diagnostic specimens are mostly submitted from patients in whom empiric treatments have failed [Although several networks collect data on selected pathogens in the region, there are no formal surveillance networks dedicated to AMR in APAC . The lace failed , leadinge failed . Also, le failed , 48.Nonetheless, many efforts have been made to improve the situation. At the regional level, the commitment to address AMR was demonstrated by the inclusion of AMR in the current Association of Southeast Asian Nations (ASEAN) five-year work programme (2016–2020). The signing of the Joint Declaration on Action against AMR by the Heads of States in 2017 and the ASEAN Plus Three Leaders’ Statement on Cooperation against AMR  in 2018 have also sent a clear signal on the recognition of a need for regional collaboration against AMR. Based on the 2017 Joint Declaration, the Philippines, with input from the other ASEAN Member States is preparing a draft Strategic Framework that will guide the ASEAN member states in planning for AMR control. The Strategic Framework is targeted to be endorsed by the ASEAN Health Ministers in the near future and will be followed up by a monitoring & evaluation framework for progress tracking.Laboratorio Nacional de Saude and National Hospital to incorporate microbiology diagnostics into daily clinical decisions as well as to capture trends of infections and AMR [At the national level, countries are making progress, albeit at varying pace. Timor Leste, a country that has emerged from a period of political instability and war in recent years, is at the start of the process in establishing a surveillance system to understand the impact of AMR in the country. Although the country’s past has been characterised by weak microbiology laboratory capacity and scarcity of data, the Timorese National Health System has made many efforts in the past few years to improve infrastructure and human resources in microbiology diagnostics and bring together the relevant stakeholders to implement a multi-centric surveillance system by 2019. The progress thus far includes the introduction of antimicrobial susceptibility testing for clinical bacteria isolates and a closer working relationship between the  and AMR . The col and AMR .E. coli and K. pneumonia and other pathogens from blood and urine specimens, and antimicrobial usage (AMU) from hospital prescription in 2016 and 2017. There are opportunities for capacity building through the establishment of external quality assurance and proficiency test in the NRL, and extension of the surveys to the broader network of hospitals including 20 national and province-level referral hospitals, and 100 regional-level referral hospitals. It was also highlighted that the development of guidelines and tools for prescription review would help to raise awareness of AMR and guide decision making to reduce the rate of empirical therapy in Indonesia.In contrast, Indonesia, the fourth most populous country in the world, is transitioning from the early phase of its efforts against AMR that started in 2017. Guidelines for surveillance have been developed but not fully implemented across the country, and quality data and analyses are far and few . NonetheIn addition to strengthening AMR surveillance for human health, countries such as Myanmar and Singapore are making progress in incorporating a multisectoral approach in tackling AMR. In Singapore, the National Strategic Action Plan overseen by the AMR Coordinating Office emphasises a One Health approach for education and training, surveillance and risk assessment, and research. In Myanmar, a multisectoral approach combining the expertise of epidemiologists, infectious disease clinicians and social scientists to perform research studies and facilitate translation of evidence to policy for AMR is underway.Innovations to Fight Antimicrobial Resistance.There are several opportunities to improve the capacity for AMR surveillance in the region as a result of new funding streams and technological breakthroughs, building on the foundation laid by past efforts in respective countries and commitment by international and regional bodies. This section briefly covers the potential of technology such as whole-genome sequencing (WGS) before a more extensive discussion on technology in the last section on The “AutoMated tool for Antimicrobial resistance Surveillance System” (AMASS) application to enable local hospitals to perform data analysis, generate report and share data .A patient-focused surveillance tool through the “A Clinically-Oriented antimicrobial Resistance surveillance Network” (ACORN) to supplement existing pathogen-focused surveillance systems.New funding streams such as the Fleming Fund aim to collate existing AMR data from the laboratories in the region and work prospectively in Indonesia, Laos, Papua New Guinea, Timor-Leste, and Vietnam to build human and laboratory capacity in order to facilitate WHO GLASS data submission . It willK. pneumoniae and Shigellae