Gibberellin‐regulated protein  was recently identified as a new marker allergen related to systemic reactions in peach allergy; however, its role in other fruit allergies is unknown.To investigate the allergenicity of Japanese apricot (JA) GRP and clarify the clinical characteristics of JA allergy patients with GRP sensitization.Seven patients  diagnosed with JA allergy based on relevant clinical history, positive skin test and/or challenge test were enrolled. JA GRP with a molecular weight of 6896.5 Da was purified by ion‐exchange column chromatography. We performed enzyme‐linked immunosorbent assay (ELISA), IgE‐immunoblotting, basophil activating tests (BATs), and skin prick tests (SPTs) with purified JA GRP. To investigate the cross‐reactivity between JA GRP and native Pru p 7 (nPru p 7), we performed ELISA inhibition tests. We measured specific IgE levels against apricot, peach, rPru p 1, rPru p 3, and rPru p 4 using ImmunoCAP.ELISA and IgE‐immunoblotting using JA GRP showed positive reactions in six (85.7%) and seven (100%) patients, respectively. Five patients who provided informed consent for BATs and SPTs using JA GRP had positive results. In four patients who underwent ELISA inhibition tests using JA GRP and nPru p 7, IgE binding to one GRP inhibited binding to the other. The positivity for specific IgE against apricot, peach, rPru p 1, rPru p 3, and rPru p 4 was 14.3%, 57.1%, 0%, 0%, and 0%, respectively. Patients developed allergic reactions that were frequently accompanied with facial swelling, especially of the eyelids, which was dependent on cofactors, such as exercise.These results indicated that GRP might be a causative allergen of JA allergy, whose onset frequently requires a cofactor, such as exercise, and might be cross‐reactive between JAs and peaches. Prunus mume) belongs to the Rosaceae family, which includes apricots (Prunus armeniaca), and is mainly harvested in Asian countries. In Japanese and Korean cooking, Japanese apricots are used in juices and sauces and as pickles. Pickled and dried Japanese apricots, umeboshi, are very popular in Japan and are eaten daily as an everyday food with steamed rice. Umeboshi has been introduced as a healthy ingredient in westernized recipes for dressings, sauces, and sushi rolls. A recent study showed that the daily consumption of JA might improve digestive dysmotility symptoms, resulting in relief from gastroesophageal reflux disease symptoms Japanese apricot  was first identified as the fifth peach allergen, Pru p 7 , in 2013 Note: The partial sequence of amino acids in GRP, along with biochemical, clinical and immunologic data, has been submitted to the WHO‐IUIS Subcommittee for Allergen Nomenclature, which assigned the designation of Pru m 7 as a new JA allergen.Seven consecutive patients diagnosed with JA allergy at the Dermatology Department of Yokohama City University Hospital in Yokohama between January 2002 and December 2012 were enrolled in the present study.Patients were diagnosed with JA allergy if they had a convincing history of allergic reactions within 2 h after the ingestion of JA and had positive results on skin prick tests (SPTs) with JA and/or challenge tests with JA. Open food challenges using JA were performed as previously described, except in cases with a recent history within 1 year of anaphylaxis after JA ingestion and SPT positive to JA In addition, we evaluated each patient's clinical history to determine the involvement of cofactors, such as nonsteroidal anti‐inflammatory drugs (NSAIDs) and exercise, which are associated with food‐dependent exercise‐induced anaphylaxis (FDEIA). The time‐period accepted for a potential relationship with a food‐allergic reaction was 2 h before food ingestion, combined with the simultaneous intake of NSAIDs This study was approved by our institutional review board. Written informed consent was provided by all patients.Prunus mume, cultivar Nanko strain) at the commercial unripe stage were obtained from a local grocery store. Whole JA was homogenized in a blender after the addition of an extraction solution  g for 15 min at 4°C. After protein extraction, the samples were centrifuged and the supernatants, representing the protein extract, were collected.JA . The purity of protein preparations was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry and Native peach GRP (nPru p 7) was purified from peach pulp by ion‐exchange chromatography following as previously described