There is no more challenging a group of pharmaceuticals than antimicrobials. With the antibiotic era came great optimism as countless deaths were prevented from what were previously fatal conditions. Although antimicrobial resistance was quickly identified, the abundance of antibiotics entering the market helped cement attitudes of arrogance as the “battle against pestilence appeared won”. Opposite emotions soon followed as many heralded the return of the pre-antibiotic era, suggesting that the “antibiotic pipeline had dried up” and that our existing armament would soon be rendered worthless.In reality, humans overrate their ecological importance. For millions of years there has been a balance between factors promoting bacterial survival and those disturbing it. The first half century of the “antibiotic era” was characterised by a cavalier attitude disturbing the natural balance; however, recent efforts have been made through several mechanisms to respond and re-strengthen the antimicrobial armament. Such mechanisms include a variety of incentives, educational efforts and negotiations. Today, there are many more “man-made” factors that will determine a new balance or state of ecological harmony.Antibiotics are not a panacea nor will they ever be inutile. New resistance mechanisms will be identified and new antibiotics will be discovered, but most importantly, we must optimise our application of these extraordinary “biological tools”; therein lays our greatest challenge – creating a society that understands and respects the determinants of the effectiveness of antibiotics. The discovery of penicillin brought great hope for the treatment of infectious diseases. However, two decades later, in the late 1940s, acceptance, mass production and distribution led to the advent of the concept with which we are all now very familiar – antimicrobial resistance (AMR). As other classes of antibiotics emerged, a similar pattern ensued from discovery through increasing utilisation to resistance. Poor infection control practices in healthcare settings facilitated the transmission of resistant organisms, amplifying the effect of antimicrobial usage. The healthcare industry responded with two major programmes – Infection Control in the 1980s and Antimicrobial Stewardship in the last decade. These programs rely on the end user; however, while the importance of such global programmes cannot be disputed, these efforts alone are inadequate against the emergence of AMR.If clinicians are to have an antimicrobial arsenal to treat infectious diseases, a combination of efforts to both preserve the value of what is currently available and to promote drug discovery is needed. Preservation of existing antimicrobials ultimately relies on minimising their use in all circumstances. In humans, there are many strategies to support this goal, including the implementation and adherence to guidelines provided by international organisations such as the World Health Organization (WHO) and the Centres for Disease Control and Prevention, by national bodies and individual institutions, as well as through the implementation of rapid (possibly point-of-care) diagnostic tests for AMR. Further, infection prevention activities also need to be implemented to minimise the need for treatment.In addition to direct human ingestion, animal husbandry is a major setting for antibiotic consumption that allows indirect human exposure to the same antibiotic or the resistant organisms (or their resistance genes) emerging via animals farmed as a source of food. The strategies to curb this are considerably more complex, requiring engagement of a number of non-medical players less appreciative of the ramifications of antibiotic misuse.Drug discovery has suffered from a lack of exploitable bacterial binding sites, leading to the slow emergence of truly novel agents. Modifying existing antimicrobial agents to achieve theoretical benefits is often seen as commercially safer. Many pharmaceutical companies have shifted towards treatment of chronic conditions in order to lessen the financial risk. Therefore, this movement needs to be opposed with financial incentives and better protection for future and more novel antimicrobial discoveries. The antibiotic pipeline needs to be restored, but whether such efforts will be successful remains to be seen.Along with the excessive morbidity and mortality, AMR poses a major economic burden, which helps justify the costs of AMR response programmes. In the United States (US), mortality from antimicrobial-resistant infections is reported at 23,000 deaths annually, with costs estimated at US$ 26 billion . In EuroHumankind has enjoyed the capacity to treat bacterial infections for over 75 years. In this commentary, we present a number of efforts intended to help maintain this capacity moving forwards. These efforts could be regarded as more of a “bundle”, where none will have significant benefit if implemented in isolation. AMR is a natural phenomenon – there are behaviours that stimulate it and responses that minimise it. The fear of “re-entering the pre-antibiotic era” is as unlikely as the eradication of antimicrobial-resistant organisms. We do, however, have some control over where on this spectrum the value of our antimicrobial armament will sit in the future. While the development of other classes of therapeutics relies on building on an existing knowledge base, in the world of antimicrobials, advances need to be made while simultaneously protecting the current therapeutic options from the very real threats to their effectiveness.Bacteria are excellent at evolving mechanisms to counter the effects of antibacterial agents and this has been the way since ancient times, long before humans and modern medicine. Much has been written on the antibiotic resistosome, which has evolved from environmental bacteria and competitive pressure from other unicellular organisms in conjunction with the ability to receive genetic material both vertically (from “parent” bacteria) and horizontally from other organisms. Confronted with many mechanisms evident against all antimicrobials, it was time for a renewed respect for our relationship between microorganisms, antimicrobials and resistance mechanisms and drivers. In the human administration of antibiotics today, this is called stewardship.Although the concept of antibiotic stewardship was raised in the 1980s, it had no validation until a randomised controlled study demonstrated that antibiotic use could be significantly reduced without adverse outcomes , 4. SincThis lack of clarity of the benefits of a solitary intervention, such as antibiotic stewardship programmes, should not deter us from the very likely benefit  of it as part of a holistic multipronged response. Although efforts have been made internationally to encourage such programs, this is still primarily limited to acute care settings and tertiary centres where there are infectious disease physicians or microbiologist champions. However, to optimise future benefits, programmes must be effective in all healthcare settings, including outpatients, aged care, peripheral/rural hospitals, the community and, importantly, animal husbandry. Stewardship practices should not be limited to antimicrobials used for treatment, but should include those used for prophylaxis, which vary considerably and are not supported by evidence. The role of good stewardship can be undermined when attention is excessively directed to seemingly more tangible strategies such as reinforcing the pipeline of new antibiotics. A balanced approach is required.Non-prescription, over-the-counter antimicrobial access occurs worldwide, particularly in Asia and Southern Europe . This isAgriculture and food animals are important sources of AMR as they are exposed to enormous quantities of antibiotics and serve as a reservoir of antibiotic-resistance genes. Antibiotics have been used in animal husbandry, veterinary medicine and agriculture to prevent, control and treat infections for over 60 years. Antibiotic use in growth promotion is, however, controversial. It was discovered that small sub-therapeutic doses of penicillin and tetracycline could enhance weight gain by about 15–20 %, although the underlying mechanism of this action remains unclear –21. SwedAntibiotic discovery was strong from the 1940s to 1960s, but slowed for some decades until the introduction of linezolid in 2000 and daptomycin in 2003 . Most ofDrug discovery and development is conventionally driven by financial motivation. For the private industry, aligning to an antimicrobial programme has more risk than other pharmacological fields. Taking new drugs to market requires considerable testing for safety and efficacy in each indication sought. Once resistance ensues, the clinical, and therefore commercial, value of the product falls. In the medical profession, physicians try to limit a new antibiotic’s utilisation to maintain its clinical value; however, paradoxically, this affects sales, in effect creating another disincentive to drug discovery. When these new drugs are used in infectious disease management they have a short-term curative application as opposed to drugs used in the treatment of chronic diseases. It is no wonder that this “antibiotic pipeline” is weak and requires support from government and non-government sectors.Recognising the waning value of the antimicrobial armament, in 2004, the Infectious Diseases Society of America proposed legislative, regulatory and funding solutions to counteract barriers in the development of new antibiotics . These iThe 10 × ‘20 campaign, launched by the Infectious Diseases Society of America in 2010, called for the development and approval of 10 novel antibiotics by 2020 . The GloMany other initiatives and collaborations have recently been established to create new models for antibiotic development via incentive strategies. These include the provision of subsidies to lower antibiotic research costs for drugs with a large market, high potential sales and low risks. Sharing of scientific databases would facilitate collaboration between developers, minimising duplication and facilitating the dissemination of new information. Funding training and development of individuals specialising in research and development of antibiotics is key, as is financial support for small companies that are likely to produce marketable products but lack the capital to do so –37. The Another strategy was to compensate successful development as a way to motivate developers. The Generating Antibiotic Incentives Now Act was ratified in October 2012 by the US government and aimed to incentivize antibiotic discovery . This inPerhaps a more well-rounded model is the WHO’s Global Consortium , 45, whiAdditionally, many business models and incentive strategies have been proposed, with some placing emphasis on investment at the beginning of drug development while others reward only successful development of a drug . NeverthStaphylococcus aureus and vancomycin-resistant Enterococcus, and are approved for treatment of acute bacterial skin and skin structure infections [Pseudomonas aeruginosa, but only ceftazidime/avibactam has activity against Class A carbapenemases.Since the approval of telavancin in 2009 and ceftaroline in 2010, five new antibiotics have been approved by the FDA, three of which have mainly gram-positive activity and two directed against gram-negative pathogens. Oritavancin and dalbavancin are long-acting, synthetic lipoglycopeptides with activity against methicillin-resistant fections –49. Tedifections , with trfections –53. BothEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp (ESKAPE pathogens), and just over a third have activity against drug-resistant gonorrhoea, Clostridium difficile and carbapenem-resistant Enterobacteriaceae, which are pathogens considered an “urgent threat” by the Centres for Disease Control and Prevention [Acinetobacter sp. or the Class D metallo-beta-lactamases (e.g. NDM-1) [In May 2016, the Pew Charitable Trust, which maintains a regularly updated antibiotic pipeline, described 37 new antibiotics in development, of which 11 were in