spp. [On the technology front, the increasing affordability of WGS presents an opportunity to enhance surveillance efforts by using molecular epidemiology data to map out the evolution, spread and transmission of antibiotic-resistant genes and drug-resistant pathogens. Previous studies that used WGS on lae spp. , 55 showlae spp. . Work inlae spp. .Using a One Health approach, samples could be collected from farm to market/restaurant and sequenced to gain insights into the transmission of drug-resistant genes and bacteria from live animals to meat and cooked food, and thus further understand the ecological drivers of AMR. The projects involving WGS will require considerable IT infrastructural investment to process, store and handle a large amount of data; develop cloud-based solutions and visual interface for data sharing; and statistical and bioinformatics expertise for data analysis. It also currently faces challenges in not having an international standardised protocol for sample and data processing, dedicated funding and personnel, and consensus for open communication as well as sharing of samples and data within and beyond countries.Lastly, building on efforts within countries and acknowledgement of the importance of collaboration with international and regional bodies, stakeholders and policymakers should consider forming a dedicated organisation like a regional Centre for Disease Control and Prevention (CDC). The significant progress achieved by the European CDC and African CDC, including the organisation of the EARS-Net and European Surveillance of Veterinary Antimicrobial Consumption (ESVAC), and launch of the AMR framework to direct efforts and resources to the threats of AMR in Africa, are exemplary of successful regionally coordinated efforts , 59. WhiThe emerging economies and growing prosperity in APAC will likely exacerbate the trend of AMR. The growth in wealth may lead to an increase in demand for animal protein and the shift to large-scale farming in countries such as China and India which are projected to double antibiotic consumption by 2030 . This trAt the population level, the accrual of risks of drug-resistant infections is not readily perceived by individuals, and at the individual level, the risk of misuse of antibiotics is often overlooked in favour of the potential benefit of recovery from illnesses. For this reason, AMR is often called the “invisible threat”. The AMR risk is further compounded by the lack of awareness of appropriate antibiotic usage. In 2015, a study by WHO revealed widespread antibiotic use across countries, with varying levels of understanding of the appropriate utilisation of antibiotics, and poor understanding of the potential consequences of AMR arising from the misuse of antibiotics in the majority of the respondents .Antibiotics taken without prescription ranged from 9 to 62% in APAC . In IndoDue to the increasing public demands for antibiotics, even in outpatient settings, the health care community in Asia increasingly prescribes antibiotics, even if inappropriate, further exacerbated by the absence or weak enforcement of policies for antibiotic stewardship , 72. In Such practices of self-medication and empirical therapy are driven mainly by poor understanding of the cause of conditions such as UTIs and the association between antibiotic use and AMR. At the same time, health systems in countries such as Singapore and Japan struggle with increasing ageing and immunocompromised populations that are more vulnerable to infections and correspondingly prescribed more antibiotics. The paradox of excessive use of antibiotics and timely access to antibiotics, however, is more prominent in LMICs. The restriction in the use and increase in access to antibiotics, without proper adaption to the local contexts, may have a detrimental effect on the control of infectious diseases and AMR .The WHO acknowledged the challenges relating to poor awareness and knowledge of AMR in the WHO Global Action Plan and is leading this front through the World Antibiotic Awareness Week that occurs in November every year . In 2018In addition to health literacy, other socio-behavioural determinants include cultural beliefs and the use of technology. Past studies have found that Asians were more optimistic about disease risks and held stronger fatalistic beliefs about prevention than the European and Americans, potentially explaining the weaker adherence of Asians to prevention recommendations . This suThe effectiveness of public health campaigns could also be augmented by policy implementation to control the use of antimicrobials where feasible. This may include introducing a requirement for a prescription to buy antibiotics, delinking the prescription and sales of antibiotics to remove financial incentives in the healthcare and food-producing sectors –86, and The ability to estimate the financial impact of AMR is essential to justify investment for interventions, especially in LMICs where insufficient resources