Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct PrtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance.  Vibrio vulnificus is an opportunistic, Gram-negative, halophilic pathogen , which are both suggested to be inhibitory factors of phagocytosis  it may not induce a pathogen to develop AMR (b) being non-antibiotic in nature, it may not disturb the native gut-microbiota, and (c) it acts solely by inhibiting virulence factors without threatening general physiology and survival of pathogen  in V. vulnificus has shown that virulence regulator HlyU is preferentially induced during infection conditions  , RtxA1 during V. vulnificus infection  and forms a bridge within DNA. As a result, it obstructs the movement or exclude the entry of RNA polymerase leading to the repression of rtxA1  and VvhA were not found to be inhibited . We showed that FTH inhibits HlyU-regulated toxin genes (rtxA1 and vvhA) at the transcriptional level without affecting the expression of hns, which acts as repressor of rtxA1 , deletion mutants ΔhlyU (ZW141) and ΔrtxA1 (MW064) of V. vulnificus MO6-24/O were used in this study  agar plate. A single colony from TCBS-agar plate was inoculated in Luria-Bertani medium supplemented with 2% sodium chloride (LBS) broth and incubated at 37°C under orbital shaking culture conditions. The growth of V. vulnificus was measured by optical density at 600 nm (OD600) and enumerated by determining the bacterial colony forming units (CFU) on LBS-agar (1.5% agar) plate. The antibiotic concentration used for the selection and/or growth of recombinant and mutant strains was 2 μg/ml and 300 μg/ml for chloramphenicol (Cm) and kanamycin (Km), respectively. E. coli DH5α was grown in LB medium (broth/agar) with the appropriate concentrations of antibiotics  to select recombinant strains harboring plasmids with corresponding antibiotic resistance genes. Unless stated otherwise, HeLa cells were cultured at 37°C with 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose, containing 10% fetal bovine serum (FBS) for routine culturing.The strains and plasmids used in this study are listed in Table ::luxCDABE . V. vulnificus genomic DNA was extracted using G-Spin genomic DNA extraction kit . High-fidelity Taq DNA polymerase was used for PCR amplification. The restriction endonuclease enzymes were purchased from New England Biolab . Plasmid extraction  and gel purification  were performed as per the manufacturers' protocols. All recombinant DNA techniques were performed according to the Standard Laboratory Manual  in V. vulnificus was PCR-amplified from genomic DNA using specific primers  were electroporated into WT and hlyU deletion mutant, ΔhlyU  supplemented with 200 mM sucrose. After washing, the cells were resuspended in 1000 μl cold GYT medium . An aliquot of 100 μl of electrocompetent cells was added to a chilled electroporation cuvette with a 0.1 cm electrode gap. Approximately 700 ng of plasmid DNA was added to 100 μl of electrocompetent cells just before the electroporation pulse. The cells were electroporated using the BIORAD Gene Pulser X-cell instrument at 750 V, 25 μF capacitance, and 200 Ω resistance. The electroporated cells were recovered in 900 μl LB for 1 h and plated on LBS-plates supplemented with 2 μg/ml Cm. By this approach, V. vulnificus WT strain harboring pBBRMC2_PrtxA1::luxCDABE (hereafter termed as WT reporter strain) under the regulation of native chromosomal hlyU was created for screening the chemical libraries. The V. vulnificus ΔhlyU reporter strain harboring pBBRMC2_PrtxA1::luxCDABE was used as a control to assess the background signal.The putative clones obtained were confirmed by plasmid isolation, followed by restriction digestion and analytical agarose gel electrophoresis. Both strands of all cloned DNA fragments were sequenced by the di-deoxy method, and the accuracy of sequences was ascertained by BLAST and peak analysis of nucleotides. The recombinant plasmids and the empty vector backbone (pBBRMCS2::yU Table  of V. vuhlyU (control) reporter strains were sub-cultured with 1% inoculum and supplemented with 2 μg/ml chloramphenicol in LBS for primary chemical screening. A total number of 1,840 small molecules  were screened. The final concentration of tested chemicals for screening was 20 μM. The luminescence and bacterial growth (OD600) of samples were recorded in multi-well plates after 6 h using a microplate reader . The primary hits were identified on the basis of reduction in the luminescence signal per unit OD600 relative to the untreated or vehicle (DMSO 0.2% v/v)-treated control. The hits were re-checked in a concentration-dependent manner, and were sorted for further analysis. The selected chemicals were purchased from Prestwick Chemicals, and stocks of 10 mM or 100 mM were prepared in DMSO or water.Overnight cultures of WT and ΔhlyU V. vulnificus were diluted to 2 × 106 CFU/ml in 3 ml fresh LBS and incubated at 37°C, 220 rpm in 14 ml polypropylene culture tubes. WT V. vulnificus was treated with the small molecule inhibitors or with DMSO (as a control) and was grown up to OD600 ~1.8–2.0. RNA isolation from V. vulnificus reporter strains was carried out with the similar bacterial cells input and incubated for 9 h in 48-well plates with or without FTH. The treated cells were harvested by centrifugation at 5000 × g for 5 min. The cell pellets devoid of supernatant were immediately treated with RNA-Protect reagent (QIAGEN). Total RNA was isolated using the QIAGEN RNeasy Mini kit as per the manufacturer's instructions. A total of 1 μg of RNA from both the control and treated samples was subjected to DNase I treatment  for 15 min at room temperature. The reaction was stopped by adding stop buffer and DNaseI was heat inactivated by incubating the samples at 70°C for 10 min. First-strand cDNA synthesis was performed using the Ecodry Premix (random hexamer) kit . DNase I-treated RNA samples were subjected to target gene PCR amplification to check for genomic DNA contamination. The primers .Overnight cultures from freshly streaked single colonies of WT or Δrs Table  used forne Table . Relativ4 cells per well in 100 μl of culture medium using 96-well plates. After 24 h of incubation, FTH was added at concentrations ranging from 1 to 2048 μM at two-fold increments. After 48 h of drug incubation, EZ Cytox kit solution was added to each well and plates were incubated for 2 h. The absorbance of dye was measured spectrophotometrically at A450 using a multi-plate reader . The mammalian cell culture media was used as blank. The IC50 was calculated using GraphPad Prism 6.01. The data were plotted, transformed to log values, and then fitted onto a non-linear curve after normalization. FTH untreated cells were considered to be 100% viable.A host cell viability assay as a measure of cell proliferation was performed to evaluate the cytotoxicity of the FTH small molecule using the EZ Cytox cell viability assay kit . HeLa cells were seeded at a density of 2 × 10V. vulnificus were inoculated in fresh LBS with or without FTH inhibitor (10–60 μM), and incubated for 3 h wherein ΔhlyU served as a positive control of hemolysis inhibition. The cell free culture supernatants were withdrawn and mixed with 1% human red blood cells (hRBCs) and were incubated at 37°C for 1 h under shaking culture conditions. The cell debris was removed by centrifugation at 1,500 rpm (0.2 × g). The hemolysis was visualized using absorption spectra (500–650 nm) with 1:1 dilution of supernatant to hRBCs. To calculate the hemolytic unit, the culture supernatants were diluted by 1/2, 1/4, and 1/8 with PBS and the remaining method was followed as described above. The hemolytic unit was expressed as the reciprocal of the dilution factor showing 50% hemolysis absorbance at 540 nm (A540) as described by Lee et al.  were cultured in 20 mm glass-bottom cell culture confocal discs for 48 h. The cells were replenished with fresh DMEM media without FBS before starting the experiment. HeLa cells were infected with 20 moi of WT V. vulnificus with or without the simultaneous addition of FTH (100 and 200 μM) and incubated for 1 h. HeLa cells were infected with 20 moi of ΔhlyU and ΔrtxA1 strains of V. vulnificus  were incubated with 0, 100, and 200 μM FTH for an hour in DMEM medium. After incubation, the cells were harvested, washed with PBS, diluted, and plated on LBS agar plates to enumerate the CFU.HeLa cells (3 × 10us Table  as contrus Table . The celV. vulnificus infection. Norfloxacin was taken as a representative of the flouroquinolone antibiotics and compared with the antivirulent FTH for the adaptive evolution of their corresponding resistance against WT V. vulnificus. Norfloxacin was tested at the concentration of 0.155 μg/ml while FTH was tested at two different concentrations (40 and 60 μM), to evaluate antivirulence resistance. The norfloxacin concentration was selected by taking the average of a range of MIC90 values (0.063–0.25 μg/ml) for various V. vulnificus strains, as reported elsewhere  was centrifuged and resuspended in a new tube containing fresh LBS supplemented with 0.155 μg/ml norfloxacin. In the case of untreated or FTH-treated samples, 1% of 18 h grown culture was sub-cultured for next transfer. The sub-culturing and transfers were continued until bacterial growth was visible in norfloxacin treated culture. The MIC assay was performed according to CLSI guidelines (2012) by the microbroth dilution method in Mueller Hinton Broth (MHB)  and were selected for further scrutiny. Chemicals showing 100% inhibition of luminescence signal due to cessation of bacterial growth were not the focus of this study , benzamil hydrochloride (BH), omeprazole (OME), fursultiamine hydrochloride (FTH), vatalanib (VAT), GBR 