In a Policy Forum, Teodora Wi and colleagues discuss the challenges of antimicrobial resistance in gonococci. Neisseria gonorrhoeae seriously compromises the management and control of gonorrhea.Antimicrobial resistance (AMR) in N. gonorrhoeae to the extended-spectrum cephalosporins (ESCs), the last-line treatment for gonorrhea, have been reported from many, particularly well-resourced, settings globally.In vitro and clinical resistance in The World Health Organization (WHO) Global Gonococcal Antimicrobial Surveillance Programme (GASP) is key to monitoring AMR trends, identifying emerging AMR, and informing regular refinements of treatment guidelines and public health policy globally.Enhanced international collaborative actions are crucial for the control of gonorrhea, including improved prevention, early diagnosis , partner management, and enhanced surveillance .Rapid, accurate, point-of-care diagnostic tests , new therapeutic drugs, and a gonococcal vaccine—which will ultimately be the only sustainable way to achieve gonorrhea control—are needed.Neisseria gonorrhoeae (gonococcus), and it is a major public health priority globally. In 2012, the World Health Organization (WHO) estimated that there were 78 million cases among adults worldwide, including 35.2 million in the WHO Western Pacific Region, 11.4 million in the Southeast Asian Region, 11.4 million in the African Region, 11 million in the Region of the Americas, 4.7 million in the European Region, and 4.5 million in the Eastern Mediterranean Region [Gonorrhea is a sexually transmitted infection (STI) caused by n Region .N. gonorrhoeae has continuously compromised the management and control of gonorrhea. Because of widespread AMR, the persistence of AMR determinants in gonococci, and the unavailability of diagnostic tests that provide AMR results at the time of treatment, clinicians resort to empiric treatment for gonorrhea. Since the introduction of antimicrobial treatment, resistance has rapidly emerged to sulphonamides, penicillins, tetracyclines, macrolides, fluoroquinolones, and early-generation cephalosporins. Currently, in most countries, the injectable extended-spectrum cephalosporin (ESC) ceftriaxone is the only remaining empiric monotherapy for gonorrhea. However, gonococcal in vitro resistance and/or treatment failures to the last-line oral ESC cefixime—and, more rarely, to ceftriaxone—have been verified in many countries [Widespread antimicrobial resistance (AMR) in highly variable strains of ountries –8. Conseountries –13.In 2016, the United Nations (UN) World Health Assembly endorsed the WHO global health sector strategy on sexually transmitted infections 2016–2021. One of the major targets is a 90% reduction in the incidence of gonorrhea globally ; to achiAdvocacy for increased awareness on the correct use of antibiotics among healthcare providers and the consumer, particularly in key populations, including men who have sex with men (MSM) and sex workers.Effective prevention, diagnosis, and control of gonococcal infections, using prevention messages and prevention interventions, and recommended adequate diagnosis and appropriate treatment regimens.Systematic monitoring of treatment failures by developing a standard case definition of treatment failure and protocols for verification, reporting, and management of treatment failure.Effective drug regulations and prescription policies.Strengthened AMR surveillance, especially in countries with a high burden of gonococcal infections, other sexually transmitted infections (STIs), and HIV.Capacity building to establish regional networks of laboratories to perform gonococcal culture with good-quality control mechanisms.Research into new molecular methods for monitoring and detecting AMR and development of new treatment options.Research into, and identification of, alternate effective treatment regimen(s) and vaccine(s) for gonococcal infections.https://www.cdc.gov/std/gisp/) [The WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP), a collaborative global network of regional and subregional reference laboratories, was initiated in 1990 to monitor gonococcal AMR worldwide . WHO GASd/gisp/) ,24, Canad/gisp/) , Australd/gisp/) , and Unid/gisp/) .There are significant variations between WHO regions with regard to the proportion of countries participating in the WHO GASP, which antimicrobials are monitored, the AMR testing methods used, and the level of quality assurance (QA). The methodologies used, including QA methods, are described in N. gonorrhoeae antimicrobial susceptibility to ESCs, azithromycin, and ciprofloxacin in 2009–2014 are detailed in The cumulative number of countries reporting gonococcal AMR data for any antimicrobial increased from 56 in 2009 to 77 in 2014. However, the number of countries reporting gonococcal AMR data for at least 1 antimicrobial each year declined, from 56 countries in 2009 to 52 countries in 2014. The WHO European Region  and Westn = 72), azithromycin (n = 58), and ESCs (n = 77), 97%, 81%, and 66% of countries, respectively, described resistant isolates (decreased susceptibility and resistance to ESCs were combined due to the different breakpoints used) for at least 1 year from 2009–2014  reported isolates with decreased susceptibility or resistance to ESCs. Notably, resistance level to cefixime  was substantially higher compared to ceftriaxone (intramuscular) because cefixime is less effective ,7,22,35.Twenty-three countries in the WHO European Region reported on gonococcal AMR. In the Euro-GASP ,22, cefiIn the WHO Western Pacific Region, decreased susceptibility or resistance to ceftriaxone was reported by 71% (5/7) of settings reporting ceftriaxone susceptibility data in 2014, 3 of which  reported decreased susceptibility or resistance in ≥5% of isolates. Prior to this, China, Mongolia, and Tonga have reported ≥5% of isolates with decreased susceptibility or resistance to ceftriaxone  of the countries  reporting ceftriaxone susceptibility data in 2014; 33%  reported ≥5% decreased susceptibility or resistance. From 2009 to 2014, 83% (5/6) of countries reported isolates with decreased susceptibility or resistance to ceftriaxone, and 50% (3/6) reported ≥5% decreased susceptibility or resistance.In the WHO Region of the Americas, decreased susceptibility or resistance to ceftriaxone was described in 40% of 10 reporting countries in 2014 . The leSurveillance for susceptibility to ceftriaxone, as well as other antimicrobials, is very limited in the WHO African Region and the WHO Eastern Mediterranean Region. In 2014, only 9% (4/47) of countries in the WHO African Region and only Pakistan in the Eastern Mediterranean Region reported on ceftriaxone susceptibility (4.9% decreased susceptibility or resistance). Of note, only 15% (7/47) of WHO African Region countries and 14% (3/22) of WHO Eastern Mediterranean Region countries have reported on ceftriaxone susceptibility for at least 1 year from 2009–2014. Only 2 of these countries (South Africa and Uganda) have reported any decreased susceptibility or resistance to ceftriaxone.In 2014, gonococcal susceptibility to azithromycin was surveyed in 45 WHO GASP countries. Twenty-three of 31 countries  and 35 of 45 countries  reported azithromycin-resistant isolates in 2009 and 2014, respectively.Among 23 reporting WHO European Region countries, 87% (20/23) reported azithromycin-resistant isolates, including 39% (9/23) reporting ≥5% resistance and 22% (5/23) reporting >10% resistance. Only 13% (3/23) of countries reported no azithromycin-resistant isolates in 2014; however, 1 of these countries previously reported azithromycin resistance .In the WHO Western Pacific Region, 78% (7/9) of countries reported azithromycin-resistant isolates, with 3 settings reporting ≥5% resistance , 4 countries reporting <5% resistance , and 2 countries reporting no resistance (Korea and the Philippines).In the WHO Southeast Asian Region, of the 6 countries reporting in 2014, 1 country (Indonesia) reported ≥5% resistance to azithromycin, 4 (67%) reported <5% resistance, and 1 country (Myanmar) reported no resistance (but only 4 isolates were tested).In the WHO Region of the Americas, only 2 countries  reported on azithromycin susceptibility, and all three had ≥5% resistance. Furthermore, South Africa reported ≥5% resistance in 2013. In the WHO Eastern Mediterranean Region, only Pakistan reported data regarding azithromycin susceptibility in 2014 (0% resistance).High levels of ciprofloxacin resistance have been reported in a majority of countries globally, especially in the WHO Southeast Asia Region and the Western Pacific Region. In these WHO regions, nearly all countries have reported high rates of resistance, including 9 countries reporting >90% resistance. Notably, a few WHO Western Pacific Region countries  have reported less than 2% resistance to ciprofloxacin. The majority of countries in the WHO European Region, the Region of the Americas, and the African Region have reported high resistance rates, which are still significantly lower than those in Asia .Treatment failures with cefixime have been identified in Japan since the early 2000s ,37, follThe vast majority of verified treatment failures are from well-resourced countries. Accordingly, these reports do not reflect the true global public health burden of ESC treatment failures, since surveillance data from resource-constrained settings are scarce. As emphasized by WHO ,33,54, iGonococcal AMR is a major problem globally, but the AMR situation varies in different parts of the world and changes over time. Sustained and quality-assured GASPs are essential but are very challenging to operate. Worryingly, gonococcal AMR surveillance remains lacking or exceedingly limited in many settings