w/v) under reducing conditions using the electrophoresis system Bolt ™  according to the method of Laemmli, following the instructions supplied by the manufacturer w/v) SDS, 20% (w/v) glycerol, 10% (w/v) 2‐mercaptoethanol, and 0.02% (w/v) bromophenol blue. The samples were denatured by heating at 70°C for 10 min. To ensure correct protein separation and visualization, proteins on the gel were stained with Coomassie Brilliant Blue R‐350 .JA crude extract (10 mg per lane) or purified JA GRP (100 ng per lane) was subjected to SDS–PAGE  by wet transfer blotting methods. The membrane was incubated in 10 mM phosphate buffered saline (PBS) (pH 7.5) containing 0.1% Tween 20 and 5% skim milk for blocking. For immunoblotting using rabbit anti‐Pru p 7 peptide antibodies, antibodies were diluted 1:1000 in the same blocking buffer as for the primary antibody. Then, horseradish peroxidase‐labeled goat anti‐rabbit IgG  was used as a secondary antibody. IgE‐immunoblotting used patient sera (1:10 dilution) in the same blocking buffer as the primary antibody. Then, horseradish peroxidase‐labeled goat anti‐human IgE  was used as a second antibody. Detection of the final signal was performed using an ECL Western Blotting Detection kit , following the manufacturer's instructions, and recorded onto X ray films .Specific IgE (sIgE) antibody to purified JA GRP was detected by enzyme‐linked immunosorbent assay (ELISA) as described in the Supporting Information ELISA inhibition assays were performed using the same method as above, except that sera (1:10 dilution) were preincubated with four progressive 1:10 dilutions of the corresponding inhibitor overnight at 4°C. Cross‐reactivity between JA GRP and nPru p 7 was tested with five individual sera, using each purified allergen as a solid phase and as an inhibitor (2 μg/mL). All assays were performed in duplicate.Serum sIgE levels  for apricot, peach, and recombinant allergens, rPru p 1, rPru p 3, rPru p 4, rBet v 1, and rBet v 2, were measured. According to the information provided by the manufacturer, the cutoff value of the assay was 0.35 kUA/L as class 1.high%. At least 500 basophils were analyzed in each assay. A sample was considered positive for basophil activation if >10% basophils were CD203chi positive and the sample induced CD203c expression to a level greater than the negative control. CD203c expression on basophils from four control subjects without JA allergy was also investigated after stimulation with JA GRP.A commercial kit  was used for quantification of basophil CD203c expression as described previously Five patients provided informed consent for additional SPT with purified JA GRP. SPTs with JA GRP (2 μg/mL in PBS) were performed and read according to a standard procedure Seven patients with a mean age of 28.0  were enrolled in this study (Table www.expasy.org). In addition, sequence similarity analysis revealed that JA GRP (accession number: XP_016648029.1) in GenPept shares 100% amino acid sequence with Pru p 7 (accession number: P86888) registered in UniProt KB  of two predicted JA GRPs registered in the GenPept database. There are no JA‐derived GRPs registered in UniProtKB  , but not in four non‐JA allergic subjects  immediately after oral challenge with 10 g umeboshi 1 h after the intake of 1.0 g aspirin, even though he had negative results for the ingestion of umeboshi alone or aspirin intake alone. Exercise and the intake of aspirin enhanced the onset of allergic reactions in five patients (71.4%) and one patient (14.3%), respectively , followed by urticaria , dyspnea , nasal obstruction , laryngeal tightness , oropharyngeal symptoms , and loss of consciousness .One patient (14.3%) had a positive ImmunoCAP for apricots and five patients (71.4%) showed positive results for peaches. ImmunoCAP to rPru p 1, rPru p 3 and rPru p 4 was negative in all seven patients. In addition, the rates of positive responses to ImmnoCAP against pollen from alder, rBet v 1 and rBet v 2 were 14.3% (1/7), 0% (0/7) and 0% (0/7), respectively. Total IgE concentration was 51–621 IU/mL .vice versa. Furthermore, the current study revealed that JA GRP sensitization might be related to the clinical type of FDEIA and specific clinical characteristics including eyelid edema, which appeared in approximately 70% of the patients.In the present study, we identified GRP as a new allergen in JA allergy. All seven patients with JA allergy were sensitized by JA GRP based on positive