12909 dihydrochloride (GBR), and mebhydroline 1,5-naphtalenedisulfonate (MND). These chemicals showed a concentration-dependent inhibition of the luminescence signal in the WT reporter strain . The remaining four chemicals were assessed for their effects on rtxA1 mRNA expression levels in WT V. vulnificus at 20 μM concentration that was used for primary screening. RtxA1 gene expression test was considered the first key step toward finding a true inhibitor, since the reporter system was designed with the promoter of rtxA1, which is directly regulated by HlyU. The molecular analysis for relative rtxA1 gene expression showed that only FTH significantly decreased rtxA1 transcription  when compared to the corresponding DMSO-treated control. The rtxA1 mRNA level was negligible in ΔhlyU, which confirmed that HlyU is the key regulator of the major cytotoxin RtxA1 in V. vulnificus  in V. vulnificus which has been recently reported  decreased the plpA2 gene expression in V. vulnificus  under its native PhlyU promoter in WT V. vulnificus  was cloned into the promoter-less luxCDABE-containing pBBRMCS2 plasmid and electroporated into WT and ΔhlyU V. vulnificus. WT V. vulnificus containing PEM7::luxCDABE was used as a non-specific control to assess the specificity of FTH toward HlyU, as HlyU drives the reporter gene expression under the PrtxA1 promoter  vs. PEM7 (non-specific) in the presence of native chromosomal HlyU, the wild type reporter strain displayed specific inhibition of luminescence with PrtxA1 in response to varying concentrations of FTH, but no inhibition was observed with the synthetic promoter PEM7 tagged with luxCDABE. This suggests that FTH specifically targets the HlyU transcriptional regulator, and attenuates the expression of the RtxA1 toxin under ambient bacterial growth conditions  by evaluating the hemolytic activity as hemolytic unit since both RtxA1 and VvhA contribute to hemolytic activity by forming pores in the cell membranes of hRBCs  merely affected 25 ± 5% host cells compared to 11 ± 4% round-cells in ΔhlyU and ΔrtxA1 mutant controls  values for six Vibrio strains (0.063–0.25 μg/ml)   strain, along with the untreated cultures, were verified by MIC assessment  but the untreated or FTH-adapted strain did not produce any colonies, even in the inoculation zone  V. vulnificus strain and the FTH-treated strain originated from the same V. vulnificus strain (MO6-24/O).Antimicrobial resistance that arises in various bacterial pathogens during antibiotic-mediated treatment is caused by adaptive evolution and clonal selection due to sustained antibiotic pressure. AMR in ) Figure . The adat Figure  and grows Figure . This was Figure . The nore Figure . The ide/O Table . SequencrtxA1 gene expression of WT V. vulnificus treated with 60 μM FTH. The qRT-PCR results showed the same level of rtxA1 transcriptional inhibition in the FTH-adapted strain as in the original stock culture of V. vulnificus  supporting pathogen entry into the host cells (b) destabilizing the cytoskeletal network of host cells to inhibit phagocytosis  evading the innate immune system for rapid dissemination into the bloodstream  to luxCDABE  promoter. Thus, several lines of experimental evidence distinctly suggest that FTH inhibits the expression of virulence factors through HlyU  infection model   Figure . Unfortuin vivo instability issue, FTH has several advantages as an antivirulence agent, such as (a) enhanced bioavailability due to its reduced absorption barrier (b) a high IC50 that poses no toxicity to the host (c) effective targeting of virulence transcription factor, HlyU (d) transcriptional inhibition of highly invasive cytotoxins, and (e) avoidance of AMR development during antivirulence strategy to tackle V. vulnificus infections presumably due to its nutraceutical nature and low selection pressure (Figures V. vulnificus pathogenesis by inhibiting the transcriptional network of cytotoxins without causing the emergence of antimicrobial and antivirulence resistance. Our results also suggest that an antivirulence strategy targeting the expression of virulence factors regulated by HlyU might be a promising approach for the treatment of infectious disease caused by V. vulnificus.Despite the  Figures . In concSI and AC performed the experiments. SI and AC wrote the manuscript with the support of KK. The experiments were designed and performed mainly under the supervision of AC and KK.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. The reviewer YM and handling editor declared their shared affiliation.


Didier Wernli and colleagues discuss the role of monitoring in countering antimicrobial resistance. Antimicrobial resistance (AMR) is developing in many pathogenic bacteria, threatening to compromise the effectiveness of crucial medical treatments.Member States of the United Nations (UN) have reiterated their commitment to tackle AMR at the UN General Assembly held in New York City on 21 September 2016. The main challenge is now implementation of the Global Action Plan (GAP) adopted by the World Health Organization (WHO) in 2015.There are currently large information gaps about the global governance of AMR regarding both the magnitude of the problem and national responses.Expanding national and subnational monitoring by integrating measurements ranging from assessments of drivers of AMR to responses can increase political buy-in, societal participation, and implementation of agreed policies.WHO should lead the way to expand monitoring of progress regarding AMR control, but a broad coalition of global health actors is needed to build a robust approach in a significant number of countries.Antimicrobial resistance (AMR) is increasing in a wide range of pathogens, causing morbidity and mortality globally, and threatening modern medicine. While the long-term impact of AMR on human societies remains uncertain , the conProgress has been accomplished regarding AMR monitoring. First, knowledge about the causes, consequences, and magnitude of the problem has improved through research and better surveillance . Second,Klebsiella pneumoniae for only 71 countries out of 194 [However, major information gaps in the global governance of AMR—i.e., how we attempt to limit AMR globally—remain. First, surveillance data are lacking for low- and middle-income countries (LMICs). There are also more epidemiological data than WHO has access to, particularly from private hospitals and laboratories, which limits international reporting . In 2014t of 194 . Second,Addressing these information gaps is critical for successful implementation of the GAP. First, harmonized data collection will provide useful measures for individual countries to benchmark their national and subnational performance against others and help them tackle the problem based on the best scientific evidence. This could stimulate both domestic and transnational policy debates and help AMR remain on the political agenda. Second, as for many problems of international cooperation in which countries can free ride on the efforts of others, improved monitoring will reduce information uncertainty about the current situation and responses and create better conditions for cooperation through joint effort to produce global public goods . This isBecause AMR control is complex, the goal should be to measure the problem and progress across its multiple dimensions while at the same time making it more tractable to policy makers and the public. Over the last decade, several instruments aimed at measuring performance in the economic, education, environmental, and health sectors have been developed . LessonsThe first is to choose an appropriate scientific approach to conceptualize AMR and related control efforts. Drawing from the growing literature on AMR determinants and interventions ,16,17, aThe second challenge is to define what to measure. As there are different levels of economic development and multiple models of organization for the health, social, and political systems, it is critical to recognize that intrinsic diversity is an important parameter. Even ecological determinants of AMR might differ significantly across countries because of climate and other causes. Given this diversity among and within countries, AMR monitoring should be based on 3 main components: (1) the epidemiological situation and its impact on human health and societies (outcomes); (2) current drivers and practices (process), including antibiotic over-, mis-, or underuse; and (3) regulations and control policies (structures) . AssessiThe third challenge is to select appropriate measurements from human, animal, and planetary health, as illustrated in Tables The fourth challenge is data. Developing surveillance capacities is one of the core legal requirements of the International Health Regulations . HoweverThe fifth challenge is reporting. Methodological issues should be addressed transparently, and results should be presented to avoid conveying simplistic and misleading policy messages. To ensure transparency, a data platform is needed in which information about each country will be collected and synthesized. A dashboard of indicators is likely to be necessary for a problem as complex as AMR. Because monitoring aims at informing policy, special attention must be devoted to communicating the findings online to make the most of data visualization for each country. Indicators—for example, those tracking countries’ actions against AMR—can be associated with appropriate visualization methods  to reflect countries’ responses so far. To increase the benefit for policy makers, countries with high or rapidly rising AMR levels will require further investigation and analysis, such as in-depth qualitative case studies that shed light on why particular AMR interventions do or do not succeed.Fostering a global transformation to deal with AMR  requiresAs the first step is to define the scope of AMR monitoring, the DPSIR framework provides an integrated approach that matches the complex nature of the problem. The creation of an independent monitoring mechanism coordinated by WHO will be the next step. The recent difficulties that have hobbled the implementation of the International Health Regulations underscore the limits of countries’ self-assessment , but theA robust approach to expand AMR monitoring demands a wide participation of global health actors. The recent launch of the “Conscience of Antimicrobial Resistance Accountability” (CARA), which aims to monitor the state of national responses, is a step in this direction . Drawing


Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis.The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as  Escherichia coli and Bacillus subtilis, constitutes one of the main bottlenecks of the rational genome engineering. The Gram-negative E. coli and the Gram-positive B. subtilis are both well-characterized bacteria used in a number of synthetic biology and biotechnology applications , glucose (0.5% w/v), casamino acids (0.02% w/v), tryptophan (20 μg/ml), and iron ammonium citrate (2.2 μg/ml). Inoculated cells were grown at 200 r.p.m. on a rotatory shaker at 37°C for 18 hours. Then, 1.4 ml of the B. subtilis culture was inoculated into 10 ml of the fresh minimal medium and grown for another 3 hours. Subsequently, 11 ml of the starvation medium composed of 5x minimal salts solution and glucose (0.5% w/v) were added to the B. subtilis culture and cells were grown for additional 2 hours and 45 mins. 0.3 ml aliquots were transferred into 15 ml polypropylene tubes and transformed with 15 μl of BAC DNA or plasmid DNA. Transformed B. subtilis cells were incubated at 200 r.p.m. on a rotatory shaker at 37°C for 1 hour prior to addition of 700 μl LB. Cells were then continued to grow for 1.5–2 hours. 20–200 μl of this culture were plated onto selection plates and grown at 37°C for 18–24 hours. Integrations into the B. subtilis chromosome were confirmed by PCR with the flanking primers and sequencing.To generate competent B. subtilis genomic DNA was obtained using the GeneJET genomic DNA purification kit (Thermo Scientific).PCR amplifications were performed in 25 or 50 μl reaction mixtures using Phusion DNA polymerase (Thermo Scientific) or Dream Taq master mix kit (Thermo Scientific) according to the supplier’s instructions. Oligonucleotide primers were synthesized by Sigma-Aldrich. PCR amplified DNA fragments were assembled in a 5.2 μl final reaction volume using the modified Gibson Assembly procedure , 23, 24.E. coli, respectively. DNA integrations into E. coli chromosome and BACs were performed using the streamlined lambda Red system-mediated method described previously  and grown at 37°C for 5 hours. The donor E. coli and the recipient B. subtilis cultures were mixed (mixing ratio 1:1) and cultivated at 42°C for 2 and 6 hours. 200 μl of the mixture was spread on LB plate with chloramphenicol to select for B. subtilis transformants. Residual E. coli was removed by inducing B. subtilis sporulation and subsequent heat treatment to kill the growing cells. Briefly, colonies scraped off the plates were resuspended in 1 ml 2 x SG medium  and grown on 2 x SG plates for 72 hours to induce sporulation. Bacteria scraped off the 2 x SG plates were washed with ice-cold water and subjected to heat treatment at 90°C for 10 minutes. 100 μl were plated on LB chloramphenicol plates and grown overnight. The average numbers of CFUs and standard errors were calculated from three independent replicates.Modified culture mix method , 18 was E. coli viability was measured with a Live/Dead BacLight™ bacterial viability kit  according to supplier’s instructions. Briefly, cells were first grown in 5 ml LB in 15 ml Falcon conical centrifuge tubes on a rotatory shaker at 200 r.p.m. and 30°C for 6 hours prior to temperature shift to 42°C. After the temperature shift cells were grown at 42°C for another 6 hours. Then red-fluorescent nucleic acid stain propidium iodide and green-fluorescent nucleic acid stain SYTO9 were added into the medium (3 μl of the stain per ml). The numbers of the dead propidium iodide-stained bacteria and live SYTO-9 stained bacteria were determined by fluorescent microscope (Nikon Microphot-SA), 5–6 hours after the temperature shift from 30°C to 42°C. The means and standard errors were calculated from three experiments.The 600 of 0.05. 200 μl were aliquoted into the microplate wells and incubated in the microplate reader  at 30°C for 24 hours or at 30°C for 3 hours, followed by incubation at 42°C for another 21 hours. Absorbance was measured with the automatically repeated protocol .Absorbance was measured in the 96 well microplates . Bacterial cultures were grown overnight and normalized to ODhttp://ncbi.nlm.nih.gov) were used to compare DNA sequences. E. coli K-12 project website (http://www.xbase.ac.uk/genome/escherichia-coli-str-k-12-substr-mg1655) was the source of the E. coli genome sequence. BioCyc (http://bsubcyc.org/) and SubtiWiki (http://subtiwiki.uni-goettingen.de/) databases were used to obtain the B. subtilis genome sequence. The sequences of DNA constructs were obtained from the Registry of Standard Biological Parts (http://parts.igem.org/Main_Page), the NCBI website and the Addgene non-profit plasmid repository (http://www.addgene.org/). Sequencing was done by Source Bioscience.BLASTN  and TBLAE. coli, there are 3 main barriers to phage release, namely an outer membrane (OM), a thin layer of peptidoglycan (PG), and a cell membrane (CM). The lysis gene cassette of the phage lambda consists of four genes, namely S, R, Rz and Rz1 regulated by pR promoter. Rz and Rz1 encode a class II CM protein and an OM lipoprotein, respectively, which form a complex spanning the periplasm of the host cell to promote fusion of the CM with the OM [S encodes the holin and the antiholin [R encodes the endolysin with lytic transglycosylase activity [Bacteriophages MS2, ΦX174 and lambda were chosen mainly due to their extensive characterization and different mechanisms of action . In E. ch the OM . S encodntiholin , while Ractivity . The endactivity . The lysactivity . The lysactivity –39. The E. coli lysis we chose the thermosensitive lambda repressor to regulate the expression of the phage lambda, ΦX174 and MS2 lysis genes. At 30°C, the expression of lysis genes from the pL promoter is inhibited by the repressor. Temperature shift to 42°C alleviates the repression and leads to cell lysis. DNA can be then taken up by other bacteria, such as B. subtilis (To construct an inducible system for controlled subtilis .http://parts.igem.org/Main_Page) [pL promoter regulated by the lambda repressor   Fig 1)..http://pe method , 27, 28.e method , 41, theE. coli cultures with the microplate reader. The absorbance of the bacterial cultures grown in both restrictive and permissive temperature for the thermosensitive repressor was compared with that of the controls grown in the restrictive temperature for 24 hours. The absorbance was significantly lower after the temperature shift when compared to the growth in the restrictive conditions for the whole 24 hours. The comparison between cultures harbouring plasmids with different combinations of lysis genes showed that the strain with lysis genes from all three bacteriophages lysed with the highest efficiency. Furthermore, lysis of strains harbouring two lysis genes was higher than that harbouring a single lysis gene (data not shown). The highest difference between restrictive and restrictive/permissive conditions was observed for E. coli with plasmid pSB1K3(FRTL123) encoding all three lysis genes.To test the effect of multiple phage lysis genes on cell lysis we first used plasmids pSB1K3(FRTL1), pSB1K3(FRTL2), pSB1K3(FRTL3), pSB1K3(FRTL12), pSB1K3(FRTL13), and pSB1K3(FRTL123), harbouring different combinations of lysis genes . The celE. coli strain Ec(Ri). The lysis efficiencies of the engineered strains Ec(RL1i), Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i) with integrated lysis genes were first calculated from the difference between the absorbance at the restrictive and restrictive/permissive conditions measured with the microplate reader , Ec(RL2i), Ec(RL3i), Ec(RL12i) and Ec(RL13i) occurred after approximately 9 hours after induction of the phage genes. Only in the strain Ec(RL123i) harbouring all three lysis genes the effect of the cell lysis on the absorbance was clearly visible after approximately 4 hours after induction. The differences in the kinetics of the cell lysis can be attributed to the variable gene components used in cell lytic systems. The previously developed systems utilized entire phages to lyse the oli cell , 18. Theoli cell . In compBacLight Live/Dead bacterial viability stain and examined by fluorescent microscopy. The survival rates of strains Ec(RL1i), Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i) after temperature shift were significantly reduced when compared to the E. coli strain with no lysis genes Ec(Ri) (E. coli cell improves lysis.The outcome of the absorbance measurement can be influenced by a number of factors (e.g. debris from the lysed cells). Therefore, to confirm the results of absorbance assays, lysis efficiencies were also investigated on the level of the individual cells. Bacteria grown at the restrictive and permissive conditions were stained with the s Ec(Ri) . Furthers Ec(Ri) . The surs Ec(Ri) , thus coE. coli cells for interchassis DNA transfer we transferred plasmid pJScav and bacterial artificial chromosome iBAC(cav)-borne