worldwide, e.g., Eastern Europe, Central Asia, parts of Latin America (including the Caribbean), the WHO Eastern Mediterranean Region, and the WHO African Region. Many of these settings also have high rates of gonorrhea, lack of  diagnosis, over-the-counter access to antimicrobials without prescription, and limited access to optimal antimicrobial treatment, such as high-quality ceftriaxone or, ideally, dual therapy with ESC plus azithromycin. These factors create the perfect conditions for rapid emergence and spread of gonococcal AMR ,6,28. AcThere are many microbiological, epidemiologic, and programmatic difficulties to the achievement of high-quality, standardized, and comparable AMR data. Between different countries and WHO regions, the number of isolates examined varies significantly. Almost half of the WHO GASP countries do not have sufficient sample sizes  to detect a 5% AMR level with high statistical confidence ,33, nor Situation analysis has revealed a lack of awareness of gonococcal AMR among policy-makers, clinicians, laboratory professionals, and patients in many settings . EngagemAdditionally, it is important to further strengthen well-established GASPs –27 by coIn many settings, there is a lack of awareness among staff of national ministries of health and among healthcare professionals that continuous AMR surveillance and/or surveys should be the foundation of a national AMR action plan, should be part of routine diagnostics and/or surveillance, and should inform refinements of the recommended treatment algorithms. In settings in which clinical practices include syndromic management of STIs, this is especially important. Accordingly, AMR surveillance is not simply a research project. Thus, in most countries, it should not require approval from an ethical committee because the collection and antimicrobial-susceptibility testing of gonococcal isolates are part of standard care, and no patient identification information is made available in AMR surveillance. Finally, there are significant delays in the release of AMR data from the current GASPs, which limits their value as part of an early warning system for AMR emergence and limits their usefulness for informing prompt refinements of gonorrhea management guidelines and public health policy. Timely reporting by GASPs will require significant improvements in the procedures for release of country-specific AMR data .Gonococcal AMR will only be effectively mitigated when the global gonorrhea burden is reduced. Improved prevention, management, and control of gonorrhea are imperative. Linking this to HIV and STI prevention in general will be essential, including education regarding symptomatic and asymptomatic STIs, promotion of safer sex behaviors including increased condom use, behavior change communication programs, enhanced partner notification and treatment, and expansion of targeted interventions for vulnerable populations .In the absence of a gonococcal vaccine, optimal public health control of gonorrhea will continue to rely on effective, accessible, affordable, and timely antimicrobial treatment in combination with prevention strategies, diagnostics , and surveillance. Appropriate gonorrhea case management is essential to reduce unnecessary or incorrect antimicrobial treatment and development of AMR. In 2016, WHO published new guidelines for the treatment of gonorrhea , based oDuring recent years, significant progress has been made in the understanding of mechanisms of gonococcal pathogenesis and molecular and phenotypic AMR determinants, and the development of new drugs and diagnostic technologies –8,57–59,Research regarding improved prevention should also be promoted. One new approach includes the use of antiseptic mouthwash against gonococci in pharyngeal infection . ApproprFurthermore, several novel gonorrhea antimicrobials are in the advanced stages of clinical trial evaluation, including solithromycin (CEM-101), gepotidacin (GSK2140944), and zoliflodacin (AZD0914/ETX0914). Several additional new antimicrobials have shown potent in vitro activity against gonococci, but clinical data are lacking. Several older antimicrobials such as spectinomycin, gentamicin, ertapenem, and fosfomycin have also been suggested for future treatment of gonorrhea –8. SinceUse of newer molecular diagnostic technologies can also reduce inappropriate treatment and slow the spread of AMR. Additional research is imperative to identify new AMR determinants  and to evaluate how molecular AMR assays can supplement phenotypic AMR surveillance, for example, by significantly increasing the sample size in surveillance. Ultimately, novel rapid molecular point-of-care gonococcal tests, which include AMR prediction, will guide personalized treatment at the first health-care visit ,57–59. Thttp://www.who.int/antimicrobial-resistance/global-action-plan/surveillance/glass/en/), to conduct regular systematic reviews, and to update treatment guidelines more rapidly. Furthermore, work is ongoing internationally to improve the regulation of drugs and prescription policies and to increase awareness about the correct use of antimicrobials. At the national level, countries need to strengthen and support their gonococcal AMR surveillance programs in the context of national AMR programs. Countries in less-resourced settings should be supported technically and funded appropriately to develop and/or enhance their national GASP and ensure timely dissemination of data as a basis for the revision of treatment guidelines and public health action. Consistent national and international financial and political commitment is urgently needed.Gonococcal AMR is a major concern that threatens our ability to treat and control gonorrhea, and its complications and sequelae, globally. WHO GASP, in collaboration with other GASPs, monitors gonococcal AMR worldwide. However, GASPs need to be substantially strengthened, especially in many less-resourced settings in Eastern Europe, Central Asia, parts of Latin America (including the Caribbean), the WHO Eastern Mediterranean Region and the WHO African Region. Building national leadership and commitment—both political and financial—to address gonococcal AMR is essential. In general, there is a need for greater involvement and increasing country-level ownership through advocacy, capacity-building, strengthened communications, and linkages with broader interventions to control AMR. Efforts are underway to ensure that gonococcal AMR is addressed in the global AMR action plan  and in thttp://www.dndi.org/diseases-projects/gardp/). In conjunction with the development of novel antimicrobials, strategies to conserve these new antimicrobials while making them accessible should be implemented.The development of novel antimicrobials for treatment of gonorrhea should be the highest priority. To facilitate the development, clinical evaluation, and registration of new antimicrobials and therapeutic regimens for gonorrhea, WHO has liaised with the Global Antibiotic Research and Development Partnership , partner management (notification and treatment), development of new diagnostics , and novel treatment options for gonorrhea should be given the greatest attention in order to achieve a 90% reduction of gonorrhea incidence by 2030, relative to the incidence in 2018 .S1 Table(DOCX)Click here for additional data file.S2 TableThe WHO regions are ordered based on estimated gonorrhea burden .(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Clozapine is the most effective anti-psychotic for treatment refractory schizophrenia, but causes significant metabolic disturbances including obesity and type 2 diabetes. The metabolic adverse reactions may be mediated in part by clozapine induced dysregulation of Glucagon-like-peptide-1 (GLP-1). GLP-1 is an intestinal epithelial derived peptide, released with ingestion of food, that triggers satiety, reduces glucagon production, promotes insulin production and slows gut motility. Clozapine has been shown to interfere with GLP-1 function in animal models, leading to metabolic dyregulation including obesity and preference for high calorie meals. Administration of exogenous GLP-1 agonists such as exenatide to animals have been shown to counter this effect of clozapine. Exenatide subcutaneous weekly injections may assist obese people on clozapine lose weight.This randomised, controlled, open-label, pilot trial aimed to evaluate the effect of exenatide on weight loss among clozapine-treated obese adults who have schizophrenia, with or without stable diabetes. Twenty-eight out-patients were randomised to once weekly extended release sub-cutaneous exenatide or treatment as usual for 24 weeks. This trial examined the safety, tolerability and acceptability of exenatide among obese people with schizophrenia on clozapine, with an evaluation of change in weight, glycaemic control, psychosis severity and metabolic parameters.All 28 participants completed the study. (Exenatide=14 (3 T2DM), control=14 (2 T2DM)). Six people on exenatide achieved >5% weight loss, compared to only 1 control (p=0.029). Mean weight loss was greater for exenatide than control at week 24  as were. BMI (-1.78 vs -0.39 p=0.019), fasting glucose  and HbA1c . There was no significant difference for other metabolic syndrome components. There were higher rates of transient nausea (n=8), vomiting (n=7) and diarrhoea (n=7) in the exenatide group.Exenatide is a promising therapeutic agent for glycaemic control and weight loss in clozapine-treated people with obesity. These results suggest good tolerability and a consistent and favourable pattern of weight loss effects with exenatide. GLP-1 agonists could assist in reducing the cardio-metabolic associated morbidity and mortality secondary to clozapine.


Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-κB pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the IκB kinase (IKK) kinase, IKKγ (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIP–IKKγ interaction both in cells and IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-κB pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKKγ. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKKγ is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP–IKKγ complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions. Kaposi's sarcoma-associated herpesvirus (KSHV) is the most common cause of malignancy among AIDS patients , 2. In aAlthough persistent activation of both the canonical and alternative NF-κB pathways by KSHV have been documented, its effects on the canonical pathway have been the most extensively characterized. Key to activation are the p65(RelA)/p50 or p65/c-Rel heterodimeric transcription factors –15. Thes–S-transferase (GST)-peptide fusion protein (GST-IKKγ) not only are competitive inhibitors of IKKγ in vitro but also, when combined with etoposide (ETO) or tumor necrosis factor alpha (TNF-α), drastically reduce the viability of BC3 and BCP-1 cells. This is a direct consequence of removing the apoptotic block imposed by the ks-vFLIP-mediated overproduction of prosurvival/antiapoptotic proteins, in which there appears to be little coinvolvement of ATG3-dependent autophagic and no involvement of important death receptor-mediated pathways. Our findings not only demonstrate ks-vFLIP as an amenable target for the treatment of PEL and other KSHV-related malignancies using gene therapies but also suggest that stapled peptides/helical mimetics could form a basis for the design of future high-potency therapeutics.In previous studies, the crystal structure of ks-vFLIP bound to IKKγ (PDB code 3CL3) was determined . PivotalAlthough it has been established that dysregulated activation of the canonical NF-κB pathway by ks-vFLIP renders mouse embryonic fibroblasts resistant to apoptosis induced by TNF-α , we sougin vitro. The original ks-vFLIP–IKKγ structure (PDB code 3CL3) adopts a heterotetrameric configuration in which a ks-vFLIP dimer associates with IKKγ in a dimeric, parallel, helical coiled-coil conformation -2-(2′ pentenyl)alanine that are subsequently cyclized to produce a constraining aliphatic linker. Such peptides have been frequently found to have enhanced affinity for their targets as well as innate cell permeability, thought to partially derive from the hydrophobic nature of the linker and favored by an overall net positive charge.The fact that we were able to at least partially substitute IKKγ for unmodified peptides led us to investigate whether the IKKγ ks-vFLIP minimal interacting motif could be engineered into a conformationally constrained, helical peptide through incorporation of an appropriate aliphatic linker or “staple” . This st23–in silico, a model peptide was constructed of the IKKγ minimal interacting motif (L230 to S248) incorporating the staple and energy minimized (MMFF94x force field) using the Molecular Operating Environment (MOE) software package . Following minimization, the helical geometry and overall configuration of the peptide were observed to remain unchanged. The model was then docked into cleft 1 of ks-vFLIP and the complex subjected to energy minimization in which the position of the protein moiety was fixed while permitting movement of the peptide. The resulting energy-minimized complex revealed that all of the important interactions remained unperturbed. Based on these findings, the modified peptide (spIKKγ  ∼ 75 μM after 24 h). Either TNF-α or ETO caused a striking enhancement of spIKKγ-induced BC3 cell death , both GST-IKKγ and spIKKγ sensitize PEL to ETO and TNF-α when ks-vFLIP is expressed.Although spIKKγ is able to directly instigate BCP-1 and BC3 cell killing, albeit at moderate concentrations  and grown overnight at 37°C. These cultures were used to inoculate 2-liter flasks containing 800 ml of 100-μg ml−1 ampicillin-supplemented LB medium and grown to an optical density at 600 nm [OD600] of 0.6 to 0.8. The flasks were subsequently induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and the temperature was reduced to 25°C overnight. The cultures were spun at 5,000 × g for 20 min and pellets stored at −80°C or resuspended in lysis buffer (20 mM Tris-HCl [pH 8.5] and 200 mM NaCl [buffer A] plus 25 mM imidazole) and subjected to two rounds of lysis using a Vibra cell sonicator. The resulting lysates were spun at 46,000 × g and supernatants passed through a 1.2-μm filter prior to being loaded onto a 5-ml His-trap column preequilibrated in buffer A. The column was washed with 20 column volumes (CV) of buffer A and GB1–ks-vFLIP eluted in buffer B (buffer A plus 500 mM imidazole) using a 5-CV step gradient. The resulting protein was then concentrated in a 10-kDa-cutoff Vivaspin concentrator and applied to a Superdex 26/60 S75 size exclusion column preequilibrated with buffer A supplemented with 250 mM imidazole and 5 mM dithiothreitol (DTT) {or 0.3 mM Tris(2-carboxyethylphosphine hydrochloride) [TCEP]}. The eluted fractions were analyzed using SDS-PAGE, and those found to contain GB1–ks-vFLIP at a purity of >95% were pooled, concentrated to 100 μM using a 10-kDa-cutoff Vivaspin concentrator, and stored at 4°C. Fractions used for crystallization trials were dialyzed into a buffer comprising 20 mM Tris-HCl (pH 8.5), 50 mM glycine, and 10 mM TCEP-HCl prior to setup.ks-vFLIP (residues 1 to 188) was PCR cloned into the EcoRI and XhoI sites of GB1-pBR22b  using the primers GB1vFLIP-Fwd and GB1vFLIP-Rev . The resDetails for the production and purification of IKKγ(150–272) and ks-vFLIP(1–178) have been previously reported by Bagnéris et al. .In order to express the IKKγ fusion peptides in Jurkat cells, a GST fusion protein was produced by first PCR amplifying the GST coding region of pGEX-4T1 using Phusion high-fidelity polymerase (New England BioLabs [NEB]) and the primers GST-Fwd and GST-Rev . The PCRLentiviral vectors expressing GST or GST fusion peptides together with green fluorescent protein (GFP), CherryFP, WT vFLIP, or vFLIPA57L (a mutant which fails to bind IKKγ) were produced as previously described . These wBC3, BCP-1, and DG75 cells were purchased for this study from the American Type Culture Collection (ATCC). They were verified as human B cell lines by surface staining for human Ig.The transfected cells, the human B cell lines BC3, BCP-1, and DG75 (a PEL cell line negative for KSHV and Epstein-Barr virus [EBV]) were treated with human TNF-α (PeproTech or Alomone Labs), ETO (Sigma), spIKKγ  system (Bio-Rad) in a 96-well plate format. Trials were performed in which 30 μM ks-vFLIP (unless otherwise stated) was combined with either IKKγ or mimetic peptides (at the specified concentrations) prior to the addition of SYPRO orange diluted in a 1:1,000 (vol/vol) ratio and thermal denaturation. The data were subsequently processed using the Bio-Rad iQ5 software. Each experiment was repeated at least 3 times. All peptides were purchased from Eurogentec.12 produced the most significant improvements and was subsequently included in repeat trials of the original condition at a concentration of 0.1 to 0.2%.spIKKγ (Eurogentec) was first dissolved in 100% dimethyl sulfoxide (DMSO) to a concentration of 2 mM. A 1:1 ratio of GB1–ks-vFLIP to spIKKγ was next combined to give a final GB1–ks-vFLIP concentration of 50 to 70 μM. A Mosquito crystallization robot was used to set up vapor diffusion crystallization trials involving a range of commercially available screens. Drops of 200 nl  were dispensed in a 1:1 ratio of complex to well solution, and after 1 week, small crystals were observed under the condition 1.2 M ammonium sulfate, 0.05 M trisodium citrate, and 3% isopropanol. In attempts to improve their size and diffraction quality, the Silver Bullet additive screen (Hampton Research) was used in conjunction with the above-described condition. Of the various additives, vitamin B12, were cryoprotected in 25 to 30% glycerol and flash frozen in liquid nitrogen prior to irradiation at SOLEIL on the Proxima 1 beamline. Data were collected to a maximum resolution of 3.3 Å on two crystals that were integrated and scaled using XDS . The effective resolution of the corrected data was reduced to 3.38 Å . The stdK values comparable to those obtained using deionized water were observed  experiments were performed using a Microcal VP-ITC micocalorimeter . For all binding reactions, syringe concentrations varied between ∼85 and 100 μM for IKKγ(150–272) and cell concentrations between ∼8 and 10 μM for GB1-vFLIP. Titrations consisted of an initial 2-μl injection, followed by 20 10-μl injections with stirring at 300 rpm. In competition assays, GB1–ks-vFLIP was first incubated with 20 μM spIKKγ and DMSO to 4% prior to loading into the cell and titration with IKKγ(150–272). All ITC binding experiments were performed in deionized water at 25°C owing to the buffer constraints of spIKKγ, GB1–ks-vFLIP, and IKKγ(150–272), where GB1–ks-vFLIP and IKKγ(150–272) were buffer matched by overnight dialysis, followed by the addition of 4% DMSO to each. It was confirmed that the use of deionized water and 4% DMSO had no discernible impact on structural integrity and/or the affinity of IKKγ(150–272) for GB1–ks-vFLIP by parallel ITC experiments in which both proteins were alternatively dialyzed into a buffer comprising 20 mM Tris-HCl (pH 8.5), 200 mM NaCl, and 0.3 mM TCEP. In vitro IKK kinase assays were performed as previously described  or normal rabbit serum as a control. Lysates and immunoprecipitates were blotted using anti-ks-vFLIP antibodies . In vitrescribed .5LDE.The coordinates determined in this study have been deposited at the Protein Data Bank under accession code


The prefrontal cortex (PFC) is indispensable for several higher-order cognitive and executive capacities of primates, including representation of salient stimuli in working memory (WM), maintenance of cognitive task set, inhibition of inappropriate responses and rule-guided flexible behavior. PFC networks are subject to robust neuromodulation from ascending catecholaminergic systems. Disruption of these systems in PFC has been implicated in cognitive deficits associated with several neuropsychiatric disorders. Over the past four decades, a considerable body of work has examined the influence of dopamine on macaque PFC activity representing spatial WM. There has also been burgeoning interest in neuromodulation of PFC circuits involved in other cognitive functions of PFC, including representation of rules to guide flexible behavior. Here, we review recent neuropharmacological investigations conducted in our laboratory and others of the role of PFC dopamine receptors in regulating rule-guided behavior in non-human primates. Employing iontophoresis, we examined the effects of local manipulation of dopaminergic subtypes on neuronal activity during performance of rule-guided pro- and antisaccades, an experimental paradigm sensitive to PFC integrity, wherein deficits in performance are reliably observed in many neuropsychiatric disorders. We found dissociable effects of dopamine receptors on neuronal activity for rule representation and oculomotor responses and discuss these findings in the context of prior studies that have examined the role of dopamine in spatial delayed response tasks, attention, target selection, abstract rules, visuomotor learning and reward. The findings we describe here highlight the common features, as well as heterogeneity and context dependence of dopaminergic neuromodulation in regulating the efficacy of cognitive functions of PFC in