phase I clinical trials, 13 in phase II and 13 in phase III . About a. NDM-1) . Of the Bacteriophage treatment is a largely untested approach to antibacterial therapy and represents a revival of a previous option. These viruses infect and rapidly kill specific target bacteria. Nevertheless, none are in therapeutic practise although many are in development and at various stages of clinical trials . BacteriIn January 2015, a group of scientists from Northeastern University, MA, described their discovery of an antibiotic, teixobactin, with a completely different chemical scaffold to that of existing antibiotics. It has gram-positive activity, which works through binding to precursors of bacterial cell wall polymers and appears to have no inducible resistance. This discovery involved a novel technology called iChip , a miniaWhen a new antibiotic reaches the end of the arduous pipeline and is registered, the traditional patent-based pharmaceutical model grants developers market exclusivity for a number of years, following which generic manufacturers are permitted development and marketing opportunities to the drug. During these early years with the patent in place, the pharmaceutical organisation has a window period to obtain financial returns for the research and development, clinical trials and notoriously expensive marketing, as well as shareholder profit; they control the market geographically and they control price. Most patients cannot benefit from drugs that are not distributed globally and financial returns are not usually found in low- and middle-income countries. In fact, most patients in high-income countries will struggle with meeting the costs as individuals; drugs not yet recognised in national pharmaceutical benefit systems or by insurers may lead to excessive costs for individuals even in these countries.It is a “double-edged sword”, where we wish to improve access, yet not to the point where excessive use drives high levels of resistance. Any programme that improves access to individuals in need should build-in measures that minimise inappropriate use. The current model potentially creates incentive to encourage any consumption of the antibiotic, but only to those individuals and countries that can pay. In the last decade or so, several systems have provided remarkable contributions to access in low- and middle-income countries on a large scale, including the Global Fund , The Presidents Emergency Fund for AIDS Relief (PEPFAR) and the Global Alliance for Vaccines and Immunisation (GAVI). Ideally, similar efforts in the name of antibiotic access could ensue via negotiation and bulk purchasing or via subsidy. The crucial effort lies in delinking profits from sales volume. The scale of the antibiotic issue is different and the situation more complex, but some efforts have been initiated; examples include the Antibiotic Health Impact Fund, Antibiotics as Public Goods, and the Rewarding Antibiotic Development and Responsible Stewardship Programme, where antibiotics could be offered at a marginal cost and overall profitability of projects is tied to global health impact , 58. WhiThere is no doubt that inappropriate antibiotic use is a significant contributor to the emergence of resistance , 60. It Antibiotics are unique therapeutics and a human resource. They are the only drug class that, when used in an individual, it can affect others. Their misuse has resulted in a shift in the ecological balance between resistant and susceptible strains in the flora of humans, animals and the environment, and there are several determinants of where the balance will settle Fig. . As healThe urgent call to curb AMR requires efforts at all levels – governments, healthcare providers, pharmaceutical companies, veterinary and agriculture sectors, and the general public. We can be very optimistic about current deliberate efforts, including the expansion of stewardship in human medicine, animals and agriculture, changing regulations, and creating incentives that promote the discovery of truly novel drugs, as well as ensuring global access to antibiotics. Global society as a collective must share the same perspective of the issues and the vision required to work towards maintaining a strong antimicrobial armament. Antibiotics are a unique biological tool that demands respect, as does any form of Nature. Humans have only recently begun to influence the ecologic balance between antibacterials, bacteria and the resistance mechanisms they display. There is an increasing current understanding that we can favourably restore the balance that preserves and enhances the value of this critical therapeutic class.FDA, Food and drug administration; WHO, World health organization


Caenorhabditis elegans. To understand and control dynamics and kinetics of the processes involved, pharmacological modulation of specific G protein pathways would be advantageous, but is difficult due to a lack in accessibility and regulation. To provide this option, we designed G protein-coupled receptor-based designer receptors (DREADDs) for C. elegans. Initially described in mammalian systems, these modified muscarinic acetylcholine receptors are activated by the inert drug clozapine-N-oxide, but not by their endogenous agonists. We report a novel C. elegans-specific DREADD, functionally expressed and specifically activating Gq-protein signalling in vitro and in vivo which we used for modulating mating behaviour. Therefore, this novel designer receptor demonstrates the possibility to pharmacologically control physiological functions in C. elegans.G-protein signalling is an evolutionary conserved concept highlighting its fundamental impact on developmental and functional processes. Studies on the effects of G protein signals on tissues as well as an entire organism are often conducted in  So far only optogenetics have been successfully utilised as a tool for the manipulation of some signalling cascades in the nematode1213Despite the power and simplicity in the use of DREADDs many physiological processes and functions associated with specific signalling cascades are not easy to delineate in mammals due to the complexity of the organisms. The application in less complex systems would be highly advantageous to overcome these limitations. Recently, the DREADD technology was transferred to C. elegans using a newly designed DREADD based on the nematode muscarinic receptor GAR-3b. In-depth pharmacological characterisation in vitro revealed that the DREADD specifically activates Gq signalling. In the nematode the DREADD is able to modulate physiological functions upon stimulation with CNO in vivo.Here, we show that the DREADD technology can be applied in C. elegans is that the synthetic ligand CNO utilised to activate these receptors does not have any adverse effects on nematodes. To elucidate the influence the compound has on C. elegans fertility, development, viability, and the neuronal system as well as certain aspects of behaviour we treated wild-type nematodes in liquid culture with varying concentrations of CNO and assayed brood size, individuals reaching adulthood, lifespan, locomotion, pharyngeal pumping, egg laying and sensitivity to aldicarb. However, none of the parameters was affected  involved in controlling this process. GAR-3 has been shown to activate a Gq cascade similar to its mammalian homolog1819q-protein cascade is evolutionary well preserved. Thus, we speculated that the DREADD (rM3Dq) which is based on the rat M3 receptor (rM3R) can activate Gq signalling in the nematode. The C. elegans strain null for gar-3, gar-3(gk305) displayed a spicule protraction defect (16gar-3(gk305) mutants     and for Gi-protein signalling (hM4Di) (data not shown).As mammalian DREADDs have been shown to be a useful tool for analyses in n defect  which wadefect 16. Consist ± 2.4%) . We test ± 5.1%) . The sam ± 5.4%) . Due to  protein  despite  protein . LikewisC. elegans is likely due to difficulties in properly expressing or processing the receptors.Therefore, we conclude that the lack of activity of mammalian DREADDs in C. elegans, we set out to generate a nematode-specific DREADD for modulation of G protein-signalling pathways in the worm. We sought to base this modified receptor on a muscarinic acetylcholine receptor similar to the mammalian DREADDs. Three G protein-linked acetylcholine receptor genes are known in C. elegans  with GAR-2 being described to bind Gi/Go proteins and GAR-3 being a Gq-coupling receptor212123q signalling cascade upon stimulation with ACh or CCh1920rd intracellular loop (ICL)C. elegans GAR-3b  and A5.46 (A237G). The DREADD was N-terminally tagged with an HA-epitope to allow for detection using commercially available antibodies.Since mammalian DREADDs did not seem to be properly expressed in s GAR-3b , but onlns GAR-2 . Therefoq, Gi, and Gs among metazoan species analyses of functional G protein-coupling of receptors from C. elegans or other invertebrate species can be performed using mammalian systems272850 value of 3.5 μM, whereas the muscarinic agonist CCh did not have an effect on receptor activity   . For furpathways . These rin vivo. To ensure that the receptor is functional we first assessed expression of ceGAR-3Dq::yfp based on genomic gar-3 driven by the gar-3 promoter which is indistinguishable from gar-3::yfp, albeit appearing to be generally weaker ;Ex[cegar-3Dq] nematodes displayed no spicule protraction upon treatment with Oxo M or CCh  similar to the effect seen in untreated males (16.8 ± 2.1%)  , indicat ± 2.1%) . These donducted . Howevertraction .gar-3(gk305);Ex[cegar-3Dq] nematodes from CNO the spicule protraction rate decreased over time  which is about 100-fold higher than for the mammalian DREADD rM3Dq determined in yeast or cell culture experiments12As the technology of mammalian DREADDs was not easily transferable to pEC50: 5.6 ± 0.43 C. elegans DREADD is probably due to the lower potency of agonists at GAR-3b compared to rM3R , Oxotremorine M (Oxo M), forskolin, 3-isobutyl-1-methylxanthine (IBMX), aldicarb, and standard chemicals were purchased from Sigma Aldrich. Clozapine-N-Oxide (CNO) was obtained from the National Institutes of Health  as part of the Rapid Access to Investigative Drug Program funded by the National Institute of Neurological Disorders and Stroke. Substances applied were dissolved in the media or buffer the according experiment was performed in.in vitro assays were generated by cloning gar-3b cDNA into the mammalian expression vector pcDNA5FRT and contained an N-terminal HA-tag which does not alter functional properties of GPCRs (pSP109)cegar-3Dq was engineered by introducing the two point mutations Y146C/A237G into pSP109 (pSP112). For generation of constructs for in vivo analyses recombineering was employed42ar-3::yfp (genomic) inserted behind the gar-3 promoter (pYL9)C. elegans-specific DREADD cegar-3Dq downstream the gar-3 promoter (pSP110) was generated by inserting the two point mutations (Y146C/A237G) into the genomic sequence of gar-3 in the vector pYL9 gar-3 promoter-driven mammalian DREADDs  and rat M3R (pSP104) were cloned by exchanging gar-3 for the cDNA of the respective receptor in pYL9. For details see GAR-3b constructs for 2. Lipofectamine 2000 (Life Technologies) was used for transient transfection according to the manufactures’ recommendations.CHO-K1 and COS-7 cells were purchased from Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Culture GmbH , HEK-GT cells were obtained from ThermoFisher Scientific . All cells were grown in F12 media or Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator with 7% CODynamic mass redistribution measurements are a label free assay system to study GPCR properties in living cells5 cells/well) and transfected with a total amount of 0.5 μg of plasmid DNA and 1.5 μl Lipofectamine per well. Subsequently, cells were incubated with 2 μCi/ml of myo-3H-inositol  for 18 hours. Thereafter, cells were washed once with serum-free DMEM containing 10 mM LiCl followed by incubation with test compounds for 30 minutes at 37 °C. Intracellular IP levels were determined by anion-exchange chromatography as described previouslyTo