genetic circuits cat and ven into B. subtilis. The genetic circuits cat and ven encode Bacillus-specific chloramphenicol resistance gene and the yellow fluorescent protein-encoding gene mVenus flanked by the integration sequences homologous to amyE target site in the B. subtilis chromosome (cat and ven we used culture mix method [E. coli strains Ec(Ri), Ec(RL1i), Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i) by transformation. Temperature shift led to E. coli cell lysis and release of pJScav and iBAC(cav) from Ec(RL1i), Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i). pJScav and iBAC(cav) were then taken up and incorporated into the B. subtilis cells cultivated in the same test tube. The resulting recipient B. subtilis colonies grew on medium containing chloramphenicol and emitted yellow fluorescent light (cat and ven in B. subtilis. Presence of amyE integration target sequences on pJScav and iBAC(cav) led to the integration of genetic circuits cat and ven into the amyE locus of the B. subtilis chromosome  between Ec(RL123i) and other investigated strains harbouring fewer lysis genes . This anB. subtilis occurred even in the absence of any phage gene expression using strain Ec(Ri). This is due to spontaneous lysis of the fraction of E. coli cell population [E. coli cells can be taken up by B. subtilis cultivated in the same test tube. The number of transformants using both pJScav and iBAC(cav) was highest with strain Ec(RL123i), thus showing that combining lysis genes from phages MS2, ΦX174, and lambda in a single cell improves transfer of genetic circuits from E. coli to B. subtilis , Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i), transfer of pJScav and iBAC(cav) into pulation . DNA relsubtilis .E. coli to B. subtilis. This is particularly important for transfer of the high molecular weight DNA molecules, such as BACs, which are prone to mechanical shearing by pipetting [E. coli cells using solely the lysis genes from multiple bacteriophages. To this end, we investigated lysis genes from phages MS2, ΦX174, and lambda. Genetic circuits L1, L2, L3, L12, L13 and L123 harbouring different combinations of phage lysis genes were integrated into the E. coli chromosome using the lambda Red recombineering approach [E. coli into B. subtilis.We set out to provide the synthetic biology community with a reliable system for the transfer of genetic circuits from ipetting , 18. As approach . Our resE. coli and DNA transfer from the lysed cells into the B. subtilis chromosome. Our system does not require the traditional DNA isolation and transformation techniques which are time-consuming. The advantages of our system over other cell lysis methods, such as enzyme degradation and mechanical disruption, particularly on the industrial scale, include lower production and product recovery costs. Furthermore, in our conditionally-inducible cell lysis system there is no need to add an inducer to initiate cell lysis. At the laboratory research scale, transfer of DNA from E. coli undergoing phage-induced lysis to B. subtilis by the co-culture method [E. coli cells can be detrimental to the quality of the transferred DNA, thus in our system we used solely the phage lysis genes. In the future, this system could be further enhanced by utilization of other lysis genes, such as those of bacteriophages T4, T7, and Mu. Other phage-encoded genes crucial for phage's life cycle could be integrated into the chromosome to improve the kinetics of cell lysis. Alternative promoters, such as PmgtB, PnisA, Pcmp or Plac-LacI [To our knowledge, this is the first study investigating synergistic effect of lysis genes from three different bacteriophages on the lysis of e method , 18 usedlac-LacI , 44 and lac-LacI  could beS1 FigFigure shows confirmation of the plasmids pSB1K3(FRTL1), pSB1K3(FRTL2), pSB1K3(FRTL3), pSB1K3(FRTL12), pSB1K3(FRTL13), and pSB1K3(FRTL123), harbouring different combinations of lysis genes using flanking primers. HyperLadder 1kb (Bioline) has been used as the molecular weight marker.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


The association of mycosis fungoides and kaposi’s sarcoma in HIV-negativepatients is a rare phenomenon. The presence of human herpesvirus 8 (HHV-8) –associated with all forms of Kaposi’s sarcoma – has also been recentlyidentified in mycosis fungoides lesions. However, a causal association betweenHHV-8 and the onset of mycosis fungoides has not been established yet. Thepresent case reports a patient who developed Kaposi’s sarcoma lesions after atwo-year UVB phototherapy to treat a mycosis fungoides. Negativeimmunohistochemistry staining for Kaposi’s sarcoma-associated herpesvirus in theinitial mycosis fungoides lesions strengthens the absence of a link betweenKaposi’s sarcoma-associated herpesvirus and mycosis fungoides. Immunosuppressioncaused by the lymphoma and prolonged phototherapy were probably the contributing factors for the onset of Kaposi’s sarcoma. The association of KS with lymphoproliferative disorders is unusual andless frequent in MF.4 Thecoexistence between MF and KS in HIV-negative patients is rare: the literaturereports only a few records of this association.5 The influence of HHV-8  on MFoccurrence has also been recently identified. However, there is no consensus aboutthe role of the virus in the pathogenesis of MF and the development of secondarymalignancies.9Mycosis fungoides (MF) is the most common form of cutaneous T-cell lymphoma. Itpresents itself mostly in the early stages. The evolution to more severe forms isvariable and its association with secondary malignancies may occur.Lymphoproliferative disorders are the most common diseases after the diagnosis ofMF.We report a 53-year-old male patient with a three-year history of mycosis fungoidesstage IB. The patient was on his 138th narrowband UVB phototherapy session (twosessions per week) with partial control of the skin condition. Two years after theinitial diagnosis, the patient developed fast-growing nodular lesions on the limbs.Dermatological examination revealed erythematous scaly plaques on the abdomen andthighs and violaceous papules on the anterior side of the right arm, lateral side ofthe left forearm and posterior side of the right leg  and 2.A biopsy revealed superficial perivascular dermatosis with some mildly pleomorphiclymphoid cells and positive immunohistochemistry for CD3, CD4 and CD5, negative forCD8, suggesting MF . HistoloWe opted for the surgical removal of the KS lesions and for the continuation ofphototherapy for MF. During follow-up, new KS lesions occurred on the lower limbs,confirmed by biopsy and immunohistochemistry. We scheduled the excision of these newlesions. After three months, the lesions to be excised resolved spontaneously.3 Hodgkin’s and non-Hodgkin’s lymphomas are the most common.Rarely, MF patients can develop cancer in the lung, colon, bladder, bile duct,vulva, skin (melanoma), and blood (acute myeloid leukemia).3 The coexistence ofMF with KS – especially in negative-HIV and non-transplant patients – is rare. Thereason for this association is not well established.5Mycosis fungoides (MF) association with other malignancies is well established. MFpatients present a 1-2 fold risk for developing secondary neoplasias.4 In the case of KS, that immunomodulation could favor theactivation and replication of HHV-8, contributing to the emergence of clones ofmalignant cells and of KS lesions.MF, even at early stages, causes impaired cell-mediated immunity by T-cellactivation. This factor could contribute to the development of otherneoplasias.8 A higher frequency of HLA-DRB1*11 was observed bothin MF patients as in KS patients.5Epstein Barr virus, cytomegalovirus and HTLV have also been identified in patientswith MF. However, there is still controversy about the role of these viruses in thepredisposition to secondary neoplasias.8Genetic factors, environmental exposures and viral infections may also contribute tothe occurrence of other neoplasias.9 HHV-8 couldact as a chronic antigen or oncogene and induce the proliferation and uncontrolledactivation of T-cells in the skin. Thus, it could contribute to the onset ordevelopment of MF.8 However,some authors believe that the presence of the virus is not necessarily related tothe pathogenesis of the disease. The detection of the virus could only indicate theexistence of opportunistic, recent, or reactivated infection.9 In the reported case, as observed in other studies, HHV-8 wasnot identified in the early MF lesions, strengthening the hypothesis that the viruspresents no relation with the development of the disease.The recent detection of HHV-8 in patients with MF lesions suggests an influence ofthe virus in the pathogenesis of the disease and a likely predisposition to developKS.9 Phototherapy induceslocal and systemic immunosuppression by DNA damage, Langerhans cell reduction andaltered cellular immunity, with consequent generation of cytokines and T-cells withsuppressing activities.10 In our case, the cumulativeexposure to UVB radiation may have been a facilitating factor and immunosuppressioninducer for the further development of KS.10Frequent exposure to treatments such as phototherapy, chemotherapy and radiotherapycould also contribute to increased risk of secondary neoplasias.In the present case, we demonstrated the absence of association between HHV-8 and MF.Immunosuppression generated by MF, even at early stages of the disease, maypredispose to the occurrence of other malignancies. Phototherapy may aggravate thisimmunosuppression when held for prolonged periods. Physicians should be alert to anychanges of cutaneous or systemic clinical pictures of their patients with MF forearly detection of other neoplasias.