health and disease. The prefrontal cortex (PFC) is a critical node in brain networks involved in complex cognitive functions and has undergone a great evolutionary expansion in primates Wise, . PFC dysThe PFC is subject to substantial neuromodulation from all major ascending modulatory systems  family comprising of D1 and D5 receptors and the Gi-coupled D2 receptor (D2R) family comprised of D2, D3 and D4 receptors  in neighboring periprincipal PFC, affecting neurons with identical delayed response activity, increased contralateral memory-guided saccade errors and reaction times (Sawaguchi and Goldman-Rakic, Another, related point of interest concerns the physiological actions of dopamine. Data gathered in macaques from a series of iontophoretic studies (Wang et al., A trivial explanation would be that these differences in behavioral consequences of D1R blockade reflect an areal difference in dopamine’s modulation of the “spotlight of attention”. Alternatively, there may be differences in dopaminergic modulation of  “immediate” vs. “delayed” selection of action targets: enhancement of response field stimulus selection could be positively modulated by D1R blockade when the stimulus is visible, whereas the introduction of a delay would cause the opposite effect, perhaps due to increases in variability of cortical representations in the intervening delay period leading to deterioration in attentional representation of the stimulus location. These divergent pharmacological effects appear to highlight the heterogeneity of D1R actions on different cortical areas. Future experiments employing similar microinjections in a task similar to Lebedev et al.  may shedDopamine has a well-established role in reward signaling in the brain (Schultz et al., gain of spatial tuning in the memory period, a phenomenon reminiscent of attentional modulation. Interestingly, Leon and Shadlen (Ventrolateral PFC neurons are modulated by reward, and spatial selectivity of neurons therein is modulated by the reward contingency (Kennerley and Wallis,  Shadlen  found th Shadlen , and the Shadlen . These f Shadlen .Two studies examined the role of PFC D1Rs and D2Rs in associative learning (Puig and Miller, Puig and Miller , 2014 alThus, injections of antagonists of both dopamine receptor families disrupted novel visuomotor learning involving associating objects with saccades in particular directions, but did not affect familiar and well-learned associations. This is interesting when considering effects of injection of these antagonists in periprincipal PFC on oculomotor delayed response performance, as discussed in the previous section (Sawaguchi and Goldman-Rakic, Another critical role that has been associated with PFC is in the maintenance of cognitive sets: learning, maintenance and application of sets or rules used to guide flexible responses to environmental stimuli (Wallis and Miller, PFC neurons maintain information about the cognitive set and the rule to be employed in a behavioral trial (Wallis et al., Recently, our group examined the influence of dopamine receptor subtypes on rule representation in PFC (Vijayraghavan et al., Using iontophoresis, we examined the effects of D1R and D2R stimulation on the activity of dorsolateral PFC neurons engaged in this task. We tested two agonists, SKF81297 and quinpirole, and examined the overall effects and dose-dependence on PFC excitability and task representation. D1R stimulation had an overall suppressive effect on the population of PFC neurons tested, while D2R stimulation had an overall excitatory effect Figure , consistOur results with dopamine receptor modulation of rule selectvity were different from those found in the study by Ott et al. . One expIn our study (Vijayraghavan et al., A previous study found that D2Rs selectively modulated the activity of neurons that were only activated during the saccade response in the spatial delayed response task (Wang et al., What then are the origins and functional role of perisaccadic activity in dorsolateral PFC, and thereby, what is the role of D2Rs in the neuromodulation of this activity?One possibility is that, D2Rs modulate the presaccadic component of perisaccadic activity that is involved in saccade target selection. PFC neurons exhibit greater representation of the contralateral visual space in their stimulus-driven activity (Funahashi et al., In this review, we have described the gamut of evidence about the role that dopaminergic neuromodulatory systems play in regulating the cognitive functions of PFC in primates. While much work has been done over the past four decades on involvement of dopamine signaling in shaping PFC cognitive circuitry during delayed response task performance, new studies performed over the past decade have begun to explore the role that dopamine plays in other cognitive and executive functions in which the PFC is intimately involved, including spatial attention, maintenance and flexible switching of cognitive sets, reward and feedback representation. While consensus is emerging about the direct physiological effects of dopamine receptor subtypes in the PFC of awake behaving primates, the complexity of dopamine’s actions on PFC task activations point to heterogeneity in the functions of cortical dopamine. Further work examining potential links between what is known about dopamine’s role in reward and reinforcement learning and its modulation of “states” of activity in well-learned tasks are needed. Such studies will be of considerable significance, given the interest in dopaminergic pharmacology in the development of treatments for enhancing cognitive outcomes in patients with neuropsychiatric diseases.SV and SE designed the experiments. SV and AJM conducted experiments and analyzed data. SV, AJM and SE wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


X-ray scattering (XRD), differential scanning calorimetry (DSC), and the tensile test were used to characterize the microstructure and performances of the nanofibers. The orientation and crystallinity of the as-spun and hot-stretched nanofibers confirmed by X-ray have increased. Differential scanning calorimetry showed that the glass transition temperature of PAN increased about 3 °C by an addition of 0.75 wt% SWNTs indicating a strong interfacial interaction between PAN and SWNTs. The tensile strength and the modulus of the nanofibers increased revealing significant load transfer across the nanotube-matrix interface. For PAN nanofibers, the improved fiber alignment, orientation and crystallinity resulted in enhanced mechanical properties, such as the tensile strength and modulus of the nanofibers. It was concluded that the hot-stretched nanofiber and the PAN/SWNTs nanofibers can be used as a potential precursor to produce high-performance nanocomposites.Partially aligned polyacrylonitrile (PAN)-based nanofibers were electrospun from PAN and PAN/single-walled carbon nanotubes (SWNTs) in a solution of dimethylformamide (DMF) to make the nanofiber composites. The as-spun nanofibers were then hot-stretched in the oven to enhance its orientation and crystallinity. With the introduction of SWNTs and by the hot-stretched process, the mechanical properties will be enhanced correspondingly. Scanning electron microscopy (SEM), transmission electron microscopy (TEM),  Carbon nanotubes (CNTs) have an intriguing application due to their appealing mechanical ,2, electPolyacrylonitrile (PAN) nanofiber is a common precursor of general carbon fibers. However, these applications of PAN nanofibers may be hindered by the poor strength, attributed to their small diameters and unoptimized molecular orientation and crystallinity in the fibers . An alteTo enhance the strength of the nanofibers, the inorganic single-walled carbon nanotubes (SWNTs) has been considered as the ideal reinforced materials due to their excellent mechanical properties, good electrical and thermal conductivity . Also, sN, N-dimethylformamide . In order to disperse the SWNTs in the organic polymer matrix in uniformity, the SWNTs were modified to form an individually polymer-wrapped structure [PAN used in this study included PAN/methyl acrylate/itaconic acid (93:5.3:1.7 w/w)  which was purchased from UK Courtaulds Ltd. The solvent was tructure . As is wtructure .A 0.75 wt% SWNTs based PAN composite solution was prepared as follows. A given weight of SWNTs was first dispersed for 2 h in DMF through mild bath sonication , which was followed by the addition of PAN (128.91 mg per milliliter of SWNTs/DMF solution). The mixture was then mechanically stirred overnight at 40 °C using a magnetic stirrer to yield a homogeneous solution. For comparison, a pure PAN solution was prepared by a similar method (without the addition of SWNTs).Then relatively aligned PAN nanofibers and PAN/SWNTs composite nanofibers were obtained by electrospinning. The voltage between the electrode and the counter electrode could be controlled by the high voltage power supply which was set at 14–16 kV. The 0.16 m perimeter collector rotated at a surface speed about 6.6 m/s, that the high speed rotating collector could align the nanofibers into the nanofiber sheets.During the electrospinning process, however, the whirlpool jet from the pinhead to the collector made it difficult to get unidirectional alignment in a large-area sheet . TherefoThe morphologies and diameters of the as-spun and hot-stretched PAN nanofibers and PAN/SWNTs composite nanofibers were observed by scanning electron microscopy . The diameters of electrospun nanofibers were analyzed with image analyzer software (Image J).The transmission electron microscopy (transmission electron microscopy (TEM), HITACHI H-800) was used to characterize the orientation of SWNTs in nanofibers, which were directly electrospun on a TEM-copper-grid.X-ray diffractometer , operated at 40 kV and 200 mA to produce CuKα radiation (λ = 1.54 Å). The percent crystallinity (PC) was obtained by extrapolation of the crystalline and amorphous parts of the diffraction pattern. The crystallite size was calculated by using the formula cL = kλ/(βcosθ), with k = 0.89, λ = 1.54 Å, β is FWHM, θ is Bragg angle [The crystallinities of the as-spun and hot-stretched PAN nanofiber were investigated with gg angle .X-ray diffractometer . The Herman’s orientation factor, f, was determined from the fully corrected azimuthal intensity distribution diffracted from the (100) reflection at d ≈ 5.30 Å. φ is the azimuthal angle between the axis of the molecular segment and of the fiber and I is the scattering intensity of the (100) reflection at that angle [The orientation of the as-spun and hot-stretched PAN nanofibers was also examined by at angle .gT of the PAN nanofiber and PAN/SWNTs nanofiber were examined using Differential scanning calorimetry . The samples were heated at a scanning rate of 20 °C/min under nitrogen atmosphere in order to diminish oxidation. The value of gT was found by differentiating the heat flow curve with the temperature.The Mechanical property testing was performed by using a LR30K Electromechanical Universal Testing Machine (LLOYD Company). There were eight specimens used for each nanofiber sample in the tensile test. The samples were prepared in 5 mm width and 20 mm length. The tensile speed in the mechanical test was 20 mm/min. The cross section areas of the samples were calculated via the weights of the samples and the densities of PAN and SWNTs. The average value of the experimental data for each specimen was selected. The tensile strength, tensile modulus, and elongation at break were obtained from the stress-strain curves.Relatively well-aligned PAN nanofibers were obtained by electrospinning with the drum rotating at high-speeds. However, the whirlpool jet from the pinhead to the collector made it difficult to get much alignment in a large-area sheet, making the subsequent hot-stretched procedure particularly useful.In order to demonstrate that the prepared nanofibers did contain some oriented SWNTs, TEM was utilized to view the alignment and orientation of SWNTs within the nanofibers produced. As seen by TEM in X-ray diffraction (XRD) patterns from the as-spun and hot-stretched PAN nanofibers respectively. Noted that the X-ray beam is directed perpendicular to normal of the nanofibers, and thus the beam is also parallel to the winding direction of the nanofibers. The nanofibers were highly oriented along the winding direction of the rotating drum collector.