measure inositol phosphate (IP) formation COS-7 cells were split into 12-well plates (1.5 × 106) and transfected with 3 μg plasmid DNA and 7.5 μl Lipofectamine the next day. 48 hours past transfection, cells were detached using Versene and labelled in DMEM with 4 μM Fluo-4 AM (Molecular probes) for 30 minutes at 37 °C. Free dye was removed by centrifugation, and cell suspensions were re-suspended into HBS buffer . The cell suspension was dispensed into black, clear-bottom 384 microwell plates  with 45.000 cells per well. Fluorescence measurements were performed in a two-step protocol using a fluorescence imaging plate reader and a robotic liquid handling station . Fluorescence intensity was corrected for the background and normalized to the initial intensities (F/F0).COS-7 cells were split into T25-flasks  was generated by the C. elegans gene knockout consortium. The strain pha-1(e2123); him-5 (e1490) gar-3(gk305) was previously described aprEx183[gar-3::yfp (pYL9) pha-1(+) pBSK], aprEx184[cegar-3Dq::yfp (pSP110) pha-1(+) pBSK], aprEx186[rM3R::yfp (pSP104) pha-1(+) pBSK], aprEx187[rM3Dq::yfp (pSP106) pha-1(+)pBSK] aprEx188[rM3Ds::yfp (pSP108) pha-1(+) pBSK] and aprEx189[hM4Di::yfp (pSP114) pha-1(+) pBSK] were generated for this study  and confocal fluorescent images were collected with an Olympus Fluoview FV1000 setup. Fluorescence signals were quantified by intensity analysis using ImageJ softwareStatistical significance of assay data from spicule protraction assays was determined using a Fisher’s exact test for each genotype and condition. For statistical analyses of brood size, individuals reaching adulthood, lifespan, locomotion, pharyngeal pumping, egg laying, sensitivity to aldicarb, and fluorescent intensity calculations a student’s t-test was performed.How to cite this article: Prömel, S. et al. Deciphering and modulating G protein signalling in C. elegans using the DREADD technology. Sci. Rep.6, 28901; doi: 10.1038/srep28901 (2016).


To elucidate the evolution of a lung-sparing strategy with sleeve lobectomy (SL) and induction therapy for non-small cell lung cancer (NSCLC).n = 54) or SL (n = 151) from 1994 to 2013. The study period was divided into four 5-year periods, and surgical trends were analyzed, focusing on the PN:SL ratio.We retrospectively reviewed 205 patients with NSCLC who underwent pneumonectomy  and 90-day  mortality than the SL group. The overall 5-year survival rate was significantly higher with SL (71.5 %) versus PN  for patients with pN0–1. The ratio of PN among total surgeries decreased significantly over the four periods  from 5.63 % to 3.17, 1.40, and 1.38 %, respectively (p < 0.0001); in contrast, the PN:SL ratio increased significantly from 1.64 to 2.50, 3.71, and 5.44, respectively (p = 0.041). During the last period, when we introduced induction therapy, 38 of 651 who received surgery underwent induction therapy. The PN:SL ratios of those who did and did not undergo induction therapy were 15  and 4.25 , respectively.PN was associated with a significantly advanced pathological stage, a larger tumor size and less pulmonary function compared with SL. The PN group had higher 30-day (3.7 vs. 0 %, A lung-sparing strategy with SL for NSCLC can decrease the PN rate to less than 2 % with less mortality. Induction therapy may facilitate SL and increase the PN:SL ratio. The purpose of surgical resection for non-small cell lung cancer (NSCLC) is to achieve complete resection of the tumor and lymph nodes to maximize the possibility of cure. Bronchial and/or pulmonary arterial sleeve lobectomy (SL) is a lung parenchyma-sparing procedure that aims for complete resection of tumors invading the central structures. SL was originally indicated for patients with reduced pulmonary function, who were intolerant of pneumonectomy (PN). However, SL is now indicated even for patients with sufficient pulmonary function to avoid PN, which causes substantial loss of pulmonary function and thus quality of life. Much evidence has been accumulated that the long-term survival after SL is favorable to that of PN, with lower mortality and morbidity –7. A recInduction therapy has been increasingly used for locally advanced NSCLC to downstage tumors and to facilitate complete resection. However, very few studies have compared outcomes of SL and PN after induction therapy , 15. MauWe perform SL to avoid PN whenever it is technically and oncologically feasible, even for patients with sufficient pulmonary function. In this retrospective study, we reviewed the outcomes of PN and SL in our institute and elucidated the evolution of a lung-sparing strategy to avoid PN with SL and induction therapy.n = 54) or SL (n = 151) at our institution from January 1994 to December 2013. During the same period, 2047 patients with NSCLC underwent surgery at our institution. The 20-year study period was then divided into four periods of 5 years each, and the trends in surgical strategy were analyzed, focusing on the PN:SL ratio. The Ethics Committee of Chiba University approved this study and granted a waiver for patient consent.We retrospectively reviewed the clinical records of 205 patients with NSCLC who underwent PN (18F]-FDG-PET) was frequently used in recent years. Bronchoscopy was performed to confirm the diagnosis of malignancy and to observe the extent of tumor invasion to the bronchus. Suspected hilar and mediastinal lymph nodal involvements by CT or FDG-PET were confirmed pathologically by endobronchial ultrasound-guided transbronchial needle aspiration [The preoperative work-up for all patients included physical examination, chest radiography, contrast-enhanced thoracic, upper-abdominal computed tomography (CT), cerebral CT or magnetic resonance imaging, and isotonic bone scanning. Positron emission tomography  if all gross disease was removed and if all surgical margins were free of tumor cells. Incomplete resection (R1 and R2) indicated that surgical margins were microscopically positive (R1) or macroscopically positive (R2).t test for continuous variables (mean and standard deviations) and χ2 analysis for categorical variables (frequency and percentages) as appropriate. Survival was calculated from the date of surgery until the date of death (due to any cause) or last follow-up (censored). Survival curves were created using the Kaplan–Meier method, and statistically significant differences between survival curves were examined using log-rank tests. A p value derived from two-tailed tests of less than or equal to 0.05 was considered significant. All data were analyzed using JMP, version 5.0 .Patient demographics were compared between groups by p = 0.057). Three patients (5.6 %) in the PN group and 31 patients (20.5 %) in the SL group were eligible for adjuvant therapy (p = 0.028).Patients’ clinical characteristics according to the type of surgery (PN or SL) are shown in Table n = 74, 49 %), followed by vascular SL  and broncho-vascular SL . Regarding lung resection, the right upper lobe was the most frequent area resected . Extended SL resection involving more than one lobe was achieved in 27 patients (17.9 %).Table p = 0.018) and 90-day (13.0 vs. 1.3 %. p = 0.0003) mortality rates than those who underwent SL. Two of 54 patients in the PN group died within 30 days: 1 patient died from a myocardial infarction followed by acute respiratory distress syndrome (ARDS), and the other died from a bronchial fistula with empyema followed by ARDS. An additional five patients in the PN group died within 90 days from ARDS in two patients, interstitial pneumonia in one patient, and tumor recurrence in two patients. Two of 151 patients in the SL group died within 90 days: one patient died from a bronchial fistula with empyema, and the other died from pneumonia. In terms of morbidity, there were no statistically significant differences in the rate of major  and minor  complications between groups.Table p = 0.0007, Fig. n = 107) and PN (n = 20) was 71.5 and 42.8 %, respectively  and PN (n = 32) for patients with pN2–3  and stage III–IV (p = 0.23).The median follow-up time for the 54 patients in the PN group was 27.2 months (range 0.6–126.5 months), while that for the 151 patients in the SL group was 44.2 months (range 0.4–162 months). The overall 5-year survival rate of the patients in the SL group was significantly higher than that of the patients in the PN group  who underwent surgery from 2003 to 2013. Eight of 19 patients in the PN group (42.1 %) had recurrence, consisting of systemic recurrence in 5 patients (26.3 %), local recurrence in 2 patients (10.5 %), and both systemic and local recurrence in 1 patient (5.3 %), while 38 of 88 patients (43.2 %) in the SL group had recurrence, consisting of systemic recurrence in 23 patients (26.1 %), local recurrence in 10 patients (11.4 %), and both systemic and local recurrence in 5 patients (5.7 %).p < 0.0001). The ratio of SL among total surgeries also decreased from the first (1994–1998) to the third (2004–2008) period from 9.20 to 7.94 and 5.20 %. These declines may reflect the increase of small peripheral lung cancer in recent years. However, the ratio of SL increased in the last period (2009–2013) to 7.53 %. The PN:SL ratio increased significantly over the four periods from 1.64 to 2.50, 3.71, and 5.44, respectively (p = 0.041) and the average was 2.80. During the last period (2009–2013), when we introduced induction therapy for patients with central tumors or with mediastinal nodal involvement, 38 of 651 who received surgery underwent induction therapy. The PN:SL ratios of those who did and did not undergo induction therapy were 15  and 4.25 , respectively (p = 0.25).Table In the present study, we reviewed the evolution of a lung-sparing strategy for NSCLC over 20 years in a single institution. The ratio of PN among total surgeries decreased significantly, while the PN:SL ratio increased significantly over the study period. Induction therapy appeared to contribute to the increase in the PN:SL ratio. Long-term survival in patients with pN0–1 was significantly better in the SL group than in the PN group, and postoperative mortality and morbidity were significantly lower in the SL group than in the PN group.The application of SL has been extended to patients with sufficient pulmonary function as a lung parenchyma-sparing strategy that aims to decrease postoperative mortality and morbidity and to improve long-term outcomes. The operative risk of PN is still high, with mortality rates ranging from approximately 5 to 10 % in recent reports, despite the improvements in surgical techniques and postoperative care –21. In aWe have been trying to avoid PN by making full use of broncho-vascular reconstruction. The ratio of PN among total surgeries for NSCLC was 2.64 % throughout the study period, and it decreased to less than 1.5 % in the past 10 years. According to recent reports using the national database, the ratio of PN among surgeries for NSCLC was 7.2–12.3 % , 19, 23.Gómez-Caro and coworkers reported their aggressive policy of PN avoidance and showed that the PN:SL ratio can be used as a quality standard and that the ratio should be at least 1.5 or 2 . RegardiOne of the major concerns with SL is locoregional recurrence. According to the recent meta-analysis by Shi et al., the pooled locoregional recurrence with SL was 14.44 % compared with 26.08 % with PN, which was not statistically significant , 6.Our study has some limitations. It is retrospective, and it had a long time interval of patient recruitment that had changes in the treatment of lung cancer. In addition, the results of recurrence were only available in 52.2 % of patients. Another limitation is that since PN was significantly associated with advanced disease compared with SL, the difference in outcomes between groups was associated with selection bias. Therefore, our findings should be interpreted with caution.In conclusion, this study demonstrated the actual evolution of a lung-sparing strategy over 20 years in a single institution. A lung-sparing strategy with SL could decrease the ratio of PN substantially, with less mortality. Induction therapy may facilitate SL and increase the PN:SL ratio.