Global attention towards antimicrobial resistance (AMR) and the threat it presents to current and future human health has soared in the last 2 years , 2. A clSepsis accounts for around 10–15% of deaths among pregnant or recently-delivered women and among neonates: virtually all of these deaths are preventable and the vast majority occur in LMICs , 5. OptiLancet series on maternal health  has also been well-charted historically; for example, the contribution to the 80% decline in maternal mortality in the United Kingdom from 1935 to 1950  was fully integrated into quality improvement, covering enhanced practices as well as environments.The risk of maternal death from iatrogenic infections at the time of birth has been known about for centuries , as well to 1950 . This reSo have these historic lessons been learnt, adapted, and applied appropriately to the maternity units in LMICs that are now the location of most of the world's births? Is the full potential from primary prevention of infections at birth through clean, quality care being realised for mothers and babies? Unfortunately, evidence from a wide variety of assessments indicates a huge missed opportunity. A WHO survey  across 5And what is the link with AMR? Again history shows how in high-income countries, a tolerance of poor hygiene in health institutions coincided with the growing reliance on antibiotics, which – in turn – perpetuated inappropriate use and poor drug stewardship, thus contributing to emerging resistance , 18. MorSo what do we know about the magnitude of AMR on maternity units? What information is available from routine monitoring? And what is the strength of the research evidence-base? In terms of routine data, several major reports , 2, 24 hStrengthening tools, metrics, and measurement systems: Practical tools, such as standardized audit forms and simple infographics software, are needed to support the tracking of antibiotic use and to measure AMR in health service settings in LMICs. These tools must be sensitive to the limited capacity of local microbiological laboratories, including specimen transport, and to the resources needed for their use. Systems for surveillance of HCAIs – both infections captured and recorded in facilities or through community follow-up – require agreed definitions and innovations in bio-sampling and informatics in order to establish pathways for infection and the burden of resistant pathogens;Developing and evaluating interventions: Health services research and innovative audits are needed on current standards of IPC in maternity units. Modalities need to be identified and tested to ensure that interventions – be these enhanced WASH, effective bed management to reduce crowding see , efficieImproving the knowledge base on human behaviour around AMR: Understanding human behaviour is key to developing sustainable, effective, and affordable interventions to prevent infections and to mitigate the threat of AMR for maternity units. Strong, in-depth, social science is essential to understand and influence key preventive behaviours and practices, such as hand hygiene, infrastructural maintenance, and facility cleaning.At the 71st session of UNGA later this month on AMR, priorities will be set. In the absence of robust evidence on the situation in maternity units, the threat from AMR and the opportunities for infection prevention and appropriate antimicrobial stewardship may simply be ignored, with serious consequences. Together with the wide variety of agencies pledging their support for global action on AMR at the UN high-level forum, such as WaterAid , we urgeWomen in LMICs have expressed their demand to deliver in health institutions, with more than half of births now taking place in maternity units . Global


As a means to decrease the amount of waste tires and to re-use tire rubber for new tires, devulcanization of ground passenger car tires is a promising process. Being an established process for NR and EPDM, earlier work has shown that for ground passenger car tire rubber with a relatively high amount of SBR, a devulcanization process can be formulated, as well. This was proven for a laboratory-scale batch process in an internal mixer, using diphenyl disulfide as the devulcanization aid and powder-sized material. In this paper, the devulcanization process for passenger car tire rubber is upscaled from 15 g per batch and transformed into a continuous process in a co-rotating twin screw extruder with a capacity of 2 kg/h. As SBR is rather sensitive to devulcanization process conditions, such as thermal and mechanical energy input, the screw design was based on a low shear concept. A granulate with particle sizes from 1–3.5 mm was chosen for purity, as well as economic reasons. The devulcanization process conditions were fine-tuned in terms of: devulcanization conditions , extruder parameters  and ancillary equipment . The influence of these parameters on the devulcanization efficiency and the quality of the final product will be discussed. The ratio of random to crosslink scission as determined by a Horikx plot was taken for the evaluation of the process and material. A best practice for continuous devulcanization will be given. Nowadays, a significant part of waste tires is recycled. However, the reuse in cradle-to-cradle loops (tires back into tires) is limited due to the low property profile of the recycled material. The decrease in the quality of the material is partly due to the fact that the rubber already suffered a whole life cycle, but it is also caused by the recycling process itself: the waste rubber is ground and re-plasticized in a process in which not only the crosslinks, but at the same time, also, the polymers are broken down.Compared to the starting material, the re-plasticized material has a different structure and properties. In order to achieve a quality of recycled rubber that is comparable to virgin material, it needs to be devulcanized, meaning that preferably only the sulfur crosslinks and hardly any of the polymers break down in the recycling process.Saiwari  has showStyrene-butadiene rubber (SBR), a major component of ground passenger car tire rubber (GTR), is in particular sensitive to shear, as shown in the work of Saiwari : during Some preliminary experiments with a high shear setup of a co-rotating twin screw extruder, using the same optimal settings as for the batch process, lead to the conclusion that the material was integrally broken down and the properties of the recycled material were rather poor due to the high shear . TherefoAdditional equipment for the devulcanization process had to be developed: a nitrogen purging system, a cooling system for the devulcanizate and swelling equipment for the blend of the particulate rubber with the devulcanization aid and plasticizer. This was finalized during the experiments. On the one hand, this introduced the possibility to study the effect of these systems on the quality of the devulcanizate; on the other hand, it created the situation that trends during the experiments could be compared, but absolute values not.The main method for analyzing the devulcanization effect is the decrease of the crosslink density determined according to the Flory–Rehner theory, combined with the Horikx presentation of the results ,6. AnothThe analysis method based on the crosslink density calculation according to the Flory–Rehner equation and allowing one to estimate the decrease of the crosslink density in a Horikx presentation is extensively described by Verbruggen , Verbrug3 as the mean value of the densities of these polymers according to the literature [To determine the amount of soluble rubber, the samples were extracted for two days with boiling acetone followed by three days in boiling tetrahydrofuran and dried at 40 rrection . , carbon black-based tread compound on a 50/50 %wt base. This material was then sheeted out, vulcanized and visually rated according to the amount and size of visible particles. As reference, a 100% virgin compound was used, and the vulcanized samples were sorted according to their visible surface roughness. The observed differences were organized into 10 classes of increasing roughness, with Class 0 representing the virgin compound. Because of the low heat transfer-rate of rubber and the low distribution rate of TDAE, and hence, the DPDS, in the rubber particles, the work hypothesis was that the visible particles consist mainly of non-devulcanized material.In an extruder, the devulcanization kinetics might be different due to differences in mechanical shearing forces, as well as heat transfer compared to an internal mixer. Therefore, an approach to re-adjust the devulcanization time was elaborated. In the earlier experiments in an internal mixer , the devA shorter residence time results in a decrease of the devulcanization efficiency, indicated by the lower degree in ‘decrease of crosslink density’ due to the shorter devulcanization process. The position of the 6-min point in the Horikx plot indicates the highest degree in devulcanization and the lowest degree of main chain scission.During the residence period, the dispersion of the devulcanization aid into the rubber, the transfer of heat into the rubber particles and the reaction of the devulcanization aids with the active chain fragments have to take place: after the scission of a crosslink, the devulcanization aid will react with the remaining highly-reactive radicals and neutralize these . Only ifBasically, in a thermo-chemical devulcanization process, there are three main factors contributing to the efficiency of the devulcanization: thermal energy, the chemical reaction and shearing forces. Initially, a