θ = 29.5° corresponding to a spacing of d ≈ 3.03 Å from the (110) reflection and another was at 2θ = 17.0° corresponding to a spacing of d ≈ 5.30 Å from the (100) reflection. The ratio of the d-spacing of these two peaks (1.74) is very close to √3:1, indicating hexagonal packing of the rod-like PAN chains [The diffraction pattern showed two equatorial peaks which one was at 2θ at 17.0°. This indicated that electrospinning of the nanofibers onto a rotating drum generated limited crystallinity. In contrast, the hot-stretched nanofibers showed two diffraction peaks indexed with values of 2θ of 17.0° and 29.5°. And it also can be found that the peak at 2θ = 29.5° became much bigger after the process of hot-stretched.The diffraction pattern of the as-spun nanofiber showed the weak peak with the value of 2f, the average crystallite sizes for PAN nanofibers. The percent crystallinity of the hot-stretched nanofiber increased about three times in comparison with those of as-spun nanofiber. The orientation factor, f, increased from 0.22 to 0.76 after the hot-stretched process. The crystallite size also increased about 162%, indicating highly oriented PAN nanofibers, which was in agreement with the results of the orientation factor [n factor . Fibers n factor .g)T of PAN and PAN/SWNTs nanofibers, as illustrated in gT = 102.3 °C), the gT was increased by about 3 °C by incorporating only 0.75 wt% SWNTs into the PAN matrix (gT = 105.4 °C). Increase in the glass transition temperature as compared to the PAN fiber provided evidence of interaction between PAN and SWNTs. The improvement in the gT stemmed from a stronger interfacial interaction and possible covalent bonding between PAN and the SWNTs. The results suggested that the mobility of PAN chains was reduced due to the constraint effect of SWNTs [Differential scanning calorimetry curves showed the glass transitions (of SWNTs .In this work, a better representation of the nanocomposites characteristics was attempted by measuring the macroscopic nanofiber sheets. Stretching a piece of the nanofiber sheet gives an assessment of the average mechanical properties of the nanofibers rather than measuring an individual segment of a nanofiber composite .The stress-strain curves of PAN nanofibers, before and after hot-stretched respectively, are presented in The stress-strain curves of PAN/SWNTs nanofibers, before and after being hot-stretched, are presented in In this study, PAN nanofibers and PAN/SWNTs composite nanofibers were prepared by electrospinning from PAN/DMF solution. Hot-stretched method was used to increase the degree of crystallinity and molecular orientation of PAN nanofibers and PAN/SWNTs composite nanofibers. Such PAN/SWNTs composite nanofiber sheets represented an important step toward utilizing carbon nanotubes in materials to achieve remarkably enhanced physical properties. An addition of only 0.75 wt% SWNTs to PAN increased the polymer mechanical properties significantly. TEM results showed that SWNTs had a high orientation in PAN/SWNTs composite nanofibers. Compared to pure PAN nanofibers, tensile strength and Young’s modulus of the hot-stretched nanofibers exhibit considerable improvement. The improvement of orientation and crystallinity, the better PAN nanofibers alignment, are all contributed to the obvious increases of mechanical properties of the nanofibers. Thus, the composite nanofibers with the component of SWNTs and the hot-stretched nanofibers can be used as the potential precursor to produce high-performance carbon nanofibers. The mechanical properties of the PAN nanofibers and PAN/SWNTs composite nanofibers can be improved more by extensive studies of electrospinning and the hot-stretching process.


The most common chromosomal abnormality due to non-obstructive azoospermia (NOA) is Klinefelter syndrome (KS) which occurs in 1-1.72 out of 500-1000male infants. The probability of retrieving sperm as the outcome could be asymmetricallydifferent between patients with and without KS, therefore logistic regression analysis isnot a well-qualified test for this type of data. This study has been designed to evaluateskewed regression model analysis for data collected from microsurgical testicular spermextraction (micro-TESE) among azoospermic patients with and without non-mosaic KSsyndrome.This cohort study compared the micro-TESE outcome between134 men with classic KS and 537 men with NOA and normal karyotype who were referred to Royan Institute between 2009 and 2011. In addition to our main outcome, whichwas sperm retrieval, we also used logistic and skewed regression analyses to compare thefollowing demographic and hormonal factors: age, level of follicle stimulating hormone(FSH), luteinizing hormone (LH), and testosterone between the two groups.A comparison of the micro-TESE between the KS and control groups showeda success rate of 28.4% (38/134) for the KS group and 22.2% (119/537) for the controlgroup. In the KS group, a significantly difference (P<0.001) existed between testosterone levels for the successful sperm retrieval group (3.4 ± 0.48 mg/mL) compared to theunsuccessful sperm retrieval group (2.33 ± 0.23 mg/mL). The index for quasi Akaikeinformation criterion (QAIC) had a goodness of fit of 74 for the skewed model which waslower than logistic regression (QAIC=85).According to the results, skewed regression is more efficient in estimatingsperm retrieval success when the data from patients with KS are analyzed. This findingshould be investigated by conducting additional studies with different data structures. The most common chromosomal abnormalitydue to non-obstructive azoospermia (NOA)is Klinefelter syndrome (KS) which occurs in1-1.72 out of 500-1000 male infants . ThisgeIt is important to evaluate the chances forsperm retrieval among KS patients and comparethem to azoospermia with normal karyotype byusing a model which provides more efficientresults for researchers. When researchers havemore efficient results they can choose the bestmethod to handle this problem and patients maynot need to undergo invasive surgeries . Onthe This cohort study enrolled 134 patients with KSand 537 patients without KS. Patients were referredto Royan Institute, a referral infertility clinic in Tehran,Iran between 2009 and 2011. We individuallymatched patients and controls according to disease duration,time of surgery, and surgeon in order to avoidconfounding factors such as surgical skills and age.The 47,XXY karyotype was confirmed by Gbandingof more than 30 peripheral blood lymphocytes.Semen analysis was performed accordingto World Health Organization (WHO) guidelinesto evaluate sperm parameters. At least two analysesconfirmed azoospermia in each patient. Bloodsamples were taken in the morning to measureFSH, LH, and testosterone levels.Microsurgical testicular sperm extraction(Micro-TESE) was performed on the patients undergeneral anesthesia as described by Schlegel .The pro1, y2, …,yn are a set of nobservations of binaryoutcomes such as successand failure in sperm retrieval, which are independentof each other; p_i is the probability of successforpatient “i”; x1, x2,…, xn are independent predictors;and β1, β2, …, βk are equal to regression coefficients,then, the binary regression model can bewritten as follows:The binary logistic regression model, as previouslyintroduced , is apprwhere: F(.) is acumulative distribution function.If F is the distribution function of normal and logistic,then the above model will be alogistic andprobit regression model, respectively.it"  using the BB package and GEE pack for asymmetric and symmetric models, respectively. The logistic regression model with the symmetric and asymmetric link was fit to the data and we compared the goodness of fit of the two models by quasi Akaike’s information criteria (QAIC) . The levThe mean age ± SD of patients with KS was 32.64 ± 0.64 years (range: 22-49) and the mean age of participants without the KS was 34.11 ± 0.27 years. The mean serum level for FSH was 34.5 mlU/m Lin KS patients and 22.6 mlU/mL in those without KS. Patients with KS had a mean serum testosterone level of 2.65 ng/mL; those without KS had a mean testosterone level of 4.04 ng/mL. Of 134 patients with KS, 38 patients had sperm retrieved; out of 537 participants, 119 had sperm retrieved. The sperm retrieval rate was 28.4 in KS patients and 22.2 in patients without KS .We performed a marginal effect logistic regression analysis with serum FSH, LH, testosterone, age, and patient groups to determine an association between the probability of sperm retrieval and the covariates during micro-TESE. Adjusted association from the model showed that probability of retrieving sperm during micro-TESE did not differ between the two groups (control and KS) after adjusting for the covariates and could not be predicted by any of the variables. On the other hand, the results obtained from skewed logistic regression showed that the probability of retrieving sperm was not the same for patients with and without KS (P=0.01). QAIC for logistic regression with the symmetric link was equal to 85, whereas for an asymmetric link it was 74. The results of comparing the regression model are shown in Table 2. The proportion of sperm retrieval was almost equal between patients with and without KS.The present study has compared sperm retrieval in azoospermia patients with and without KS by two regression models. Sperm from KS patients is typically retrieved by conventional TESE and micro-TESE techniques. Although both techniques have successfully retrieved sperm in 44% of patients, a comparison between conventional TESE and micro-TESE showed that micro-TESE had a higher success rate (55%) compared to conventional TESE (44%) . Sperm rIn some studies it was found that the sperm recovery rate in men with KS reduced with increased age, however there were no effects on serum FSH, LH, or testosterone levels -25. MediWe used logistic regression with symmetric and asymmetric links for data analysis. To the best of our knowledge, this was the first study that applied a regression model with asymmetric link in reproduction research and in this group of infertile patients. The incorrect use of asymmetric link instead of an asymmetric link could lead to a poor fit and result in biased estimates of the regression coefficients. Chen’s model that has been used in this study was more flexible than common models. Therefore, the QAIC value was lower and has shown a better fit than logistic regression with a symmetric link , 28. In Our findings have revealed that the logistic regression model with an asymmetric link is more flexible and a better fit than the conventional regression model. Since this is the first time a skewed regression has modeled data from infertility studies, we recommend that additional studies and analyses be conducted to evaluate how well this model fits a set of observations. It is also should be notice, sperm could be retrieve of non-mosaic KS as well as without KS patients.