and competing demands exist. However, current projections of the economic impact of AMR rely greatly on simplified assumptions and data inputs due to limited data on AMR , 89, 90.More importantly, hospital data does not account for the pervasive impact AMR has on society and the future. A broader assessment including the loss of productivity due to morbidity and mortality arising from AMR, loss of revenue in travel and trade due to fear of infections , reductiAntimicrobial Resistance Surveillance touched on the potential of technology such as WGS to understand the transmission dynamics of drug-resistant genes and pathogens and highlighted the potential for new therapeutic, diagnostic and surveillance innovations to bolster the fight against AMR.The earlier section One of the most promising areas in which technological innovations can be used to tackle AMR is to minimise empirical therapy, which has contributed to the unnecessary prescription of antibiotics for viral infections such as URTI. Among the factors promoting the practice of empirical therapy is the lack of affordable and simple point-of-care (POC) diagnostics that can distinguish bacterial from other infections and readily determine the antimicrobial susceptibility for the former. A variety of approaches to developing novel POC diagnostic tools have emerged to increase the throughput, speed, and cost-effectiveness to diagnose infectious diseases and determine the antibiotic susceptibility profile. Recent new developments include the combination of microfluidics to integrate DNA and RNA extraction, and PCR amplification with technologies such as micro-optics to create optofluidic platforms to enable single-cell analysis and pathogen identification , 100. AnThe application of engineering design principles to create affordable, fast, and accurate medical diagnostics is also gaining prominence –105. ThiAnother potential development that could complement pathogen-specific tests is the use of systems-level “omic” analyses that profile host-derived response markers that are predictive of the infections. Initial successes in using integrated omic analyses to identify human biomarkers have been shown in studies that accurately differentiate sepsis from systemic inflammation , differeIn addition to diagnostics and surveillance, innovative approaches could also help to overcome the barriers in developing new drugs. The high cost of research and development and uncertainties in the return of investments have caused most large pharmaceutical companies to abandon development of new antimicrobials , 110, shUnderstanding of the human microbiome as being important in the establishment of reservoirs of drug-resistant pathogens is emerging , 116. Thhttp://microbiomeconservancy.org). Building on an initial collection of 7600 gut bacterial isolates and their genomes from healthy and urban North Americans, the Global Microbiome Library expanded the collection with more than 4000 additional strains from under-represented populations in Arctic regions, and East and Central Africa. Defining the genomic sequences and the presence of AMR genes in this growing strain collection has relied on recent innovations in WGS technology, while the identification of sequences of individual microbes among the thousands in a single faecal DNA sample requires even more sophisticated sequencing innovations, such as epicPCR for profiling genetic traits such as AMR [The latter problem is being addressed by a team of scientists that founded the Global Microbiome Conservancy, which has the goal of culturing and sequencing more than 10,000 bacterial strains from the human gut in populations from more than 30 countries  to treat chronic, drug-resistance infections by ifficile . Previou studies , presumabacteria .Box 3 IThree recurring themes emerge from the discussions above. The first is that there is no single solution or path to AMR control in the region, given the socioeconomic and cultural diversity within APAC, rapid changes in the region, and the multifaceted nature of the challenges of AMR. The second is the need for multisectoral and One Health approaches to synergise the efforts and harness the expertise and experiences in surveillance, sociobehavioural, economics and innovations. The third is the value of inter-country collaborations in the region and the necessity to elevate the issue of AMR control beyond just national agendas. After all, progress achieved in one country could be off-set by failures in another country. It is an opportune time to put together a regionally coordinated effort that is target-driven, sustainable, and builds on a framework that facilitates communication and consistent governance to strengthen the fight against AMR.This report neither provides an extensive review of AMR in the region which has been captured exceptionally well in existing literature , 43 nor