devulcanization temperature of 220 The expected trend, that an increasing temperature improves the devulcanization efficiency, indicated by an increasing ‘decrease of crosslink density’, is not found. On the contrary, the data points show a tendency towards the ‘random scission line’, indicating that the quality of the devulcanizate decreases: at 280 In a thermo-chemical devulcanization process, the devulcanization agents are added in order to scavenge radicals formed during this process. However, at very high devulcanization temperatures, i.e., above 220 Therefore, it is to be concluded that the devulcanization temperature of 220 Compared to the material used in the earlier experiments  with a sWith the surface/volume ratio of particles with a diameter 3.5 mm set to one, the ratio for particles of 2 mm is 1.75. Hence, when distributing the devulcanization aid over the particles, 2 mm particles will have 1.75 times the amount compared to the larger ones, absorbing a disproportional higher amount of devulcanization aid compared to the relatively small fraction they constitute. It can be calculated and has been experimentally verified that for the main fraction of particles with a diameter of 2–3.5 mm to adsorb a TDAE concentration of 5 %wt, the amount of TDAE calculated for an average concentration of 5 %wt over the whole batch  used in this investigation was obtained from Genan, Dorsten, Germany. It is a commercial passenger car tire grade, thus mainly based on synthetic rubber, and had a particle size of 1.0–3.5 mm. This GTR type was chosen due to its purity: a higher particle size material contained a considerable amount of fibers; a lower particle size granulate contained fine metal dust worn off of the grinding equipment and fine-ground stones, glass, etc.Ambiently-ground car tire powder with a maximum particle size of 0.35 mm was used as a comparative material for a study on the effect of the visible particle cores. It was devulcanized and re-vulcanized similar to the coarser GTR. As this material behaved differently in the extruder compared to the 1.0–3.5 mm material, these results cannot be compared to the results of the granulate, other than the roughness of the re-vulcanized samples.As devulcanization agent diphenyl disulfide (DPDS) was used and as the anti-oxidant, tris phosphite (TDTBP). Both were supplied by Sigma Aldrich Cooperation, Zwijndrecht, The Netherlands. The processing oil treated distillate aromatic extract (TDAE) was supplied by Hansen & Rosenthal, Hamburg, Germany.The solvents used for the analysis were acetone , length 40D with D = 25 mm, facilitating 3 de-aeration positions between the supply funnel and outlet. The screw design was based on a minimal shear concept, as shown in When checking the progress of the devulcanization in the extruder with Screw 1 via the de-aeration openings in the barrel of the extruder, it appeared that the GTR kept its granulated shape up to halfway the extruder, after which the consistency changed into a smaller size granulate embedded in a plastified matrix at the end of the middle section of the extruder. Based on the observation in the internal mixer experiments that the devulcanization activity is decreasing considerably at temperatures below 180 In the supply and mixing zone, a mixing element was installed with the necessary preceding pressure elements. At the discharge side of the screw, pressure elements were added to be able to discharge the devulcanized product. In the intermediate section, a mix of several kinds of transport and pressure elements was used to create gentle mixing and shear due to varying degrees of filling of the screw in this zone (see It is important to notice that in both the supply and mixing zone and the intermediate zone, the GTR is still present as particles. The behavior of these GTR particles in the extruder differs from the models concerning transport and mixing for high viscous liquids, and these models are therefore not applicable for the design of alternative screw configurations ,18,19. DAfter the mixing step as described above, the material was milled on a two-roll mill with a nip-width down to 0.5 mm. During milling with this nip-width, it was observed that the visible particle size decreased considerably. However, the material did not become completely smooth; this indicated that, although the material is devulcanized, it preserved a granular structure up to rather high shearing forces. Because of the amount and size of the visible particles, additional screws were designed see : Screw 2The temperature of the devulcanized material was measured at the outlet of the extruder with an integrated sensor in the extruder, which measured the temperature at the surface of the devulcanizate. For each different condition of the extruder, the temperature of the core of the devulcanizate was measured at the outlet of the extruder with an independent thermocouple-based thermometer. The core temperature was kept at the set temperature of the experiment The order of the experiments was designed to first optimize the devulcanization temperature and subsequently the residence time, the concentration of the devulcanization aid and, at last, the screw configuration. For Screws 2 and 3, the residence times turned out to be similar to that of Screw 1 at the conditions used for the comparison of the screw performances.The die of the extruder is a rounded elongated slit of 20 mm The possibility to quench the rubber after devulcanization, thus to rapidly cool it down to a temperature low enough to significantly reduce oxidation, proved to be one of the main factors contributing to a high devulcanizate quality in the batch process.In the earlier work with an extruder, the devulcanizate was quenched into a water bath. Consequently, an extra drying step for the extruded devulcanizate was needed. This additional step at elevated temperature for a rather long period in the presence of oxygen resulted in low properties of the devulcanizate. Therefore, a cooling calender was designed and built to cool the devulcanizate directly after the discharge of the extruder.The supply-funnel, extruder and extruder output piping were flushed with nitrogen during production.2. Manual counting of visible particles was done on a surface area of 2 cm2.The devulcanizate was blended with a virgin rubber compound in a ratio of 50/50 %wt, based on polymer content, and vulcanized according to the recipe in -Temperature profile: The main factor determining the devulcanizate quality is the temperature: if it is too low, devulcanization is too slow; if it is too high, the polymer degrades. The optimum within the temperature window investigated here was 220 -Time: The residence time in the extruder is another important influencing factor for the product quality: shorter times set by higher screw speeds result in insufficient devulcanization and uncontrolled breakdown of the polymer due to the higher shearing forces. The optimal condition for a screw configuration like Screw 3 is a 6-min residence time. -Devulcanization aid concentration: The optimum concentration of DPDS was found to be 18 mmol/100 g GTR. Increasing the concentration to 30 mmol/100 g GTR decreased the quality of the devulcanizate.Devulcanization process conditions: The extruder conditions turned out to be comparable to the parameters found for the Brabender internal mixer, as shown in The earlier established parameters of the devulcanization process in a small-scale, discontinuous batch mixer with a capacity of 15 g per batch were transferred to a 40D 25 mm co-rotating twin screw extruder process with a capacity of 2 kg/h. Within this scale-up of the devulcanization process, the following parameters were fine-tuned for the extruder-based process:-Screw speed: For the given extruder and screw configuration and within a range of 10–120 rpm, 10 rpm leading to a residence time of 6 min was optimal.-Screw configuration: The low shear concept has been shown to be a useful approach. From the three screw variations tested, Screw 3, -Fill factor: Being a paramount parameter for the amount of shear in an internal mixer, for the extruder screw, it appeared to be of less importance.Extruder parameters: The extruder parameters are interdependent: The screw configuration determines the amount of shear and the residence time, at a certain screw speed. For a small speed range, both residence time and shear rate can be assumed to have a linear relation with the speed; however, this is not valid for larger differences in screw speed.Still visible particles: For non-optimal devulcanization parameters, large particles remain, which mostly consist of non-devulcanized material. However, even with the use of a small-sized passenger car tire powder, small visible particles remain in the devulcanized material. The first analysis indicates a high amount of silica in these particles, but additional investigations are necessary.The conditions defined for the Brabender batch reactor were also valid for the extruder process. Looking at the degree of devulcanization, an extruder is more efficient than a batch mixer: while the maximum degree of devulcanization in the batch mixer was less than 80%, the percentages found after extruder devulcanization were between 80% and 90%. Higher degrees of devulcanization cannot be established, as some polymer is bound to the filler and is therefore not soluble; furthermore, the present devulcanization process is not efficient for monosulfidic crosslinks.To be able to run the extruder under the best conditions for both the product and the environment, additional ancillary equipment is needed: swell equipment, nitrogen supply and a cooling calender.