A medical emergency team (MET) was introduced in our institution in January 2012 to provide timely response to the needs of acutely ill inpatients and cardiac arrest calls. The MET assesses the patient and prescribes a management plan for the responsible team to follow; promptly stabilising and transferring patients to a place of safety where required. We aimed at evaluating the effects of introducing the MET on clinically relevant processes and outcomes.Prospective data were analysed using STATA 10.1. The primary outcome measure was immediate mortality ; the secondary outcome measures were admission to critical care after a MET call and cardiac arrest.P = 0.18) and secondary outcomes .A total of 5,763 MET calls were made between 9 January 2012 and 4 March 2014, of which 5,310 (92.1%) were MET calls, 349 (6.1%) cardiac arrest calls, 36 (0.6%) false alarms and 68 (1.2%) unclassified. The number of calls increased by 32.7% from 2,255 in 2012 to 2,993 in 2013, with all month-specific comparisons showing significant increases in MET activity (ranging from 0.5% to 103.6% increases). MET activity displayed cyclical yearly changes, with the winter months and the month of August (junior doctors' changeover period) being particularly busy. Median response time (interquartile range) was 1 (1 to 2) minutes, with 99.1% calls attended to within 3 minutes. There were 210 (3.64%) immediate deaths (with no significant differences between years), 112 (1.9%) patient transfers to critical care, 233 (4%) patients were transferred to other locations  while 4,697 (81.5%) patients remained on the ward of origin. In 408 cases (7.1%) a do-not-resuscitate order was instituted. On multiple logistic regression analyses, when the type of call was taken into consideration, the response time had no influence on primary (mortality OR = 0.83, 95% CI = 0.63 to 1.09, The MET has been successfully implemented, with demand for its services having increased by 32.7% in 1 year. The unadjusted immediate mortality rate of patients for whom a MET/cardiac arrest call is activated is 3.64%. Response time had no influence on mortality, most probably due to the rapid response time. Immediate mortality was low, probably as a result of early adequate intervention. Further evaluation of overall hospital mortality is warranted for future studies.


Approximately 200 participants from several studies were included in this research. STATA 11.2 software  and Meta-DiSc 1.4  were used to perform the meta-analysis.Nine studies were included in the meta-analysis. The pooled diagnostic odds ratio was 18.94 . The sensitivity was 58.80%  and the specificity was 90.20% . The positive and negative likelihood ratios were 3.12  and 0.26 , respectively. The area under the curve of the summary receiver-operating characteristic was 0.88.+/IgG+ ratio of plasmacytic infiltration is modestly effective in diagnosing IgG-related disease.To conclude, the IgG4 Few studies have focused on the incidence or prevalence of IgG4-RD, but recent epidemiological studies have shown that approximately 6700 to 26,000 patients have developed IgG4-RD over the past 20 years in Japan.2Immunoglobulin G (IgG)4-related disease (IgG4-RD) is a systemic immune-mediated disease characterized by storiform fibrosis, infiltration of IgG43 Furthermore, elevated serum IgG4 is not exclusively observed in IgG4-RD, and 30% of IgG4-RD patients indeed present normal IgG4 levels.5 Histopathological evidence has shown higher sensitivity and specificity for steroid therapy, and is widely accepted as the gold standard for the diagnosis of IgG4-RD.6Diagnosis of IgG4-RD mainly depends on 4 criteria: histopathology, imaging, serology, and response to steroid therapy. However, these criteria are insufficient for diagnosing IgG4-RD. Imaging of involved organs cannot efficiently differentiate tumors and IgG4-RD.+ plasma cells are the most important features of IgG4-RD, especially the IgG4+/IgG+ ratio. Over the past decade, numerous studies have evaluated the IgG4+/IgG+ ratio for its ability to accurately diagnose IgG4-RD. However, inconsistent conclusions relating to the diagnostic performance of IgG4+/IgG+ ratio antibodies have been drawn.15 The aim of this study was to systematically review the literature to determine the diagnostic performance of the IgG4+/IgG+ ratio in patients with IgG4-RD.Histopathological findings of IgG4+ plasma cells, we searched for the follow terms: “IgG4/IgG ratio,” “IgG4+/IgG+ ratio,” “ratio of IgG4+/IgG+,” and “IgG4+/IgG+” combined with IgG4-related diseases. No limits were placed on ethnicity or geographic region, and all documents were updated to September 2014. Additional relevant references cited in searched articles were also selected. All analyses of this systemic review were based on previous published studies, thus no ethical approval and patient consent are required.Studies were identified in the EMBASE, ISI Web of Knowledge, PubMed, and the Cochrane Library databases. To retrieve all relevant publications related to histopathological findings of IgG4+ plasma cells with an IgG4+/IgG+ ratio ≥0.40 and IgG4+/high power fields (HPFs) >10 for IgG4-RD; sufficient data reported to construct 2-by-2 tables; no criteria for published language; and studies based on animals or cell cultures and case reports; conference abstracts without subsequent publication in full text were excluded. In the case of overlapping studies, only the study with the largest sample size was included in our analysis.Studies meeting the following criteria were eligible for inclusion: those that assessed the diagnostic accuracy of histopathological findings of IgG4Data were extracted from all selected studies by 2 independent investigators. Interresearcher disagreements were resolved by consensus or by a third investigator. The following data were collected from each selected study: first author's name; publication year; country in which the study was performed; study design; and study results. Study quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool. Authors of the identified studies were contacted via e-mail if further study details were needed.Q-statistic as well as I2-statistic. A P value >0.10 in Q-statistic indicated lack of heterogeneity among studies. I2 <25% was considered low heterogeneity, 25% to 50% moderate, and >50% a high degree of inconsistency. Finally, the overall or pooled diagnostic odds ratio (DOR), sensitivity, specificity, positive likelihood ratio (LR+) and negative likelihood ratio (LR–), and their 95% CIs were obtained by a random-effects or a fixed-effects model in the presence (P ≤ 0.10 or I2 > 50%) or absence (P > 0.10 and I2 ≤ 50%) of heterogeneity, respectively. The area under the summary receiver-operating characteristic (SROC) curves represented the overall performance of the detection method. A P value <0.05 (2 sided) was considered significant. Evaluation of threshold effect and publication bias was also undertaken.Statistical analysis was performed using STATA 11.2 software  and Meta-DiSc 1.4 . Heterogeneity between studies was evaluated by Cochrane 15Electronic and manual searches yielded a total of 388 potentially eligible articles. A flow chart of screening articles for meta-analysis is illustrated in Figure 8 6 in Japan,15 and 1 in Korea.12 Assessment using QUADAS indicated that the studies were of medium quality, with positive results for at least 6/14 items . The sensitivity was 58.80%  and the specificity was 90.20% . The LR+ and LR− were 3.12  and 0.26 , respectively. The area under the curve (AUC) of the SROC was 0.88. The forest plots and SROC are shown in Figure The sensitivity of the IgG4+/IgG+ ratio of plasmacytic infiltration with IgG4+ plasma cells/HPF, we performed meta-analysis of IgG4+ plasma cells/HPF based on the articles included in this research. The sensitivity of IgG4+ plasma cells/HPF ranged from 38.00% to 97.60%, and reported specificity ranged from 50.00% to 81.80%. The pooled DOR was 26.16 . The sensitivity was 72.20%  and the specificity was 72.50% . The LR+ and LR− were 3.08  and 0.09 , respectively. The AUC of the SROC was 0.91. The forest plots and SROC are shown in Figure In order to compare the IgG4P = 0.009; the slope (b) of the regression equation did differ from zero (P = 0.412)].A Spearman rank correlation was performed to confirm the threshold effect; indication of no threshold effect was found [Spearman correlation coefficient = −0.77, P < 0.05) was considered to indicate possible bias. We conducted funnel plots that represented a somewhat symmetric curve  but the sensitivity was higher (72.20%). The AUC of SROC was 0.91. Primary comparison revealed that the diagnostic performance of IgG4+/HPF was better than that of the IgG4+/IgG+ ratio.The IgG4+/IgG+ ratio . In order to explore the heterogeneity found across studies, publication bias and Spearman rank correlation were performed. No statistical difference was found using Spearman rank correlation, which means that there was no threshold effect among these studies. However, publication bias was found in this meta-analysis, which could introduce heterogeneity.Heterogeneity has been found in the pooled DOR of the IgG4+/IgG+ ratio remains somewhat controversial, and exploring its clinical significance might help further our understanding of the IgG4+/IgG+ ratio; however, relatively few studies have focused on this aspect. Second, meta-analysis of IgG4+ plasma cells/HPF only considered the studies that we included for the IgG4+/IgG+ ratio, and thus, requires further research.Some limitations in this meta-analysis need to be noted. The cutoff value of the IgG4+/IgG+ ratio is a modestly effective diagnostic biomarker for IgG4-RD, with a low sensitivity but a high specificity.In conclusion, the IgG4