It was proved that even despite evident deformations of the molecular structure, involving twisting of the porphyrin's central plane, the coupled π-bonding system remains flexible and stable. DFT calculations were applied to a number of selected porphyrins representative for the reviewed compounds to emphasize the relevance of theoretical methods in structural investigations of complex macrocyclic molecular systems. Experimental and DFT-simulated IR spectral data were reported and analyzed in context of the individual molecular features introduced by the meso substituents into the porphyrin moiety base. Raw experimental spectral data, including 1H- and 13C-NMR, UV-Vis, FTIR, XRD, and other relevant physicochemical details have been provided for a specially chosen reference zinc porphyrin functionalized by tert-butylphenyl groups.Structural parameters of a range of over 100  Handbook of Porphyrin Science Cl2 as standard.Fluorescence spectra were recorded in toluene for λPhilips FTIR PU 9800 spectrometer was used to measure the FTIR spectrum and the zinc porphyrin sample (ZnTBPP) was analyzed in a KBr disc.1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance II 400 Hz spectrometer.2/min) and at a heating rate of 10 deg/min.Universal V3.0G device (TA Instruments) was used to register the DSC  and TGA  runs, under constant nitrogen flow (40 mL Nmeso-substituted zinc porphyrins constitutes a very stable and flexible molecular set-up. Both the crystallographic and IR investigations have indicted that the considered meso-substituents practically do not affect the strength of the porphyrin core. DFT theoretical investigations appeared very useful in analyzing both structural and IR-spectral properties. It must be emphasized, that the IR spectra reported in this work well conform to the quoted references concerning both the experimental data and theoretically derived IR vibrational frequencies. Diverse structural effects observed in real Zn-porphyrin crystals may appear crucial to the catalytic activity of such compounds. The flexible porphyrin core allows the molecule to accumulate extra energy resulting from intermolecular interactions, which could even produce structural deformations but not disruption of the bonding system. Thus, under certain circumstances one may expect such compounds to exhibit enhanced reactivity. Therefore structural investigations are of principal meaning particularly in molecular designing of porphyrin solids considered for application in heterogeneous systems.The bonding system in tetra-


Whisker-type poly (P3HT) nanofibers were aligned by restricting their growth direction using an approximately 100–1000 nm wide narrow groove fabricated by thermal nanoimprinting. In grooves made of an amorphous fluoropolymer (CYTOP™) with widths of less than 1500 nm, the nanofibers oriented uniaxially perpendicular to the groove and their length was limited to the width of the groove. This result indicates that the nucleation of nanofibers tends to be selectively promoted near the interface of CYTOP™ with fluoro-groups, and nanofiber growth perpendicular to the wall is promoted because P3HT molecules are supplied more frequently from the center of the groove. Furthermore, the orientation induced anisotropic conductivity, and the conductivity parallel to the oriented nanofibers was more than an order of magnitude higher than that perpendicular to the oriented nanofibers. Molecular electronics, which are positioned to be the bottom-up technology, have attracted significant interest because they are constructed via a self-assembly approach, which allows for the possible elimination of a vast number of lithography steps ,2. Conduca. 10 nm, in addition to a higher degree of crystallinity and a higher carrier mobility than conventional P3HT films [The carrier transport properties of conducting polymers are strongly affected by the degree of crystallinity and chain orientation. One effective approach for improving the degree of crystallinity is to use a whisker-type nanofiber structure made by crystallization in an adequate solvent. This method has been intensively investigated, particularly with regioregular poly (P3HT) . These PHT films ,12,13,14The electrospinning (ES) method can be used to align P3HT nanofibers uniaxially by a collector/electrode modification ,16,17 orIn this study, whisker-type P3HT nanofibers were crystallized in a narrow groove with a width of approximately 100–1000 nm to align nanofibers by restricting their direction of growth. The preferential orientations for polymer crystals of syndiotactic polystyrene (sPS) , polyethMw = 87,000) was purchased from Sigma-Aldrich Co., Inc.  and used without further purification. In our previous report, the nanofiber was crystallized from a solution of approximately 0.05 wt % P3HT in a mixture of anisole and chloroform as the solvent [m-xylene was used because nanofiber crystallization in a narrow groove requires a small volume of solution and the solvent evaporates immediately. The P3HT solution was prepared by stirring at a high temperature (ca. 70 °C). After the reflux was stopped, the