results of at least two further analyses  with JA GRP. In addition, ELISA inhibition assays using sera from JA allergy patients indicated cross‐reactivity between JA GRP and Pru p 7, suggesting that patients sensitized by GRP from JA might have cross‐reactivity to peaches and GRP is a small, basic, cysteine‐rich protein with a molecular weight of approximately 6.9 kDa. GRP functions as an antimicrobial peptide and the first isolated homolog from potato, snakin‐1, was active against plant pathogens, such as bacteria and fungus We identified GRP as the first allergen, Pru m 7, in JA allergy. To date, few cases of JA allergy have been reported and the causative allergen was not identified The present study is the first to show cross‐reactivity between GRPs involved in food allergies and derived from different fruits. ELISA inhibition tests using the respective serum of all four patients showed IgE binding inhibited IgE binding to another GRP. The cross‐reactivity between JA GRP and Pru p 7 might be explained by high similarity of their amino acid sequences. Despite the high identity of their amino acid sequences, sIgE levels against JA GRP tended to be higher than against Pru p 7. In addition, the inhibition rate of Pru p 7 by JA GRP was higher than that of JA GRP by Pru p 7. We did not determine the reason for the different inhibition rates between JA GRP and Pru p 7. However, Tuppo et al. reported that heat denaturation affects the immunological properties of Pru p 7 Rosaceae family fruits, it was presumed that apricots may cause allergic reactions, ranging from mild symptoms such as oral allergy syndrome There have been a few reports regarding the clinical features of apricot allergy, including JA. Based on clinical experiences of other Interestingly, physical exercise and/or intake of medication, such as aspirin, were required as cofactors to develop allergic reactions after the ingestion of JA in six of seven patients (84.6%). Indeed, two previous cases with JA allergy were also diagnosed with FDEIA Pru p 7 is stable to heating and simulated gastric digestion because of its high cysteine content  similar to LTP, which forms six disulfide bridges that stabilize the small molecule Recent reports demonstrated BATs were superior to other diagnostic tests when discriminating between peanut allergy and tolerance, particularly in difficult cases, and therefore reduced the need for oral food challenges This study had some limitations including small number of subjects enrolled. To date, only a few cases of JA allergy have been reported. JA allergy might be underestimated because of the low reproductivity of allergic reactions in a cofactor‐dependent manner. In addition, information is available on GRP derived from JA but not from apricots in all databases. Therefore, determining the clinical features and allergens of JA allergy might help diagnose patients with allergy to JAs and apricots. However, further investigation of a large number of subjects is required to clarify whether GRP sensitization might be involved in JA allergy provoked by cofactors.In conclusion, our results indicated that GRP is a cross‐reactive allergen between JA and peaches, and might be a causative allergen of JA allergy, especially FDEIA. If patients are suspected to have JA allergy, the possibility of cofactor involvement in anaphylactic reactions caused by JA and cross‐reactivity should be considered.All authors have no financial supports or relationships that may pose a conflict of interest.Additional supporting information may be found in the online version of this article at the publisher's web‐site.Figure S1. Purity of Japanese apricot (JA) and reactivity of polyclonal antibodies to Pru p 7 and Pru p 3 peptides. (A) SDS–PAGE of JA extract, 10 μg/lane (lane 1) and the purified JA gibberellin‐regulated protein, 10 ng/lane (lane 2). (B) Separation of proteins in JA extract, 10 ng/lane (lanes 1 and 2) and the purified JA gibberellin‐regulated protein, 10 ng/lane (lanes 3 and 4). Proteins were electro‐transferred to PVDF membranes and incubated with anti‐Pru p 7 peptide antibodies (lanes 1 and 3) or anti‐LTP peptide Ab (lanes 1 and 3).Click here for additional data file.Figure S2. Mass spectrometry measurements. Annotated mass spectrum of Japanese apricot gibberellin‐regulated protein, indicating an average mass of 6896.5 Da.Click here for additional data file.Supporting Information S1.Click here for additional data file.