Heroin use is closely associated with emotional dysregulation, which may explain its high comorbidity with disorders such as anxiety and depression. However, the understanding of the neurobiological etiology of the association between heroin use and emotional dysregulation is limited. Previous studies have suggested an impact of heroin on diffusivity in white matter involving the emotional regulatory system, but the specificity of this finding remains to be determined. Therefore, this study investigated the association between heroin use and diffusivity of white matter tracts in heroin users and examined whether the tracts were associated with their elevated anxiety and depression levels. A sample of 26 right-handed male abstinent heroin users (25 to 42 years of age) and 32 matched healthy controls (19 to 55 years of age) was recruited for this study. Diffusion tensor imaging data were collected, and their levels of anxiety and depression were assessed using the Hospital Anxiety and Depression Scale. Our findings indicated that heroin users exhibited higher levels of anxiety and depression, but the heroin use-associated left uncinate fasciculus was only related to their anxiety level, suggesting that association between heroin and anxiety has an incremental organic basis but that for depression could be a threshold issue. This finding improves our understanding of heroin addiction and its comorbid affective disorder and facilitates future therapeutic development. In previous studies, anxiety sensitivity has been reported to be associated with substance use, and the prevalence of anxiety disorders among heroin users is more than half.16 Importantly, neuroimaging findings using diffusion tensor imaging (DTI) have suggested a substantial overlap between changes in white matter (WM) structural connectivity among heroin users and anxiety disorder or depression. DTI was used to investigate WM structural connectivity by quantifying the degree and directionality of diffusion of water molecules along their fiber tracts and local neural tissue via four diffusion indices. In general, fractional anisotropy (FA) has been suggested to characterize WM integrity, mean diffusivity (MD) has been suggested to characterize changes in diffusion tensors, and axial diffusivity (AD) and radial diffusivity (RD) have been suggested to be specific for axonal and myelination changes, respectively.17Several neuroimaging studies have aimed to explore heroin users' brain changes and their corresponding impairments in cognition and behavior, the results of which revealed that heroin addiction resembles a disorder of neural connectivity.18 emotional regulation and memory.19 These widespread disruptions in WM connectivity were consistently observed in the WM connecting the frontal, cingulate, corpus callosum and temporal regions, with more widespread damage among those with more prolonged heroin usage.22 In addition, individuals with generalized anxiety disorder or major depressive disorder often exhibit differences in WM connections involving frontal and limbic regions; for instance, the uncinate fasciculus that connects frontal regions to temporal regions, including the amygdala, was reported to be different.25 Patients with social anxiety disorder were reported to exhibit anomalies in WM and deficits in the left insula and the left inferior frontal gyrus were associated with the severity of social anxiety.26 At the subclinical level, individual differences in trait anxiety were also associated with WM connectivity in the left temporal cortex and the uncinate fasciculus.29 Specific impairments were observed in the corpus callosum and the uncinate fasciculus in adolescence-onset patients.30 WM matter deficits in connections involving cingulate region and the parahippocampal area were identified in young adult31 and late-life depression patients.33 The severity of depression was suggested to be associated with the WM deficits in connectivity involving the frontal, limbic and cingulate cortices and the corpus callosum, including WM deficits in the anterior cingulate cortex in subclinical depression.34 These studies have indicated close associations between WM connectivity, specifically in the uncinate fasciculus, and the levels of anxiety and depression in both subclinical and clinical populations. Particularly, the overlap of the changes in structural connectivity between heroin use and anxiety disorder and depression suggests that the effects of heroin on structural connectivity might disrupt emotional regulation. Therefore, the next logical step would be to investigate whether the effect of heroin on the neurobiological emotional regulatory system is general or specific. We hypothesized that (1) the microstructural WM in frontal, cingulate and temporal regions in heroin users are associated with their duration of heroin use and that (2) the connectivity between these regions, as reflected by diffusivity indices, are related to the severity of both anxiety and depression in heroin users.Using DTI, studies have revealed aberrant WM structural connectivity in chronic heroin users and have associated these WM microstructural damages with their impairments in risk taking,19 As polydrug use was very common among drug abusers, our heroin subject group was defined as those using heroin at >50% of their overall drug use and diagnosed with heroin abuse or dependence based on a structured clinical interview. Age-matched healthy controls without any history of substance abuse were recruited using advertisements in the local communities. No participants exhibited any neurological or psychiatric disorder. Raven's Progressive Matrices,35 a test of nonverbal intelligence, was administered to the participants to ensure that the intellectual capacity of the heroin users and the healthy controls was matched. Their body mass index was also measured and matched accordingly. This study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all the participants.Twenty-six male abstinent heroin users with mean age of 34.23 years (s.d.=3.85) and thirty-two age-matched healthy controls with mean age of 31.84 years (s.d.=11.08) participated in this study, which was approved by the Institutional Review Board of The University of Hong Kong and the Hospital Authority (The West Cluster). The heroin users were recruited from two drug rehabilitation centers, and those who had any history of replacement therapy were excluded from the present study to avoid medication effects.36 This scale consists of 14 items on a four-point Likert scale measuring the level of distress in the subjects. Two separate scores of their anxiety and depression levels were obtained and analyzed in this study.The Hospital Anxiety and Depression Scale (HADS) was used in this study.b=0) reference using the following imaging parameters: TR=10 000 ms, TE=88.4 ms, flip angle=90°, phase encoding direction=COL and voxel size=0.94 × 0.94 × 4.00mm. T1-weighted structural images were also obtained, and the corresponding parameters were: TR=9.464 ms, TE=3.892 ms, flip angle=20°, phase encoding direction = ROW and voxel size = 0.90 × 0.47 × 0.47 mm. The anterior commissure, the posterior commissure and the mid-sagittal plane were manually defined in the T1-weighted images and were used to convert the T1-weighted images to the conventional anterior commissure–posterior commissure-aligned space via rigid-body transformation.DTI data sets were obtained using a 3T GE Signa Propeller HD MR Scanner  equipped with a standard multichannel head coil. They were acquired in 25 diffusion gradient directions with a nondiffusion weighted  using a seventh-order b-spline algorithm. Then, the diffusion tensors were fitted to the re-sampled DTI data using a robust least-squares algorithm and outliers from the diffusion tensor estimation were removed. From the diffusion tensor model, four diffusivity measure maps for each subject were computed. FA and MD are summative measures that describe a normalized s.d. of the three diffusion directions and average total diffusion, respectively; whereas AD and RD describe diffusion parallel and perpendicular to the principal diffusion direction, respectively. The units for MD, AD and RD would be mm2s−1. The four diffusivity maps  of each subject were further masked, of which any undefined values in the diffusivity maps were excluded using FMRIB Software Library v5.0.37All DTI data were preprocessed using the VISTA Lab  diffusion MRI software. The DTI images were first aligned to the t-tests were used to compare the characteristics and the HADS scores between the groups. For the DTI data sets, voxel-wise statistical analysis of the resulting FA, MD, AD and RD maps were performed using Tract-Based Spatial Statistics (TBSS)38 in FMRIB Software Library v5.0. TBSS, a method of voxel-wise statistics that is performed on an alignment-invariant tract representation, was used to investigate the association between heroin use and the anxiety and depression levels in our sample. This method is more sensitive and objective for analyzing diffusion images than the conventional structural voxel-based morphometry analysis, as the latter is susceptible to the generation of false positives.39 All the FA images were aligned to the target FA image and were transformed to the standard Montreal Neurological Institute (MNI) space during nonlinear registration. Then, a mean FA image was produced and thinned to generate a mean FA skeleton using FA >0.2 as the threshold. Next, the individuals' FA, MD, AD and RD maps were projected onto the mean FA skeleton, and all diffusivity maps were subsequently used for voxel-wise statistical analyses for comparisons between the abstinent heroin users and the healthy controls  were identified according to the probabilistic tractography atlas (the Johns Hopkins University WM tractography atlas). The age, the Raven's Progressive Matrices score, the duration of abstinence and the average diffusivity indices  for each TOI were demeaned and then included as independent variables in multiple linear regression models and the HADS anxiety and depression scores as dependent variables for further analyses of the heroin users. It was performed to investigate how the heroin users' HADS anxiety or depression levels changed when the diffusivity of a specific TOI varied.P>0.05; The age, body mass index, and Raven's Progressive Matrices score of the heroin users and the healthy controls were matched P>0.05; . The uset=9.27, P<0.001) and depression .Heroin users were also found to experience elevated levels of anxiety  and in RD of clusters peaking at left superior longitudinal fasciculus . RD of clusters within uncinate fasciculus was also positively associated with the heroin use . No significant association was observed between the AD or the RD of the TOIs and the HADS anxiety or depression score (P>0.05).Eight WM TOIs were selected  according to the peak voxels in the WM clusters, which were identified to be associated with the duration of heroin use based on TBSS. The age, the Raven's Progressive Matrices score, the duration of abstinence and the average diffusivity measures for the given TOI  were included as independent variables in multiple linear regression models of the HADS anxiety and depression scores for further analyses of the heroin users. It was found that the anxiety level significantly changes when MD of the left uncinate fasciculus varied  and temporal  regions were found to be associated with the duration of heroin use. In previous studies, reduced FA in bilateral frontal sub-gyral regions were identified in current heroin users;12 Our findings suggested that the change in the left uncinate fasciculus related to prolonged heroin use was associated with the level of anxiety of heroin users. The uncinate fasciculus, which connects the anterior temporal lobe  to the medial and lateral orbitofrontal cortex (OFC), is particularly important for emotional processing and regulation.43 Patients with social anxiety disorder were reported to display WM anomalies in the inferior frontal gyrus and the middle temporal gyrus,26 and individual differences in trait anxiety were also associated with deficits in WM connectivity in the uncinate