The widelyvariable phenotypic spectrum and the different severity of symptoms in men with Klinefelter syndrome (KS) suggest a role for epigenetic mediators. Therefore, the aim of this study is to evaluate the possible involvement of miRNAs in the clinical manifestations of KS. To accomplish this, we performed a transcriptome analysis in peripheral blood mononuclear cells (PBMCs) of 10 non-mosaic KS patients, 10 aged-matched healthy men and 10 aged-matched healthy female controls with normal karyotype. After RNA extraction from PBMC and the preparation of RNA libraries, the samples were sequenced using next generation high-throughput sequencing technology. Expression profiling analysis revealed a significant differential expression of 2 miRNAs in KS compared to male controls. In particular, MIR3648 resulted significantly  down-regulated by −19.084- fold, while MIR3687was strongly down-regulated  considering KS patients. These results were confirmed by qRT-PCR. The functional analysis of the two transcripts showed that they seem to play a role in breast cancer, hemopoietic abnormalities, immune defects and adipocyte differentiation and fat cell maturation. Therefore, we speculate that both miRNAs may play a role in the immune and metabolic disorders and in the risk of breast cancer development in men with KS. However, most of the men with KS remain undiagnosed or are being diagnosed later in life3. KS is characterized by a 47,XXY karyotype in about 80–90% of men, whereas the remaining cases are given by chromosome mosaicis.369*m , additional sex chromosomes  or X chromosome structural abnormalities 4. The lowtestosterone levels observed in a high proportion of these men could explain the classical characteristics of KS, such as tall stature, gynecomastia, and infertility5, as well as some of other disorders that have an increased prevalence in KS, including osteoporosis6, metabolic syndrome, and type 2 diabetes mellitus7. The other disorders associated with KS, such as an increased incidence of mediastinictumors, neurocognitive and psychiatric disturbances, are less readily explained by hypotestosteronemia8. The wide phenotypic spectrum and the different severity of symptoms in non-mosaic KS patients suggest a role for epigenetic factors.Klinefelter syndrome (KS) is a common sex-chromosome aneuploidy with an estimated prevalence of 1 inevery 660 male births9. However, data on the role of X-linked genes on testicular function are inconsistent10. Therefore, evaluation of molecular regulators of gene expression, such as microRNA (miRNAs), could help to understand the molecular background in KS men. Nevertheless, so far,only few studies have explored the role (if any) of miRNAsin KS men.In recent years, a number of studies have investigated gene expression profiles of KS to improve the evaluation of the molecular basis of KS phenotype. Nevertheless, the mechanisms leading to germ cell degeneration and consequently azoospermia are still unclear. It has been hypothesized that an altered gene dosage, escaping inactivation on the supernumerary X-chromosome, might affect the development or cause degeneration of germ cells12. Abnormal miRNA expression has been reported to be involved in the occurrence and development of various diseases, such as cancer, cardiovascular disease, mental retardation, fetal growth restriction18. Growing evidence has shown that miRNAs play critical roles in regulating male germ cell development and are essential for epigenetic regulation of mitosis, meiosis and spermiogenesis21.miRNAs are non-coding small RNAs (about 22 nucleotides) that play a role in the post-transcriptional regulation of gene expressionand protein translationOn this basis, the present study was undertaken to evaluate the expression of miRNAs in men with KS by high-throughput sequencing technology and to assess the possible involvement of miRNAs in the pathogenesis and clinical manifestations of KS. miRNA expression was compared to that of healthy age-matched men and women with normal karyotype.We performed a transcriptome analysis on peripheral blood mononuclear cells (PBMCs) of 10 non-mosaic KS patients and 10 male controls. Expression profiling analysis revealed the significant differential expression of 73 transcripts in KS men compared to controls, highlighting two clusters of differentially expressed genes composed by 60 down- and 13 up-regulated transcripts in KS.Among the down-expressed genes, we found a significant differential expression of two miRNA precursors in KS compared to controls Table . MIR3648The results obtained by NGS analysis were confirmed by qRT-PCR. Indeed, miRNA 3648 and miRNA 3687 were significantly under expressed in KS compared to both male -positive breast carcinoma25. The expression characteristic of miRNA during the development of Down syndrome (DS) fetuses showed that miR-3648 and miR-3687 were up regulated more than 50% in the DS fetal cord blood mononuclear cells. This suggests that the differentially expressed miRNAs may be involved in the hemopoietic abnormalities and the immune defects of DS fetus26.Men with KS have an increased risk of developing breast cancer and several studies have addressed this issue. The mechanism is not well understood. Several publications have looked at the miRNA expression profiles in breast cancer cell lines and tumor tissues. A closer inspection of the patterns of deep sequencing reads mapped to the miR-3648 suggests that it may not be a 6. Insulin resistance and metabolic syndrome are present in 24% and 7%, respectively, of 89 KS children as young as 4–12 years of age27. Both studies showed that KS patients had truncal obesity. miRNA have been suggested as therapeutic targets in metabolic syndrome due to their role in the maturation of fat cells and the differentiation of adipocytes from mesenchymal stromal stem cells. Recently, the role of metformin to suppress the differentiation of human preadipocytes was evaluated. After 2 weeks of 5 mM metformin treatment, 27 miRNAs were significantly upregulated and 6 miRNAs were down regulated in human visceral preadipocytes (HPrAD-vis) compared to cells control. In particular, the expression of miR-1246 and miR-3687 increased following metformin administration, while the expression of miR-378 family members decreased. This finding suggest that adipocyte differentiation in regulated by miRNAs. This result identifies miR-1246, miR-3687 and miR-422a as promising therapeutic targets for the treatment of visceral obesity28.Adults with KS have five-fold increased risk for developing metabolic syndrome compared to age-matched controls29. Certain allelic variations in this gene increase the risk of development of obesity.To date, several genes have been identified as targets of miR-3687, including PPARGC1B (PPARG coactivator 1β), MEMO1 (mediator of cell motility 1) and GDF7. PPARGC1B encodes a protein that stimulates the activity of several transcription factors and nuclear receptors, including ERα, nuclear respiratory factor 1 and glucocorticoid receptor. The encoded protein may be involved in fat oxidation, non-oxidative glucose metabolism and the regulation of energy expenditure. This protein is downregulated in prediabetic and type 2 diabetes mellitus patients30.MEMO 1 is a copper-dependent redox protein with an essential role in migration and metastasis. It is involved in breast carcinogenesis via regulating insulin-like growth factor-I receptor-dependent signalling events31.GDF7 encodes a secreted ligand of the transforming growth factor-β proteins superfamily. It is required for normal seminal vesicle development, branching morphogenesis and cell differentiation. Mice lacking a functional copy of this gene exhibit absence of some spinal dopaminergic neurons and brain defects, male sterility, and premature deathIn conclusion, NGS analysis showed that non-mosaic KS men have a lower expression of MIR-3648 and MIR-3687 compared to normal controls. The functional analysis of the deregulated transcripts suggests that these miRNAs may play a role in the molecular mechanisms involved in germ cells degeneration, neurological disorders and the risk of development of cancer and obesity that are more likely to occur in KS patients. Results are to be considered preliminary because gene expression was measured in blood cells. Further validating and functional studies are needed to confirm these findings and to better understand the molecular basis of the variable phenotype found in KS patients.This study was approved by the Ethics Committee of University teaching Hospital of “Policlinico-Vittorio Emanuele, University of Catania, , trial registration number 49/2015/PO (Register of the Ethics Committee opinions). All methods were performed in accordance with the relevant guidelines and regulations. All participants were asked for and provided their informed consent. We performed a transcriptome analysis on PBMCs of 10 non-mosaic KS patients and 10 aged-matched healthy controls. All KS patients (mean age 32.5 ± 3.0 years) had 47,XXY karyotype as shown by cytogenetic investigation conducted on at least 50 metaphase and five out of ten were treated with testosterone enanthate. Normozoospermic controls (mean age 32.0 ± 3.0 years) had a negative history for genetic disease, normal testicular volume and normal reproductive hormones  levels. Ten healthy age-matched women (mean age 32.0 ± 2.9 years) were also recruited as female controls. All KS and male and female controls were Italians (Eastern Sicily)  were obtained using Ficoll-Paque . Total RNA was extracted using the standard RNA extraction method with TRIzol . Before use, RNA concentration in each sample was assayed with a ND-1000 spectrophotometer (NanoDrop) and its quality assessed with the Agilent 2100 Bioanalyzer with Agilent RNA 6000 nano kit .Indexed libraries were prepared from 1 µg/ea purified RNA with TruSeq Stranded Total RNA (Illumina) Library Prep Kit according to the manufacturer’s instructions. Libraries were quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies) and pooled such that each index-tagged sample was present in equimolar amounts, with final concentration of the pooled samples of 2bnM. The pooled samples underwent cluster generation and sequencing using an Illumina HiSeq. 