solution was immediately introduced by capillary flow into the narrow grooves on the substrate, which was covered with a glass slide. The sample was then cooled to −15 °C to crystallize the nanofibers. The nanofibers were used in the undoped state.Regioregular P3HT , was spin-coated at 3000 rpm onto a Si substrate with a 255 nm thick thermally grown SiO2 layer. A mold  and the substrate were heated at 170 °C (CYTOP™) or 135 °C (PMMA), and the mold was pressed onto the polymer for 5 min at a pressure of 4.5 MPa using a thermal nanoimprinter . After gradually cooling to room temperature and demolding, the residual layer was completely removed by reactive ion beam etching  with O2 gas for 8 s (CYTOP™) or 12 s (PMMA), according to the etching rates determined from preliminary measurements.The narrow grooves were fabricated by thermal nanoimprinting , as follThe width of the groove varied from approximately 600 nm to 3 μm and tended to be slightly larger than that of the mold due to the residual layer etching process. The height of the wall was approximately 100 nm. The groove pattern and the crystallized nanofiber in the pattern were observed using SFM  in intermediate tapping mode. To analyze the nanofiber orientation in the SFM images, we calculated the autocorrelation images as follows. Initially, the SFM images of the nanofibers observed in the groove were clipped out and binarized with an appropriate threshold. Autocorrelation images were then generated using image analysis software . Finally, the autocorrelation images observed at 6–11 different locations were averaged to generate an averaged autocorrelation image.χ/φ) measurements was crystallized at −15 °C on a nonreflecting Si plate  that was covered by a thin film of CYTOP™ and then dried. In the in-plane measurements, the grazing incident angle ω and diffraction angle 2θ were fixed at 1.0° and 2.0°, respectively, and ω was slightly larger than the critical angle of the non-fibrillar P3HT film.Wide angle X-ray diffraction (WAXD) measurements were performed using an X-ray diffractometer . The nanofiber for out-of-plane (θ/2θ) and in-plane  in a cryogenic probing station . Pt electrodes were sputter-deposited through a metal mask by RIBE, and the width and gap of the electrodes were 40 and 600 μm, respectively. To investigate the anisotropic conduction, electrodes were deposited parallel and perpendicular to the grooves. The electrodes perpendicular to the groove were formed using an oblique deposition method to deposit metal on the groove wall, which prevented division of the electrode into isolated segments by the grooves.Conductivity measurements were carried out using a two-probe method in a vacuum below 102 gas to remove the residual layer. In the wider gaps of 2400 and 3000 nm, no obvious uniaxial orientation of the nanofibers was observed. On the other hand, the nanofibers formed in narrower gaps of 600, 900, and 1500 nm seemed to be oriented almost perpendicular to the grooves. Nanofibers grown in a narrow groove are generally expected to orient parallel to the groove because nanofibers can grow to more than several micrometers in length in free space, whereas their growth perpendicular to the groove is restricted by the wall. However, the experimental results indicate that the nanofibers did grow perpendicular to the groove, and that their length was limited by the width of the groove.Nanofibers could be formed in grooves made of CYTOP™ with gap widths of (a) 600; (b) 900; (c) 1500; (d) 2400; and (e) 3000 nm, as shown by the SFM images in To precisely evaluate the degree of orientation, averaged autocorrelation images were calculated for various groove widths, as shown in To investigate the effect of the groove wall material, we observed the orientation of nanofibers crystallized in grooves made of PMMA with a gap width of 800 nm. χ/φ) displacement. d100 ≈ 16 Å. This is almost the same distance as the sum of twice the molecular length of the tilted side chains in an all-trans conformation and the width of the polythiophene backbone. Furthermore, the higher-order peaks indicated the periodic structure with interlayer distances corresponding to the (200) and (300) diffractions. On the other hand, the broad peak at 2θ ≈ 23° in the in-plane measurements of nanofibers crystallized on CYTOP™  and in-plane  and higher order in the out-of-plane measurement and of (020) in the in-plane measurement suggests that the P3HT molecules of the nanofiber have an edge-on arrangement on CYTOPTM, which was the same as that for nanofibers cast on Si after crystallization in solution. Therefore, the CYTOPTM surface had no specific effect on the orientation of P3HT nanofibers.The indices for the nanofibers crystallized on CYTOPAlthough the CYTOP™ surface itself had no specific effect on the P3HT nanofiber orientation, the nanofibers oriented themselves only in narrow grooves made of CYTOP™. Careful observation of the SFM images shown in I–V characteristics are shown in perp) and parallel (σpara) to the groove were estimated to be 1.61 × 10−4 and 8.45 × 10−6 S·cm−1, respectively, where the width and length of the conductor region contributing to the conductivity corresponded approximately to the width and gap of the electrodes, and the thickness was estimated as an average from SFM images of nanofibers. σperp, directed along the oriented nanofiber, was more than an order of magnitude larger than σpara, perpendicular to the oriented nanofiber. Although there are groove walls, which obviously hinder conduction perpendicular to the groove, σperp was much larger than σpara, which indicates that anisotropic conduction was achieved by the controlled orientation of the nanofibers.The uniaxial orientation achieved in a narrow groove can lead to anisotropic electric conduction, which is the purpose of this study. The anisotropic conductivity of nanofibers oriented in the groove was measured by depositing electrodes parallel (to measure the conductivity perpendicular to the groove) and perpendicular  to the groove. Schematic images of the electrode placement are shown in The crystallization of whisker-type P3HT nanofibers in a narrow groove was shown to be an effective method of aligning the nanofibers. This was suggested to result from the selective promotion of nucleation near the interface of CYTOP™ with fluoro-groups. This method of orienting nanofibers was successfully used to achieve anisotropic conduction with a greater than ten-fold difference in conductivity between the directions parallel and perpendicular to the aligned fibers.


Healthcare-associated infection (HAI) is a major challenge for patient safety worldwide, and is further complicated by antimicrobial resistance (AMR) due to excessive antimicrobial use in both humans and animals. Existing infection prevention and control (IPC) networks must be strengthened and adapted to better address the global challenges presented by emerging AMR.In June 2017, 42 international experts convened in Geneva, Switzerland, to discuss two key areas for strengthening the global IPC network: 1) broadening collaboration in IPC; and 2) how to bring the fields IPC and AMR control together.SAVE LIVES: Clean Your Hands initiative is an example of a successful collaboration between multiple global stakeholders including academia and international public health organisations; it can be used as a model. IPC-strategies are included within the four pillars to combat AMR: surveillance, IPC, antimicrobial and diagnostic stewardship, research and development. The prevention of transmission of multidrug-resistant microorganisms is a patient safety issue, and must be strengthened in the fight against AMR.The US Centers for Disease Prevention and Control, the European Centre for Disease Prevention and Control, and the World Health Organization (WHO) convened together with international experts to discuss collaboration and networks, demonstrating the participating organizations’ commitment to close collaboration in IPC. The challenge of emerging AMR can only be addressed by strengthening this collaboration across international organisations and between public health and academia. The WHO The working group determined that international organisations should take the lead in creating new networks, which will in turn attract academia and other stakeholders to join. At the same time, they should invest in bringing existing IPC and AMR networks under one umbrella. Transmission of multidrug-resistant microorganisms in hospitals and in the community threatens the success of antimicrobial stewardship programmes, and thus, research and development in IPC should be addressed as an enhanced global priority. Under the auspices of the US Centers for Disease Control and Prevention (CDC), the World Health Organization (WHO), and the University of Geneva Hospitals and Faculty of Medicine (HUG), an international panel of 42 experts with backgrounds in infection prevention and control (IPC), microbiology, infectious diseases, public health, psychology, medical technology, and social sciences, convened for 2 days at the Geneva Think Tank on IPC and antimicrobial resistance (AMR) in June 2017. The aim was to develop a global vision on IPC and AMR, and to agree on a road map for research and public health activities. Three dimensions of IPC and AMR were discussed: 1) implementation of IPC and antimicrobial stewardship; 2) technology in IPC and AMR; and 3) broadening the global IPC network. This is the final paper in a series of three summarising the discussions during the meeting, and addressing the current and future role of networks in IPC.Two key areas for strengthening the IPC networks globally were addressed: 1) broadening collaboration among IPC organisations around the world; and 2) improving and aligning IPC and AMR control efforts. Two impulse talks for each of the areas set the stage for a moderated plenary discussion. Writers took notes. This paper is the summary of presentations and notes taken from the