fasciculus.29 Along these lines, the impairment in the uncinate fasciculus observed in our heroin sample may explain the emotional dysregulation exhibited by heroin abusers44 and the alteration in the reward circuit characterized by drug dependence.46Our heroin user sample, relative to the healthy control sample, exhibited higher anxiety and depression levels. This result is in accordance with previous studies that reported a high comorbidity between heroin use and anxiety and depression.47 Within the temporal region, the pivotal role of the amygdala in drug addiction has been illustrated,48 and studies have reported that heroin acutely affects negative emotional processing and regulation by modulating amygdala responses.49 Our present findings confirmed widespread damage to WM microstructures due to heroin use, and this damage was located in not only the amygdala but also its projections to OFC regions, which was in accordance with another study that reported that opioid-dependent patients exhibited alterations in the affective network, including connections projected from the amygdala.50 The OFC has a key role in responses to reinforcement by evaluating rewarding and punishing reinforcers.52 One study has investigated heroin addicts using PET and observed that the regional cerebral blood flow in the OFC was associated with their urge to use heroin.53 Abnormal patterns of fMRI activation in the bilateral OFC of heroin users during decision-making were reported; furthermore, a strong association between the duration of heroin use and the activation of their left OFC was detected.54 The MD of the uncinate fasciculus in our heroin users associated with their anxiety level, indicating the close association between prolonged heroin use-related microstructural changes in these WM tracts and their anxiety level. Our finding supports the concept that heroin use may be associated with the users' elevated anxiety level by damaging the WM in the uncinate fasciculus, although this speculation requires verification in future studies.In a previous study, substance addicts with affective disorder displayed significantly reduced brain activity in their right frontal and left temporal regions.56 Our findings suggest that the prolonged heroin use-induced changes in WM tract diffusivity were associated with elevated anxiety on an incremental organic basis but not with depression. We propose that heroin use may induce elevated anxiety by affecting the uncinate fasciculus, whereas elevated depression may be observed in heroin users when sufficient dosages of heroin were taken.In contrast, heroin users' depression level did not change while the diffusivity of WM tracts varied. In previous reports, only few animal studies have reported on the heroin-induced anxiety or depression levels.49 Heroin users might ultimately adapt to the dampening of anxiety by continuously using heroin and might develop an unhealthy mechanism of managing increased anxiety, paired with structural changes in underlying brain connectivity. Therefore, the elevated anxiety was related to changes in the uncinate fasciculus that was associated with chronic heroin use, as demonstrated in our study. However, prospective studies are required to confirm these results and to further investigate the relationship between depression and heroin use.Stress hormone release with respect to acute heroin use was evaluated recently and a close association between reduced left amygdala responses, lower anxiety levels and lower concentrations of stress biomarkers, such as adrenocorticotropic hormone, serum cortisol and saliva cortisol, was identified after administration of heroin.43 Therefore, the current findings pointed to the direction that prolonged heroin use may damage users' uncinate fasciculus, which is related to aberrant top-down emotional regulation. Such heroin-induced emotional dysregulation depended on the users' elevated anxiety; however, the elevated depression levels experienced by the heroin users appeared to be more of a threshold issue. Our findings also support the theoretical model explaining how heroin intake increases anxiety via cognitive and biopsychological mechanisms.57 We believe that our findings have increased our understanding of substance addiction and its comorbid affective disorder, facilitating future therapeutic development for heroin abuse.On the basis of the findings of this study, heroin users exhibited compromised emotional regulation, as reflected by their elevated anxiety and depression levels. Particularly, elevated anxiety was related to the uncinate fasciculus that was associated with heroin use. The uncinate fasciculus connects frontal to temporal regions and is responsible for top-down emotional regulation.


Vibrio cholerae can enter into a viable but non-culturable (VBNC) state in order to survive in unfavorable environments. In this study, we studied the roles of five physicochemical and microbiological factors or states, namely, different strains, growth phases, oxygen, temperature, and starvation, on the development of VBNC of V. cholerae in artificial sea water (ASW). Different strains of the organism, the growth phase, and oxygen levels affected the progress of VBNC development. It was found that the VBNC state was induced faster in V. cholerae serogroup O1 classical biotype strain O395 than in O1 El Tor biotype strains C6706 and N16961. When cells in different growth phases were used for VBNC induction, stationary-phase cells lost their culturability more quickly than exponential-phase cells, while induction of a totally non-culturable state took longer to achieve for stationary-phase cells in all three strains, suggesting that heterogeneity of cells should be considered. Aeration strongly accelerated the loss of culturability. During the development of the VBNC state, the culturable cell count under aeration conditions was almost 106-fold lower than under oxygen-limited conditions for all three strains. The other two factors, temperature and nutrients-rich environment, may prevent the induction of VBNC cells. At 22 or 37°C in ASW, most of the cells rapidly died and the culturable cell count reduced from about 108 to 106–105 CFU/mL. The total cell counts showed that cells that lost viability were decomposed, and the viable cell counts were the same as culturable cell counts, indicating that the cells did not reach the VBNC state. VBNC state development was blocked when ASW was supplied with Luria-Bertani broth (LB), but it was not affected in ASW with M9, suggesting that specific nutrients in LB may prevent the development of VBNC state. These results revealed that the five factors evaluated in this study had different roles during the progress of VBNC induction. Changing a single factor could influence and even block the development of the VBNC state. These findings provide new insight to help design further studies to better understand the mechanisms which trigger the development and regulation of the VBNC state. Vibrio cholerae cells in the VBNC state can be resuscitated by introduction in a rabbit ileal loop and in the intestines of human volunteers  state is defined as a state where bacteria are metabolically active but lack the ability to reproduce on routine culture media . Many balunteers . For othperature .Since the first report of VBNC in V. vulnificus with or without starvation  alone could induce the VBNC state in arvation . Howevers at 5°C . Furthers at 5°C . These fs at 5°C . ComparaV. cholerae. In a laboratory setting, VBNC is usually induced in V. cholerae by incubating the culture in artificial sea water (ASW) at 4°C  at 4°C . Some miVibrio cholerae O1 serogroup classical biotype strain O395 and two O1 El Tor biotypes strains, namely, C6706 and N16961 (preserved in our laboratory) were used in this study. These strains were initially stored in 20% (v/v) glycerol at -80°C, then cultured on nutrient agar.600 = 1.0 (approximately 1 × 109 CFU/mL). Finally, the cultures were inoculated at a final concentration of 1 × 108 CFU/mL into different media for use in subsequent experiments.Three single colonies of each strain were picked and suspended in Luria-Bertani broth  and cultured with shaking (200 rpm) at 37°C overnight. The cultures were then diluted in fresh LB broth , incubated with shaking at 200 rpm at 37°C and grown to mid-exponential-phase and stationary-phase. The cultures were then washed twice with ASW and diluted to OD4Cl as the nitrogen source.Three culture media  were used in this study. ASW was made of 40 g/L sea salt  and sterilized by passing through a 0.22 μm membrane filter . ASW-LB was prepared by mixing ASW with 10× of LB  to make a final concentration of 1% tryptone and 0.5% yeast extract. ASW-M9 was prepared by mixing ASW with 10× of M9  to obtain a final concentration of 0.4% glucose as the carbon source and 0.1% NHTable 1 presents the different culture conditions studied to determine their effect on VBNC induction. Each factor was studied individually and a control was set up for each strain  for comparison. Oxygen limitation was achieved by filling 2 mL vials to the brim with the cultures and closing the lid to exclude air. At every time point, a new vial of each strain was used for the enumeration, and then disposed to prevent aeration. For presence of oxygen, each strain was incubated in a T75 flask with a 0.2 μm vent cap to facilitate aeration.Supplementary Data Sheet 1).Culturable cells were enumerated by plating on tryptic soy agar  supplemented with 0.1% sodium pyruvate . The first time point of non-culturable cells could be formed from a 1 mL microcosm was considered as the time taken to lose culturability in 100% of the cell population (LCT100). The time taken for more than 50% and more than 90% of cells to lose their cultivability were termed LCT50 and LCT90, respectively  was constructed by introducing the DNA fragment into the pMD19 T-Vector (TaKaRa) according to the manufacturer’s instructions. The DNA copies recorded in this study were computed from the calibration curves generated from the dilutions of the plasmid DNA standard ranging from 1 × 107 to 1 × 101 DNA copies/μL by the Bio-Rad CFX manager 3.0 (Supplementary Data Sheet 1).As described in our previous study , viable Live/dead staining was performed as described before . We centThe figures were drawn using GraphPad Prism software from three replicate values from three different colonies of each strain. Each replicate value represented the mean value from a triplet measurement. The error bars represented mean with standard errors of measurement (SEM).V. cholerae strains, including the classical biotype strain O395 and the El Tor biotypes C6706 and N16961. The cells were cultured under the control condition .To test if there were any differences in development of the VBNC state among the different strains, we analyzed the VBNC development curves for three Figure 1A, the culturable cell counts of all three strains decreased gradually over the 40 