Chitin nanofibers were isolated from the cell walls of five different types of mushrooms by the removal of glucans, minerals, and proteins, followed by a simple grinding treatment under acidic conditions. The Chitin nanofibers thus obtained have a uniform structure and a long fiber length. The width of the nanofibers depended on the type of mushrooms and varied in the range 20 to 28 nm. The Chitin nanofibers were characterized by elemental analyses, FT-IR spectra, and X-ray diffraction profiles. The results showed that the α-chitin crystal structure was maintained and glucans remained on the nanofiber surface. Chitin occurs mainly in the exoskeleton of shellfish and insects and the cell wall of fungi . Chitin β-1,3 glucans as shown in Recently, we have succeeded in isolating chitin nanofibers from the exoskeleton of both crab and prawn ,5,6. Chiβ-1,3/1,6-glucans lie on the wall that connects the cells together since glucan mucilage is poorly organized while α-1,3 glucans serve as an amorphous matrix which interdigitates with the inner side of the β-1,3-glucan layer. The component β-1,3-glucans are embedded in crystalline chitin nanofibers [In this study, we also applied the isolation method of chitin nanofibers from crab and prawn shells to fruiting bodies of mushrooms. However, the morphology of a mushroom cell wall is very different from that of crab and prawn shells. Highly branched nofibers . ConsidePleurotus eryngii, Agaricus bisporus, Lentinula edodes, Grifola frondosa, and Hypsizygus marmoreus widely used for human consumption were selected to isolate chitin nanofibers in this study. The chitin nanofibers were obtained from these mushrooms by a series of purification and chemical treatments described in Five different species of mushrooms: The extraction step with sodium chlorite and acetic acid can bleach the pigments in the sample. For the final stage, the sample was treated with sodium hydroxide again to eliminate and remove the residual glucans, including trace amounts of proteins. The drying process causes strong hydrogen bonding between chitin fibers after removal of matrix substances which makes it difficult to obtain chitin in the form of nanofibers . TherefoThe N and C contents of chitin nanofibers determined by elemental analysis are listed in −1), N-H stretching (3270 cm−1), amide I (1660 and 1620 cm−1), and amide II (1560 cm−1) are prominent bands observed in the FT-IR spectra of the chitin nanofibers and are similar to the commercial chitin.Hypsizygus marmoreus, which has the lowest N content , Agaricus bisporus (common mushroom), Lentinula edodes (shiitake), Grifola frondosa (maitake), and Hypsizygus marmoreus (buna-shimeji) were cultivated products of Japan. Chitin powder from crab shells was purchased from Nacalai Tesque. The other chemicals were purchased from Kanto chemical and used as received.2, and 1% (v/v) AcOH , and the mixtures were stirred at 100 °C for 1 day to bleach the pigment compositions in the sample followed by washing with distilled water. After the extraction with NaClO2, the samples were treated again with 2% (w/v) NaOH (1L) at 100 °C for 1 day to remove residual proteins and glucans. After the series of chemical treatments, the samples were filtered and washed with distilled water until the residues were neutralized. The extracted wet chitin was dispersed in water in the concentration of 1 % w/w and acetic acid was added to adjust to pH3. The sample was passed through a grinder  at 1500 rpm. Grinder treatment was performed with a clearance gauge of −1.5 (corresponding to a 0.15 mm shift) from the zero position. The position was determined as the point of slight contact between the two grinding stones. The yields of chitin nanofibers thus obtained were calculated based on the dry weight of each species of mushroom chitin nanofibers and the results are listed in The five different types of mushroom species were purified to extract the chitin component by a series of chemical treatments based on the method described previously . AccordiI110 − Iam) × 100/I110, where I110 is the maximum intensity of I110, and Iam is the intensity of the amorphous diffraction at 16° [Infrared spectra of the samples were recorded using potassium bromide pellets with an FT-IR spectrometer . X-ray diffraction profiles of the nanofibers were obtained with Ni-filtered CuKα from an X-ray generator  operating at 40 kV and 30 mA. The diffraction profile was detected using an X-ray goniometer scanning from 5° to 40°. The crystalline index (CI) was determined by the following equation: CI = (n at 16° . The preChitin nanofibers were prepared from the cell wall of a number of edible mushrooms by a series of chemical and grinding treatments. The chitin nanofibers thus obtained have uniform structures. Depending on the species of mushroom, the width of chitin nanofibers varied in the range 20–28 nm. The simple method developed enabled the manufacture of homogeneous chitin nanofibers which are important dietary fibers. The dietary nanofibers thus obtained had very long fiber length, high dispersibility in water, and high viscosity. It was noticed that the chitin nanofibers formed a complex with several glucans on the surface of the fibers. As polysaccharides, the chitin nanofibers with special physical properties can have antitumor application and immuno-modulating activity. With the development of our mushroom nanofiber research, in addition to crab and prawn shell nanofibers, the mushroom nanofibers have now been added to the list as functional food ingredients as well as useful for medical applications.


During minimally invasive pulmonary resection, both limited visualization and tactile feedback can make localization of pulmonary nodules and assessment for synchronous disease challenging. Intraoperative molecular imaging is an emerging technology that can enhance a surgeon’s ability to detect cancers at the time of resection.In this report, we describe the application of a folate receptor-targeted, near infrared optical contrast agent (OTL38) for the detection of an invasive pulmonary adenocarcinoma. During molecular imaging, an otherwise undetectable synchronous nodule was also identified. This finding resulted in intraoperative upstaging and operative plan modifications.This report marks the first successful utilization of a targeted, near infrared intraoperative molecular imaging probe useful for thoracic malignancies. This rapidly evolving technology may enhance the surgeon’s ability to perform a number of oncologic procedures including tumor localization, margin assessment and intraoperative staging.The online version of this article (10.1186/s13019-017-0664-7) contains supplementary material, which is available to authorized users. During minimally invasive pulmonary resection, both limited visualization and tactile feedback can make localization of pulmonary nodules and synchronous disease challenging. Current techniques to improve intraoperative detection include intraoperative ultrasound, radionucleotide imaging, and CT-guided or spiral wire localization . There a. This second area was not concerning on preoperative imaging and displayed no other obvious visual or palpable irregularities intraoperatively. The identified synchronous left lower lobe lesion was wedge resected using real-time fluorescence guidance. Frozen section analysis revealed a 0·4 cm invasive pulmonary adenocarcinoma, and final pathology confirmed folate receptor-alpha expression. Due to identification of this synchronous adenocarcinoma, the left upper lobe nodule wedge resected rather than removed by lobectomy as originally planned. In addition to intraoperative plan modification, identification of the occult left lower lobe adenocarcinoma upstaged the disease and the subject thus received systemic platinum-based chemotherapy following resection. After one year of follow-up, the patient has had no evidence of disease recurrence or drug toxicity.A 72-year-old female with a 60-pack-year smoking history presented with an incidentally identified 2·3 cm solitary pulmonary nodule of the left upper lobe. Preoperative PET-CT confirmed FDG uptake, no suspicious lymphadenopathy or additional pulmonary pathology was observed Fig. . Four hoAdditional file 1:Intraoperative molecular imaging allowed for identification of an additional malignant pulmonary nodule in the left lower lobe. Identification of this synchronous malignancy upstaged the patient, modified operative planning, and altered postoperative adjuvant course. (MP4 49087 kb)Intraoperative molecular imaging, also commonly referred to as fluorescence guided surgery, is a rapidly evolving technique that can enhance the surgeon’s ability to perform a number of intraoperative tasks including nodule localization, metastasis evaluation, and margin assessment. In this report, we demonstrate utility of NIR molecular imaging using the FRα-targeted contrast agent, OTL38.61H63N9Na4O17S4 ; molecular weight: 1414.42 Da) is a folate analogue conjugated to the NIR fluorescent dye, S0456 [ex 774 and λem 795) [OTL38 (chemical formula: CThis report highlights several important lessons involving molecular imaging with OTL38. First, we found molecular imaging with OTL38 feasible. To elaborate, OTL38 delivery occurs 3–6 h prior to resection and imaging, thus can be delivered in the preoperative holding area. Alternative agents are delivered days prior to resection . FurtherThis report marks the first successful utilization of a targeted, near infrared intraoperative molecular imaging probe useful for thoracic malignancies. In the future, this technology may enhance the surgeon’s ability to perform a variety of oncologic procedures including tumor localization, margin assessment and intraoperative staging.