2500 System (Illumina) in a 2 × 100 paired-end (RNA-seq) format.To validate the results obtained by Next-Generation Sequencing (NGS) analysis, we compared qRT-PCR in 10 KS patients and 10 normal subjects. In this second step, the subjects studied in the NGS analysis were included. Once again, KS cases and controls were recruited after family and/or personal informed consent. RNA extraction from leucocytes of peripheral blood was performed using RNeasy Mini Handbook , following the manufacturer’s protocol. Retro-transcription of 650 ng of total RNA from each sample was then performed in a final volume of 30 μl and the cDNA generated was used as a template for real-time quantitative PCR analysis with gene expression products. For each sample qRT-PCR reactions were carried out in duplicate using 4 μl of cDNA and QuantiTect Probe PCR Master Mix Kit  in a total volume of 25 μl. QRT-PCR experiments were performed using the Light Cycler 480 . The target MIR3648, MIR3587 and the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assays were obtained from Applied Biosystems .The thermal cycling conditions consisted of one cycle for 2 min at 50 °C, 1 cycle of 15 min at 95 °C and 42 cycles for 15 s at 94 °C followed by 1 min at 60 °C. The amplified transcripts were quantified using the comparative CT method and relative quantification analysis data were played using the comparative ΔΔCt method. The MIR expression level was normalized to GAPDH level and Target Mean Cp definition was used to indicate the mean normalized cycle threshold.In our case–control study with qRT-PCR we have used all cases and controls and specifically we obtained a mean FC Fig. . The meahttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and the quality checked reads were trimmed with cutadapt32 v.1.10 and then aligned to the human genome (hg19 assembly) using STAR v.2.5.233, with standard parameters. Differentially expressed mRNAs were identified using DESeq. 2 v.1.1234. Firstly, gene annotation was obtained for all known genes in the human genome, as provided by Ensembl (GRCh37). Using the reads mapped to the genome, we calculated the number of reads mapping to each transcript with HTSeq-count v.0.6.135. These raw read counts were then used as input to DESeq. 2 for calculation of normalized signal for each transcript in the samples, and differential expression was reported as fold change along with associated adjusted p-values (computed according to Benjamini-Hochberg). Differential expression data were further confirmed using Cuffdiff36. Raw data are available in ArrayExpress database repository (https://www.ebi.ac.uk/arrayexpress/) with accession number E-MTAB-6107.The raw sequence files generated (.fastq files) underwent quality control analysis using FastQC (


The use of HS allows us to fabricate carbon nanotube-reinforced nanofibers (CNT-reinforced nanofibers) by addressing three significant challenges: (i) the difficulty of forming nanofibers at high concentrations of CNTs, (ii) aggregation of the CNTs, and (iii) control of the orientation of the CNTs. The handspun nanofibers showed better physical properties than fibers fabricated by conventional methods, such as electrospinning. Handspun nanofibers retain a larger amount of CNTs than electrospun nanofibers, and the CNTs are easily aligned uniaxially. We attributed these improvements provided by the HS process to simple mechanical stretching force, which allows for orienting the nanofillers along with the force direction without agglomeration, leading to increased contact area between the CNTs and the polymer matrix, thereby providing enhanced interactions. HS is a simple and straightforward method as it does not require an electric field, and, hence, any kinds of polymers and solvents can be applicable. Furthermore, it is feasible to retain a large amount of various nanofillers in the fibers to enhance their physical and chemical properties. Therefore, HS provides an effective pathway to create new types of reinforced nanofibers with outstanding properties.The novel method,  The electrospinning (ES) technique is an effective and attractive method that has been investigated extensively during the last few decades to fabricate nanofibers from a polymer solution12457Recently, we reported a simple and innovative method for fabricating nanofibers, called as handspinning (HS). This method shows promise for overcoming the challenges in ESet al.Carbon nanotubes (CNTs) have attracted significant scientific and technological attention due to their exceptional mechanical1718There have been three significant challenges in the fabrication of CNT-reinforced polymer composites, i.e., (i) the difficulty of concentrating the CNTs in a matrix, (ii) the agglomeration of the CNTs, and (iii) controlling the orientation of the CNTs. Incorporation of a high concentration of CNTs in a polymer matrix without aggregation and localization of the CNTs is difficult because they tend to make a bundle, resulting in their being poor dispersed in the polymer matrix and the deterioration of the properties of the materials2123242529The microstructure of the nanofibers was investigated further using TEM. i.e., electrospinning or handspinning, due to their relatively low crystallinity. Thus, the HS process is beneficial in that it enhances the tensile strength of the fibers by inducing polymer chain alignment, which the ES process cannot do. Also, the incorporation of CNTs should have a significant effect due to the resulting enhancement of the mechanical properties of the nanofibers. Increasing the amount of CNTs in the PVAc matrix dramatically increased Young’s modulus and tensile strength , which could provide better understanding the interaction by the hydrogen bonding of the carbonyl group in the PVAc chains to CNTs. g over the entire CNT concentration range. As shown in g of about 6 °C, which was attributed to the addition of inorganic filler to the polymer412 composite membrane, the increase of Tg that occurred when TiO2 was added occurred due to hydrogen bonding between the hydroxyl groups on surface of the TiO2 and the sulfone or ether groups of PVAcg also indicated that the CNTs were dispersed well in the PVAc fiberg as concentration of CNTs increased. At concentrations greater than 0.5 wt%, the CNTs aggregated, as observed in the morphological studies, leading to a reduction in the value of Tg similar to the trend for the mechanical properties. However, the decrease in the value of Tg was not observed in the handspun nanofibers; the addition of 0.5 wt% CNTs led to an increase in Tg from 42 to 48 °C, and then, in spite of further addition of CNTs up to 10 wt%, Tg became saturated at ~47 °C. These results strongly suggested that (i) the CNTs interacted strongly with PVAc and (ii) the CNTs were well-dispersed in the PVAc nanofiber, in good agreement with electron microscope study.Differential scanning calorimeter (DSC) was performed to determine the glass transition temperature  for 3 hr at ~50 °CTo improve the miscibility of CNTs with PVAc, pristine MWCNTs  were added and sonicated in a mixture of sulfuric acid and nitric acid (3:1 H2). In this work, PAS, PAD, and PA were controlled accurately to be 40 cm/s, 13 cm, and 66 cm2, respectively. The typical volume of the solution was 10 mL. The resulting samples were dried under vacuum overnight. To optimize the processing conditions, we conducted the handspinning process by varying PAS from 30 to 50 kV, and the PAD was varied from 10 to 18 cm. All of the samples of the composite nanofibers were prepared under optimum conditions. All samples of the nanofibers were prepared with the same process parameters. At the conditions specified above, confirmed the repeatability of the handspinning process to fabricate uniform nanofibers with multiple spinning operations.For ES, a high-voltage power supply , capable of generating voltages up to 80 kV, was used as the source of the electric field. The PVAc/CNT solutions were supplied through a plastic syringe to a capillary tip with an inner diameter of 0.6 mm. A copper wire connected to a positive electrode (anode) was inserted into the PVAc/CNT solution, and a negative electrode (cathode) was connected to a metallic collector. The voltage was fixed at 10 kV, and the distance between the capillary tip and the metal collector was 15 cm. The resulting samples were dried under vacuum overnight. For HS, we built the apparatus shown in Figure S1The morphologies of both HS and ES PVAc/CNTs nanofibers were observed with scanning electron microscopy . The average diameter and distribution of the nanofibers were measured from the SEM micrographs using image analysis software . To obtain the distribution of diameters and their average value, at least 50 points in each SEM image of the corresponding sample were selected randomly and evaluated in the diameter range of 100–1100 nm. Transmission electron microscopy (TEM)  was utilized to examine the CNTs inside the resulting nanofibers. The mechanical properties of single nanofibers were characterized using a specially-developed tensile test machine . The glass transition temperatures of the nanofibers were measured with a differential scanning calorimeter  in argon gas with a heating rate of 5 °C/min from 20 to 80 °C. The wide-angle X-ray diffraction (XRD) experiments were performed at room temperature using a Rotaflex RTP300 X-ray diffractometer  operating at 50 kV and 200 mA. Nickel-filtered Cu Kα radiation was used as the X-ray source, and diffraction was detected over an angular range of 5 to 40°.How to cite this article: Lee, H. et al. Handspinning Enabled Highly Concentrated Carbon Nanotubes with Controlled Orientation in Nanofibers. Sci. Rep.6, 37590; doi: 10.1038/srep37590 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.