days of incubation on TSA-SP agar with different progress curves. The time taken to reach 100% loss of culturability (LCT100) for O395, C6706, and N16961 were 15, 30, and 20 days, respectively. During the first 5 days of VBNC induction, the classical strain lost the ability to form colonies at a faster rate than the El Tor strains . The LCT50 and LCT90 of the El Tor strains occurred both 1 day later than the classical strain. The progress curves of the El Tor strains were similar during the first 5 days. On day 40 after culture, the viable cells of all three strains were confirmed by PMA-qPCR and live/dead staining . The mean viable cell counts, which were estimated from the DNA copies of viable cells, slightly decreased from 108.33-8.46 to 107.84-7.97 copies/ml, while no cultivable cells could be found.As shown in V. cholerae might progress differently depending on the strain. The culturability of the O1 classical strain O395 decreased more quickly than that of the O1 El Tor strains C6706 and N16961. Even within the same biotype of El Tor strains, C6706 lost 100% culturability 10 days after N16961, although no significant differences were found between the progress curves of the two strains from the early stages of VBNC induction.These results suggested that the development of the VBNC state of Figure 2A). The LCT100 for stationary-phase C6706 and N16961 cells was 40 days, while for exponential-phase C6706 and N16961 cells it was 30 and 20 days, respectively. During the VBNC incubation for these two strains, there were approximately 100-fold more culturable cell counts from stationary-phase cells than from exponential-phase cells from the 10th day of the incubation to the 20th day. Although, the growth phase showed a milder effect on VBNC development in the O395 strain, the LCT100 for stationary-phase cells was still 5 days later than exponential-phase cells.To study the effect of growth phase on progress of the VBNC state, we compared the effect of stationary and exponential-phase cells when used as the starting cells in the VBNC experiment. All three test strains from the stationary-phase were found to maintain culturability for a longer time than cells from the exponential-phase . The LCT50 and LCT90 for the El Tor strains and the LCT90 for O395 were all delayed when exponential phase cells were used. Together with the results of LCT100, as only a small proportion of cells could keep their culturability for a longer duration , the heterogeneity of cells should be considered. The results of PMA-qPCR and live/dead stain showed that all the strains from both phases entered into VBNC state and no significant differences was found .However, in all three strains, most cells lost their culturability faster in the stationary-phase than in the exponential phase at the early stage of the induction (Table 1).To evaluate the effect of oxygen on the development of the VBNC state, the three strains were incubated in ASW at 4°C in the presence of oxygen or in oxygen-limited conditions  condition, respectively . The live cell counts determined by PMA-qPCR showed that cells in both conditions entered the VBNC state, and there were no significant differences between the two conditions after 40 days of incubation . The VBNC state was confirmed by live/dead staining .During the first 20 days of the incubation, the mean culturable cell counts of strain C6706 slightly decreased from 10Figure 3). These data suggested that oxygen limitation had little influence on the VBNC outcome, but could postpone the progress of VBNC development.The N16961 and O395 strains showed a similar progress under the oxygen and oxygen-limited conditions (Table 1).The effects of the incubation temperatures for the induction of VBNC state were also compared by incubating the three strains in ASW at 4, 22, and 37°C . Then, cells maintained culturability to at least 40 days at these two temperatures. PMA-qPCR results revealed that the live cell counts decreased to about 106 copies/mL and 105 copies/mL at 22 and 37°C, which were at the same level of the culturable cell counts after incubation for 40 days . The total cell counts calculated in terms of total DNA copies by qPCR (without PMA) were significantly decreased at 22 and 37°C compared to 4°C, indicating that large numbers of cells decomposed at the two higher temperatures .We found that temperature was a crucial factor for the formation of the VBNC state. Compared with the control condition, the culturable cell counts of all three strains rapidly dropped from about 10These results suggested that a higher temperature might prevent VBNC development. At 22 or 37°C in ASW, most of the cells died rapidly and only about 0.1–1% of the cells could maintain their culturability for a longer time.Table 1).To test the effects of nutrient rich environment on VBNC development, the three strains were incubated at 4°C in ASW supplied with LB . After 40 days, PMA-qPCR showed that the DNA copies of viable cells dropped from about 108 to 106 copies/mL in ASW-LB, while no live cells could be found by live/dead stain. The other strains showed similar results .There was a much slower decline in the culturable cell counts of strain C6706 in ASW-LB than in ASW. At day 30, when there were no culturable cells in the control condition (ASW at 4°C), 104 and 103 copies/mL in ASW-LB condition, respectively. These results suggested that the VBNC cells can remain viable in ASW-LB; however, the “viable” cells calculated by the PMA-qPCR in ASW-LB condition were not VBNC cells but dead cells that had gradually decomposed .Cells in the VBNC state are supposed to maintain their viability for a longer time. To verify whether the cells cultured in ASW-LB had achieved the VBNC state after incubation for 40 days, LB broth was added to the control medium (VBNC-ASW-LB), and the cells were then incubated at 4°C for another 40 days. qPCR was then performed with and without PMA . The results showed that the cell counts in both ASW and VBNC-ASW-LB were similar, while the cell counts declined to about 10V. cholerae strains cultured in ASW-M9 medium were also determined. The strains achieved the VBNC state in this medium, and there were no significant differences in the viable cells compared with the control condition (in ASW) after 80 days of incubation .To test whether the presence of carbon and nitrogen sources could impede the VBNC phase, V. cholerae serogroup O1 strains.The VBNC state is the response of bacteria to rigorous environments such as low nutrition, low temperature, and unsuitable pH. In this study, we determined the effects of different culture conditions on the development of the VBNC state among toxigenic Viable but non-culturable development progresses with loss of culturability and preservation of viability . TherefoOf the five induction factors studied, strain, growth phase, and oxygen level did not affect the outcome of the VBNC state in ASW at 4°C. When no culturable cells were found in 1 mL microcosms, 28.7–43.8% of the cells from the initial population maintaining viability. However, all the three factors influenced the speed of formation of non-culturable cells.V. cholerae. V. cholerae strain O395 exhibited a more rapid loss of culturability than the V. cholerae O1 El Tor strains. Within the same biotype, C6706 needed longer time to achieve total loss of culturability than N16961 in ASW at 4°C, although there were no significant differences in the LCT50 and LCT90 between the two strains. These findings suggested that the differences were likely related to the similarity of the genetic backgrounds of the V. cholerae strains.Several studies have reported differences in VBNC progress across different bacterial strains . We founAeromonas hydrophila and E. coli have also pointed out that the stationary-phase could delay the development of the VBNC state (8/mL, this proportion of cells cannot represent the total mass. Therefore, although we found that the growth phase may influence the progress of VBNC, there is insufficient data to confirm whether the stationary-phase causes delays in reaching the VBNC state in V. cholerae.Exponential-phase cells were found to enter the non-culturable state faster than stationary-phase cells. Similarly, previous studies of NC state . HoweverCampylobacter jejuni reported that aeration could accelerate the development of the VBNC state (Salmonella enterica and E. coli from the VBNC state, although the influence of oxygen on VBNC development was not mentioned in the studies (6-fold in all the three strains, suggesting that aerobic metabolism or oxygen injury might play a part in the mechanism of VBNC development.Not much is known about the effect of oxygen on the development of VBNC. To our knowledge, the only two studies investigating this have presented contradictory results. One study on NC state , whereasNC state . Previou studies . In the V. cholerae in ASW. It is interesting that temperature alone may work as a “switch” to control the outcome of VBNC induction in ASW. It would be useful to find out the exact temperature that initiates the VBNC process. Comparative studies over a small range of the temperature might provide helpful clues to the genetic regulation behind the VBNC development.The other two factors, temperature and starvation, have been most often used in previous studies to induce the VBNC state . It is wV. cholerae. VBNC development did not occur when the cells cultured in ASW at 4°C were supplied with LB; however, VBNC induction was not affected when cells were cultured in ASW with M9 at 4°C. Comparison of the ASW-LB and ASW-M9 results suggested there should be some differences between the two conditions that may have resulted in a failure of cells to enter the VBNC state. The different components of LB should be added to ASW-M9 individually to determine which component affects the cells and VBNC state, in order to obtain more clues into the development of VBNC.Starvation was not necessary in order to attain the VBNC state for V. cholerae, providing useful data for and impetus for further studies into how the VBNC state is triggered and the regulatory mechanisms.The knowledge of the VBNC state is to date extremely limited. One reason for this is the complicacy of inducing conditions. Without the awareness of the role of each individual factor composing the inducing condition might result in the instability of outcomes. Understanding the role of each factor, including both those facilitating and those blocking the VBNC state, will assist our understanding of VBNC development. In this study we estimated the role of the growth phase, oxygen, temperature, and nutrient starvation in the development of the VBNC state in BW contributed to the study design, the experiment operation, data analysis, and paper writing. WL contributed to the study design, experiment operation, and data analysis. BK contributed to the study design, data analysis and paper writing.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.