Our consideration clearly demonstrated that the interplay between length scales of static range ordering and dynamical heterogeneities as well as internal molecular arrangement seem to be the key to understanding the molecular dynamics of different materials characterized by varying degree of disorder in the vicinity of the glass transition temperature.In this letter, we reported thorough the structural and molecular dynamics studies on 1,6-anhydro-β-D-glucose, the second compound reported so far that is capable to form rotator and supercooled liquid phases. In contrast to the data presented for ethanol, temperature dependences of structural dynamics in both phases are very comparable. On the other hand, X ray measurements revealed unusually long range ordering/correlations between molecules in the ODIC ( Tg, probed by different spectroscopies, including mechanical, dielectric or dynamic light scattering ones1235689m), is much smaller in the case of plastic crystals. The best illustration of this relationship is ethanol, the only liquid that can form PC and SL phases at the same thermodynamic conditions. Over the years, this alcohol became a canonical reference material for the discussion of the data measured for other plastic crystals. Moreover, taking into account the behavior of ethanol, low fragility of plastic crystals was related to the reduced density of minima in the potential energy. However, it is worthwhile to recall that there are some controversy concerning assignments of the main structural process observed in this compound. Consequently, previously derived conclusions might be biased.The molecular origin of the glass transition phenomenon seems to be one of the most challenging issue of condensed matter physics. The special effort is put to understand the microscopic nature of this peculiar phase transition as well as to explain enormous increase of the viscosity or relaxation times in the close vicinity of the glass transition temperature, et al. have proposed a new, alternative explanation of low fragility of plastic crystalsNc) is much weaker in such materials with respect to the ordinary glasses. What is more, it was also shown that even for the large Nc, the energy barrier for the reorientational motions is pretty low in cyclooctanols, the prototypical ODICs. They rationalized that the lattice strain, which underlined molecular correlations, decreases the effective barrier for the reorientation of the neighboring molecules. However, it should be stressed that authors compared materials differing in chemical structures and intermolecular interactions. That seems to be an important aspect preventing formulation of more general conclusion about the behavior of plastic crystals and ordinary glass formers.Very recently Michl et al.et al.14Herein, we report thorough the structural and molecular dynamics studies on 1,6-anhydro-β-D-glucose (levoglucosan), a second compound capable to form ODIC, orientational glass, supercooled liquid and ordinary glass at the same thermodynamic conditions. Herein, it should be mentioned that the molecular dynamics and calorimetric studies on this compound have been reported previously by some of usT = 163 K–298 K), in a frequency range from 10−2 to 106 Hz, using an Alpha spectrometer (Novocontrol). The sample of 99% purity was obtained from Aldrich.X-ray diffraction measurements were carried out for various forms of levoglucosan, namely polycrystalline, plastic crystal, orientational glass, liquids and glass using a laboratory X-ray Rigaku-Denki D/MAX RAPID II-R powder diffractometer attached with a rotating silver anode, an incident beam graphite monochromator and an image plate as two dimensional detector. The experimental set up and the data processing are described in details in refs et al.et al.T = 383 K upon heating of the crystal or cooling of the liquid. However, it was also concluded that it is not possible to obtain structural glass in this carbohydrate. We decided to continue their research and found that after application of very fast cooling (molten sample has been directly immersed in liquid nitrogen) levoglucosan supercools and forms a structural glass. Therefore, it is just a second compound that can be prepared as a crystal, plastic crystal, orientational glass, supercooled liquid and ordinary glass.As discussed above, Rocha θ is the scattering angle, λ is the wavelength, n indicates the number of atomic species in the investigated sample, ci is the concentration and fi is the atomic scattering factor of the ith element, respectively. The diffraction data were then converted to a real space representation in the form of the pair distribution function Q, achieved in the experiment (here 20 Å−1) and In Q range from 4 to 20 Å−1. For the levoglucosan crystal, sharp diffraction peaks are superimposed on the broad features seen for the other forms of saccharide. The long-period oscillations seen at high Q-values can be related to intra-molecular correlations in the atomic arrangement leading to very similar features of the pair distribution functions, PDFs, in the range of 0–4.5 Å. These results show that the intra-molecular structure of the levoglucosan molecule remains practically unchanged in the crystal, plastic crystal, orientational glass, liquid and glass states. It is important to note that the PDFs for the plastic crystal and orientational glass phases exhibit oscillatory behavior, which extends to about 100 Å indicating longer range ordering of these forms of 1,6-anhydro-β-D-glucose  were described by the Vogel-Fulcher-Tammann (VFT equation2223Tg ≈ 245 K), can be detected. Just to mention that herein Tg was defined as temperature at which τα = 100 s. On the other hand, the dynamics of dominant relaxation process in ethanol is much different in ODIC and supercooled phases. Although similarly as in the case of 1,6-anhydro-β-D-glucose, the transition to the glass and orientational glass occurs at the same temperature Tg = 97 KTg to the degree of disorder in given material.To gain deeper insight into dynamics of PC and SL phases, the measured loss spectra were fitted to Havriliak-Negami (HN) functionm, of examined carbohydrate as well as ethanol prepared in both forms. In fact, using the following definition of fragility:Different temperature dependences of the structural relaxation times in ODIC and SL states indicate the difference in the fragility, m to be equal to 92, 102 and 40, 60et al.Nc. To verify this experimental finding and seek for a closer relation between long range ordering , dynamical heterogeneities and molecular dynamics, we decided to utilize two methods proposed by Berthier et al.2627et al.29χ4(t) related to the four point correlation function G4 can be done with the use of derivative of the two point correlation function. Once KWW function [exp(−(t/τα)βKWW)] is applied to describe Φ(t), the number of molecules involved in correlated motions Nc can be calculated directly from the formula given by Capaccioli et al.we obtained Cp is a difference between heat capacity of the supercooled liquid or plastic crystal phase and glass or orientational glass at given T, kB is a Boltzmann constant and βKWW is a stretched parameter. It should be added that ΔCp  were estimated from Differential Scanning Calorimetry measurements (see inset to panel (b) of where: ΔNc evolves with temperature in a similar way in both phases reaching value 105 ± 12 and 102 ± 12 at the glass transition for the supercooled and ODIC levoglucosan respectively. Hence, the observed difference seems to be within the experimental uncertainty. It should be mentioned that very similar Nc (88 ± 9 and 96 ± 9) were determined for leveglucosan prepared in both phases from Donth approach.The temperature dependences of the number of dynamically correlated molecules are presented in ξ, which provides information about the length scale of dynamically correlated molecules characterized by the same dynamics or relaxation time. For that purpose following equation that links four point dynamic susceptibility χ4(t)   with cora (diameter) is equal to 5 Å and d defines the shape of the dynamically correlated regions. One can mention that in literature cooperatively rearranging regions of different compact or fractal geometries are considered323334d varies in the range 2–4. Although, d = 3, which reflects cubic or spherical geometry of the dynamically correlated regions is generally taken into account. Due to this reason we assume d = 3. In the inset to ξ is presented. It is well seen that ξ ≈ 2 nm at the glass transition temperature for levoglucosan prepared in both SL and PC phases As a complementary, this parameter was also evaluated for ethanol ξ = 1.8 nm at Tg using Nc = 195 molecules at Tga = 3 Å. Our results indicated unquestionably that in contrast to the conclusion derived by Michl et al.Nc as well as ξ evolve in the same manner in PC and SL phases at the same compound. Moreover, taking into account structural and dynamical studies carried out for 1,6-anhydro-β-D-glucose and ethanol, we have an unique opportunity to understand the real molecular mechanism that underlies difference in dynamics of plastic and ordinary glasses appeared. As demonstrated above, the static long range correlations between molecules oscillates around 3–4 nm and 9 nm for supercooled and ODIC states of 1,6-anhydro-β-D-glucose, respectively. What is more, the molecular arrangement in both studied phases is fairly the same. Hence, by simple comparison of length scales of static long range order and dynamical heterogeneities in PC and SL carbohydrate, one can find that the latter quantity is few times lower than the former in both phases. Hence, the length scale of static long range ordering, exceeding significantly ξ, seems to be a major factor controlling molecular dynamics of supercooled and plastic phase in levoglucosan. Completely different situation is noted for ethanol. In this alcohol, the length scale of dynamical heterogeneity is almost twice larger than the static intermolecular long range correlations between molecules. Moreover, the significant decrease in orientational correlations was reported when passing from plastic crystal to supercooled state in this material. Therefore, it can be concluded that not heterogeneity alone but rather interplay between length scales of static long range ordering and dynamical heterogeneities as well as internal molecular arrangements and correlations are the most important factors influencing molecular dynamics of supercooled and orientationally disordered crystal states.where: In this letter, the molecular dynamics and structural studies were performed on 1,6-anhydro-β-D-glucose, an unique material that form both supercooled liquid and plastic phases at the same thermodynamic conditions. Our combined investigations indicated that the interplay between length scales of static long range inter-molecular correlations and dynamical heterogeneities as well as internal molecular arrangements determines degree of deviation of molecular dynamics of PC and SL phases. This finding is crucial for better understanding of the relationship between varying disorder, static range correlations and molecular dynamics in various materials in the close vicinity of the glass transition temperature.How to cite this article: Madejczyk, O. et al. Interplay between the static ordering and dynamical heterogeneities determining the dynamics of rotation and ordinary liquid phases in 1,6-anhydro-β-D-glucose. Sci. Rep.7, 42103; doi: 10.1038/srep42103 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Dopamine is thought to directly influence the neurophysiological mechanisms of both performance monitoring and cognitive control—two processes that are critically linked in the production of adapted behaviour. Changing dopamine levels are also thought to induce cognitive changes in several neurological and psychiatric conditions. But the working model of this system as a whole remains untested. Specifically, although many researchers assume that changing dopamine levels modify neurophysiological mechanisms and their markers in frontal cortex, and that this in turn leads to cognitive changes, this causal chain needs to be verified. Using longitudinal recordings of frontal neurophysiological markers over many months during progressive dopaminergic lesion in non-human primates, we provide data that fail to support a simple interaction between dopamine, frontal function, and cognition. Feedback potentials, which are performance-monitoring signals sometimes thought to drive successful control, ceased to differentiate feedback valence at the end of the lesion, just before clinical motor threshold. In contrast, cognitive control performance and beta oscillatory markers of cognitive control were unimpaired by the