plenary discussion.CDC, the European Centre for Disease Prevention and Control (ECDC), and the WHO convened together and with international experts to exchange their visions about collaboration and networking, and demonstrated their commitment for close collaboration in IPC.A number of international IPC and AMR networks are already in place Table . The WHOSAVE LIVES: Clean Your Hands campaign exemplifies the success of such collaboration, where the campaign principle is that public health and IPC experts work hand-in-hand to drive improvement [Experts in IPC and professional societies may not have a public health perspective, but the role they play in driving innovation and generating ideas and solutions cannot be dismissed given the impact of such efforts on future public health decision making. Ideally, expert-to-expert collaboration would include international public health bodies. Any resulting innovations would be more likely to incorporate global or higher-level policy perspectives, with the potential impact extending beyond research to become standard in patient care. The WHO rovement . In addirovement . ThroughA challenge is the fact that research, and consequently the guidance based on scientific findings, is typically generated in high income countries. More research in low-and-middle-income countries (LMICs) is needed, but until this is available, at a minimum, the applicability of recommendations to LMIC contexts should be assessed. The WHO core components for IPC are a good example of guidelines based on data primarily from high income settings that have been integrated with experience and additional evidence from the field gathered in LMIC settings according to a solid methodology. In collaboration with CDC, WHO has developed implementation tools with special focus on low resource settings, to support the adaptation and adoption of the guidelines into the local context , 4. ThesEmerging AMR is a global problem, which can only be controlled if stakeholders are working together at an international level. AMR control relies on four pillars: 1) surveillance ; 2) IPC (prevention of AMR transmission); 3) antimicrobial stewardship; and 4) research and development. It is well accepted that all four pillars are needed to both define the problem , and to solve it .Preventing the spread of multidrug-resistant organisms depends predominantly on good IPC practice in healthcare facilities, particularly hand hygiene, institutional environmental cleaning, and isolation precaution measures. Prevention of spread in the community setting is also predicated on the existence of adequate water, sanitation and hygiene measures. Additional measures include screening of patients at risk for carriage of multi-resistant organisms according to defined strategies that are adapted to individual types of AMR-pathogen combinations. There is sufficient evidence that such strategies work, the recommended actions are straight-forward, and they can be applied immediately. If cross-transmission is not contained, other AMR strategies such as antimicrobial stewardship or the development of new antimicrobials will be very limited in both effect and sustained impact.IPC as a core prevention measure has neither been sufficiently recognized nor prioritized at the global level to adequately address the scope of the AMR problem. The field of IPC is not adequately interconnected with the AMR community. This dichotomy is rooted in history. Research in AMR is infectious diseases and microbiology-driven, often focusing on resistance mechanisms and genetic relatedness. Infection prevention and control has a more practical attitude focusing on the prevention of cross-transmission by promoting best practice procedures. Scope and professional backgrounds of stakeholders in IPC and AMR are often different. However, the need for collaboration between the two has become more urgent. International organisations such as CDC, ECDC, and WHO could take a lead on bridging the gap between IPC and AMR experts. ECDCs ARHAI network and CDC’s Antibiotic Resistance Solutions Initiative are examples where activities on AMR and IPC are under the same umbrella.International organisations should take the lead in both strengthening existing and creating new networks, which will attract academia and other stakeholders to join. At the same time, they should invest in bringing existing IPC and AMR networks under one umbrella. Transmission of multidrug-resistant microorganisms in hospitals and in the community threatens successful combating of AMR, and thus, measures to interrupt transmission i.e. effective IPC strategies and programmes supported by effective international and national networks should be given more priority globally.The working group determined that international organisations should take the lead in creating new networks, and invest in bringing existing IPC and AMR networks under one umbrella. Research and development in IPC should be addressed as an enhanced global priority.