Introduction. The relationship between cervical spine injury (CSI) and helmet in head injury (HI) patients following motorcycle crashes is crucial. Controversy still exists; therefore we evaluated the effect of various types of helmets on CSI in HI patients following motorcycle crashes and researched the mechanism of this effect. Patients and Methods. A total of 5225 patients of motorcycle crashes between 2000 and 2009 were extracted from the Head Injury Registry in Taiwan. These patients were divided into case and control groups according to the presence of concomitant CSI. Helmet use and types were separately compared between the two groups and the odds ratio of CSI was obtained by using multiple logistic regression analysis. Results. We observed that 173 (3.3%) of the HI patients were associated with CSI. The HI patients using a helmet (odds ratio (OR) = 0.31, 95% confidence interval (CI) = 0.19−0.49), full-coverage helmet , and partial-coverage helmet  exhibited a significantly decreased rate of CSI compared with those without a helmet. Conclusion. Wearing full-coverage and partial-coverage helmets significantly reduced the risk of CSI among HI patients following motorcycle crashes. This effect may be due to the smooth surface and hard padding materials of helmet. Motorcycles are a crucial means of transportation in Taiwan . AccordiHead injury has been regarded as a critical cause of death and disability among victims of motorcycle crashes –5, and pIn Taiwan and other emerging economies people frequently used motorcycles as daily transportation tools, and the financial and social burden caused by HI and CSI following motorcycle crashes is important for both clinical physicians and public health providers. Therefore the issue of the effect of helmet on CSI was crucial for the victims of HI following motorcycle crashes. Because controversy still exists on the relationship between motorcycle helmet use and CSI, and which helmet type is superior, we utilized the data from Head Injury Registry in Taiwan and performed a multicenter case-control study to evaluate the effect of various types of motorcycle helmets on CSI in HI patients following motorcycle crashes and researched the mechanism of the effect of motorcycle helmets on CSI. The Institutional Review Board of Taipei Medical University granted approval for this study (Ref. number 201308033).Data were extracted from the Head Injury Registry in Taiwan. This Head Injury Registry is an electronic database funded by the Taiwan Government since the 1990s that contains over 100 000 cases of HI in Taiwan . Data weData provided by 25 hospitals in northern Taiwan were obtained from the Head Injury Registry and included in our study for analysis. To minimize the influence of the Taiwan Mandatory Helmet Law which was implemented nationwide in 1997, we only included the patients from 2000 to 2009. The inclusion criteria of this study were (1) patients with ICD-9 codes 800–804 and 850–854, covering the diagnoses of brain concussion, intracranial hemorrhage, and skull-bone fracture; (2) motorcycle crash; and (3) being over 17 years of age. Any cases with missing data on helmet use, helmet type, or CSI were excluded.The occurrence of CSI after motorcycle crashes was the outcome variable in our study. CSI was defined as cervical spinal fracture. The predictor variables in our study were helmet use and helmet types, including full coverage and partial coverage. A full-coverage helmet  protects the ears, cheeks, and back of the head, but it lacks a lower chin bar. A partial-coverage helmet  features the same front design as a full-coverage helmet, but it lacks a lowered rear. The enrolled patients were divided into case group, consisting of patients with CSI, and control group, consisting of patients without CSI, and differences of motorcycle helmet use between case and control groups, including various helmet types, were compared.Fourteen crucial demographic and clinical variables described by previous studies were included as covariates for testing the construct validity of the study , 22–24. r = 0.75; P < 0.001), these 2 variables were analyzed using separate models (Model 1 and Model 2) while the remaining variables were held constant in both models. These 2 models were used to estimate odds ratios (OR). SPSS Version 18.0  statistical software was used to perform all statistical analyses. A P value less than 0.05 was considered statistically significant.We first performed univariate analysis to assess the differences in helmet use, helmet types and above-mentioned covariates between case and control groups. Then we conducted multiple logistic regression analyses to assess the association of CSI and motorcycle helmet use as well as helmet types in HI patients after adjusting the effect of the above-mentioned covariates. Given the collinearity between helmet use and helmet types  with CSI were included in the case group, whereas the remaining 5052 cases (96.7%) without CSI constituted the control group .In Previous studies reported that CSI occurs in 4%–8% of patients with HI –27. In tNo evaluation of helmet type was reported in the Crompton study because of the lack of related information in the National Trauma Databank (NTDB) . Full-faThe results of previous biomechanical analyses may explain the phenomenon of lower risk of CSI in HI patients wearing helmet during motorcycle crashes. These biomechanical analyses showed that the excellent flexibility of healthy cervical spine can deflect the head out of the path of the moving torso when the head came into contact with another object. The experiments by using cadaver indicated that CSI caused by head impact was highly correlated with the friction between the head and the surface of the object and surface padding characteristics –35. ThesThere were limitations in this study. Firstly the result of the effect of helmet use on CSI cannot apply to all victims of motorcycle crashes because victims without HI were not included in this study. As retrospective study, the insufficiency of the socioeconomic and environmental information, such as the crashes scenario, including road conditions, vehicle speed, full-face helmet, cord-injury symptoms, was considered as limitation in our study. Since we restricted our study population to northern Taiwan to decrease the bias caused by the aforementioned insufficiency, the result of our study should be used cautiously when applied to the whole country. Although the impact angle, a crucial factor of CSI in head injury, was not included in the dataset , 36, theUsing full-coverage and partial-coverage motorcycle helmets exhibited a significantly decreased risk of CSI among HI patients following motorcycle crashes. According to the results of previous biomechanical analyses, the design of helmet, including smooth surface and hard linings of padding materials, was considered as the cause of the effect. Further study should be performed to clarify the effect of full-face helmets on CSI. The result of this study supports helmet use among motorcycle riders.


Neonates are vulnerable for systemic infections and wise use of antibiotics is recommended for the group to cut down the risk of development of antibiotic resistance, a global threat. There is a general lack of studies that present antibiotic prescribing practices at neonatal intensive care units (NICUs) specifically from the countries with high birth rate such as India.To compare and describe the situation of antibiotic prescribing at neonatal intensive care units of two private, tertiary care hospitals of Ujjain district in Central India.A cross-sectional study was conducted from 2008 till 2011using customized form. Antibiotics were classified based on WHO anatomical therapeutic chemical (ATC) classification and diagnoses were classified using International Classification of Diseases-10.rd generation cephalosporins (28%) in the NTH and 3rd generation cephalosporins and aminoglycosides (36% each) and FDCs (12%) in the TH. The adherence to the WHO’s model list of essential medicines and the Indian national list for children were higher in the teaching hospital than in the non-teaching hospital, 85% and 50% respectively (p<0.01).Of total 1789 neonates, 1572 were admitted at the NTH and 217 at the TH. Sepsis was most common diagnosis in both hospitals (>30%). Antibiotics were prescribed to higher percentage of neonates with sepsis (NTH-97% and TH-94%) than to the rest . Most frequently prescribed antibiotics for this group were fixed dose combinations of antibiotics  and 3Broad-spectrum antibiotics; including new FDCs were prescribed in both hospitals but extensively at the NTH. The adherence to available essential medicines lists was significantly lower at the NTH. An unnecessary exposure of neonates with the higher classes of antibiotics might be seen as a threat for the development of antibiotic resistance.There is an urgent need to develop; a diagnoses specific antibiotic prescribing guidelines for neonates and measures to reduce the prevalences of infectious diseases using antibiotic stewardship programs.None declared.