lesion. The differing dynamics of these measures throughout a dopamine lesion suggests they are not all driven by dopamine in the same way. These dynamics also demonstrate that a complex non-linear set of mechanisms is engaged in the brain in response to a progressive dopamine lesion. These results question the direct causal chain from dopamine to frontal physiology and on to cognition. They imply that biomarkers of cognitive functions are not directly predictive of dopamine loss. Neural recordings during dopaminergic depletion shed doubt on our current understanding of the direct link between frontal dopaminergic inputs, frontal neurophysiology, and cognitive performance. To successfully complete a task, we need to monitor our performance. If performance drops, we need to change our behaviour. We do this by adjusting cognitive control, an ensemble of processes through which behaviour is adapted to suit the task. In this study, we first used chronic recordings in the frontal lobe of macaque monkeys to characterise neurophysiological markers that reflect these processes: a brain potential reflecting performance monitoring and a sustained oscillatory signal reflecting cognitive control. It has been suggested that cognitive control, performance monitoring, and their neurophysiological markers are under the influence of dopamine. To understand how the input of dopamine is critical, we followed changes in the markers and performance during slow dopaminergic depletion. This protocol doubles up as a study of the early phase of Parkinson’s disease, when dopaminergic cells are dying but motor symptoms have yet to emerge. Whilst the performance monitoring potential attenuated at the end of the depletion, the performance itself did not. The oscillatory signals showed only subtle changes in comparison, despite the depletion. Together these results bring into question the simple idea that dopamine directly modulates frontal cortex, which in turn directly modulates cognition. We consider how the brain may compensate for a dopamine lesion, and whether the markers measure what we think they do. Our results question a current idea that neurophysiological markers can be directly used to predict dopamine loss in patients with conditions like Parkinson’s disease. Successful and adaptive completion of cognitive tasks requires tight integration between performance monitoring ,2, whichError- and feedback-related potentials  recorded over the medial part of the frontal lobe in electroencephalography (EEG) –7, electA second key constituent of this integrated system implements the chosen level of control and is associated with lateral prefrontal cortex (PFC). Control implementation is signaled, for example, in modification of classical working memory delay activity , and hasDopamine (DA) is proposed to have a critical role in regulating these systems and the related behaviour , and theProper testing of this working model requires a systemic approach combining dopaminergic modulation, neurophysiology of frontal mechanisms, and related cognitive control performance. This approach is absent from the literature, and yet this test is a mandatory step to understanding dopamine-neurophysiology-cognition links, the role of dopamine in driving frontal functions, and how to target treatments of the relevant conditions.Here, we reveal the dynamic of performance monitoring, cognitive control, and their neurophysiological markers during a progressive lesion of the dopamine system. In particular, we use a test of cognitive control  known toWe tested the effect of progressive dopaminergic loss on electrophysiological markers of performance monitoring and cognitive control, using chronic ECoG recordings in monkeys. Signals were acquired over extended periods  whilst inducing a very slow progressive dopaminergic lesion with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We compared this with a substantial pre-lesion baseline period  in a within-monkey design.Two monkeys learned the problem-solving task PST, , 18], a, a18], a = 638, p < 0.0001; Monkey S: F = 563, p < 0.0001,  = 59, p < 0.0001; Monkey S: F = 236, p < 0.0001, In the BL period, monkeys were tested for at least 10 weeks with sham injections to establish baseline performance and neurophysiology. During this time, monkeys’ performance approached optimality. Their level of non-optimal responses . This FRP therefore reflects critical information that might be required to adapt behaviour in the following trial, as best indexed by the difference curve shown in In BL, a medial frontal evoked response after feedback differentiated correct (COR) from incorrect (INC) feedback, with a significant response difference between 50 and 200 ms  trials from each problem, excluding repeated COR feedback, for which the monkey has a higher expectation than CO1. In SEA, the probabilities of observing negative or positive feedback (INC and CO1) are roughly equivalent on average (p > 0.05). Note that the term “beta” refers to power of high beta oscillations, and never to any form of model beta .During the delay epoch at the start of a trial, the monkey can prepare the upcoming choice based on previous choices and outcomes. Both monkeys showed strong induced oscillations in the beta band (15–30 Hz) throughout the delay . We anal = 370, p < 0.0001; Monkey S: F = 39, p < 0.0001). In addition to cognitive control, two factors potentially related to motivation had significant impact on beta power. First, beta power significantly increased with time “within-session”—that is, in a given session, power correlated with the time the monkey had spent continuously working  = 209, p < 0.0001; Monkey S: F = 22, p < 0.0001). We have previously linked this within-session change to an increase of attentional effort of the monkey during sustained work, not least because the power increase is “reset” by a voluntary pause in work PE2I) provided good indication of striatal DAT levels, but in cortex, DAT levels are low and only cingulate DAT binding was significant. We have confirmed that MPTP at this dose induces a putative compensatory increase in DAT, followed by a global loss of DAT binding once motor symptoms becomes significant PE2I) throughout the protocol. [11C]PE2I has high affinity and selectivity to DAT and is used to index the integrity of the DA pathway PE2I as a bolus followed by a saline flush through a cannula in the femoral vein. Radioactivity was measured in a series of 24 sequential time frames of increasing duration .We anaesthetised monkeys with 15 mg/kg Zoletil  after premedication with 0.1 mg/kg atropine sulphate, and placed them in the scanner in an MRI-compatible stereotaxic frame . We injected [We used the anatomical-MRI and maximum probability atlas  to defin11C]PE2I non-displaceable binding potentials (BPND). Because this procedure is semi-automated, we repeated it within and between two experimenters independently in order to account for within-measure variance. For each monkey, a PET scan was acquired in the BL period (prior to MPTP) as well as at regular intervals throughout, and once for each monkey during the 3-week “full dose” period. As in the previous study, cortical levels of [11C]PE2I-BPND were much lower than striatal levels, but the distributions of cortical and striatal DAT did overlap . These highest cortical DAT binding values were in the cingulate cortex ROI of PE2I-BPND is a specific measure of the presence of the dopamine transporter, and cannot be considered as a direct index of dopamine levels. Note also that there are more PET scans for Monkey S as a result of the longer treatment period necessary to induce threshold symptoms. Future research on this topic will be required to link the neurophysiological and behavioural results here with more detailed consideration of cortical DA function under MPTP lesion.Given the importance of ongoing recording of neurophysiology and cognitive performance to the protocol, and the necessity of anaesthetising the animals, PET scanning was limited to a single radioligand and to three to eight weekly intervals. Note that PE2I-BPND is a specific measure of the presence of the dopamine transporter and cannot be considered as a direct index of dopamine levels.Each point represents the BPer et al . These der et al  by showi(TIF)Click here for additional data file.


Encephalartos natalensis and Ginkgo biloba. This was followed by transcriptome assembly, after which these data were used to build paralog age distributions. Based on these distributions, we identified remnants of an ancient whole genome duplication in both cycads and ginkgo. The most parsimonious explanation would be that this whole genome duplication event was shared between both species and had occurred prior to their divergence, about 300 million years ago.Contrary to the many whole genome duplication events recorded for angiosperms (flowering plants), whole genome duplications in gymnosperms (non-flowering seed plants) seem to be much rarer. Although ancient whole genome duplications have been reported for most gymnosperm lineages as well, some are still contested and need to be confirmed. For instance, data for ginkgo, but particularly cycads have remained inconclusive so far, likely due to the quality of the data available and flaws in the analysis. We extracted and sequenced RNA from both the cycad  Whole genome duplications (WGDs) have been prevalent during the evolutionary history of flowering plants, and have even been linked to their origin as well as their fast rise to ecological dominance –3. FurthWelwitschia mirabilis is the only gymnosperm showing evidence for a relatively recent WGD event [Welwitschia from its closest relative, Gnetum (135–110 Mya) [Ephedra, the third Gnetales genus, also lacks evidence of WGD events [et al. [Ginkgo lineage, attributing it to the ancient WGD event proposedly shared by all seed plants [Contrary to the many WGD events recorded for angiosperms, the history of the non-flowering gymnosperms paints a very different picture. Although far fewer gymnosperm species exist today compared to the angiosperms, and as such many lineages containing evidence for WGD events could have been lost, polyploidy events, ancient or more recent, in these seed plants seem rare. Thus far, GD event , 14, pos110 Mya) –17, the , Gnetum 5–110 Mya, Gnetum 5–110 MyaD events –20. Li e, Gnetum 5–110 Mya, Gnetum 5–110 Myad plants , resultiGinkgo biloba, preceding the divergence of these lineages.The cycads were widespread during the Jurassic–Cretaceous, reaching their greatest diversity ~200–65 Mya –24. TodaEncephalartos natalensis  and Ginkgo biloba, and assembled high quality low-redundancy transcriptomes [E. natalensis and G. biloba assemblies contained 22,204 and 23,845 transcripts with average sequence lengths of 1,097 and 1,259 bases and average GC contents of 44.23 and 42.52%, respectively. Based on age distributions of paralogs inferred from synonymous substitutions per synonymous site, or so-called KS distributions [KS of ~0.8 was identified for E. natalensis  than the divergence between cycads and ginkgo  and G. biloba . The de novo assemblies were constructed with Velvet v1.2.10 [RNA-seq libraries from these tissues were used to construct  v1.2.10  followed v1.2.10  with a k v1.2.10 .E. natalensis and G. biloba [KS age distributions for E. natalensis and G. biloba were constructed as described previously [−10, followed by gene family construction and prediction using the mclblastline pipeline  [KS estimates for all pairwise comparisons in gene families, maximum likelihood estimation was performed using the CODEML program of the PAML package (v4.4c) [KS estimates between members did not exceed a value of 5. To correct for the redundancy of KS values (a gene family of n members produces n(n–1)/2 pairwise KS estimates for n–1 retained duplication events), a phylogenetic tree was constructed for each subfamily using PhyML [m KS estimates between the two child clades were added to the KS distribution with a weight of 1/m (where m is the number of KS estimates for a duplication event), so that the weights of all KS estimates for a single duplication event sum up to one. One-to-one orthologous pairs between E. natalensis and G. biloba were created by performing a reciprocal best hit BLASTP analysis of the longest translated transcripts from one species against the other. Valid orthologous pairs were then identified as having at least 30% identity over 150 amino acids.TransDecoder v.3.0.0 was used to predict coding regions in the transcriptomes of . biloba , after weviously . Brieflyorg/mcl) . Each ge (v4.4c) , 55. Genng PhyML  under dehttps://www.ncbi.nlm.nih.gov/) under the submission number SUB2337915.Raw reads of both transcriptomes are available at the National Center for Biotechnology Information (