Procalcitonin (PCT), a precursor protein of the hormone calcitonin, is a sensitive inflammatory marker in human medicine, which is primarily used for diagnosis of bacterial sepsis, but is also useful in diagnosis of exacerbation of asthma and COPD. In this study, PCT was evaluated as a potential biomarker for different chronic pneumopathies in the horse using an equine specific ELISA in comparison to established clinical markers and different interleukins.n = 17) and in the cell-free supernatant of bronchoalveolar lavage (n = 64). PCT concentrations were correlated to interleukins IL-1ß and IL-6 in BALF, clinical findings and BALF cytology.Sixty-four horses were classified as free of respiratory disease, recurrent airway obstruction (RAO), inflammatory airway disease (IAD) or chronic interstitial pneumopathy (CIP) using a scoring system. PCT concentrations were measured in plasma (n = 7) compared to controls  and correlated to PCT in BALF supernatant (rs = 0.48). Compared to controls , median PCT concentrations in BALF supernatant correlated to the overall clinical score  and were significantly increased in RAO  and IAD , while no differences were found for CIP . No significant increases were found for IL-1 and IL-6 between controls and respiratory disease in general as well as different disease groups.The median PCT concentrations in plasma were increased in respiratory disease (174.46 ng/ml, Although some correlations were found between PCT in plasma, BALF supernatant and clinical scores, PCT in BALF does not seem to be a superior marker compared to established clinical markers. PCT in plasma seems to be more promising and a greater number of samples should be evaluated in further studies. Since the 1970s, hypocalcemia and its correlation to sepsis have been in the focus of research .During sepsis, PCT is found in high concentrations in blood and almost all tissues , but PCTEquine RAO (recurrent airway obstruction) is known for parallels to human asthma  as well as COPD , resembling what is called “wheezy bronchitis” in men . During In this study, we aimed to compare ePCT concentrations in BALF with clinicals findings, BALF cytology and other biomarkers, namely interleukins 1ß and 6, which have been shown to stimulate hyperprocalcitonemia in other species , 23. AddA total of 71 horses were examined, of which 15 had no clinical signs or history of respiratory disease and 56 were presented to the clinic with a history of chronic lower airway disease.n = 7). In detail, groups were defined as follows:2 ≤ 8 mmHg, exclusion of acute signs of infection .Group I: No history of respiratory disease, clinical score < 2, no tracheal secretions, low cellular density and neutrophils ≤ 8% in BALF, AaDO2 > 8 mmHg, exclusion of acute signs of infection  according to Robinson [Group II: History of recurrent cough or dyspnea, clinical score > 6, high amount or viscosity of tracheal secretions, high cellular density and neutrophils ≥ 25% in BALF, AaDORobinson .Couëtil et al. [Group III: History of cough or exercise insufficiency, clinical score 2–6, low to moderate amount or viscosity of tracheal secretions, increased cellular density and neutrophils ≥ 8% or mast cells ≥ 2% or eosinophils ≥ 0.1% in BALF, exclusion of acute signs of infection  according to l et al. .Dieckmann et al. [Group IV: History of exercise insufficiency, clinical score 2–6, low to moderate amount or viscosity of tracheal secretions, increased cellular density and ratio of macrophages:neutrophils ≥ 2.5:1 in BALF, increased interstitial opacity of thoracic radiographs, exclusion of acute signs of infection  according to n et al. .The pre-participation examination included anamnesis documentation, clinical examination, exercise test, blood gas analysis, bronchoscopy, BALF cytology and thoracic radiography. Using a modified clinical score system , 25, endDuring endoscopy, 20 ml of 2% lidocaine  were infused around the tracheal bifurcation. The catheter  was wedged into the bronchus by mean of an air balloon. Five hundred milliliters of pre-warmed phosphate buffered saline  were infused as recommended by the International Workshop on Equine Chronic Airway disease  and immeBALF was divided into 2 portions for cytological and biochemical examination. After centrifugation at 1500 rpm for 10 min at 4 °C the cell-free supernatant was stored at −80 °C until assayed. Cytology was performed using Wright-Giemsa staining and counting 500 cells at 500× magnification.As the ELISA for equine PCT was only validated for plasma, it was adapted to BALF according to the guidelines of the American Food and Drug Administration for Validation of bioanalytic methods 2013) by partial validation which was done here by reanalyzing intra-assay (IaA) and inter-assay (IeA) coefficients of variation (CV) and recovery in BALF matrix , PCT was measured in plasma using a specific ELISA for equine PCT . As thisIn 64 horses, PCT was measured in undiluted BALF supernatant samples using a specific ELISA for equine PCT  after adIn 64 horses, interleukins was measured in undiluted BALF samples using commercially available specific ELISAs for equine IL-1ß and 6 (ELISA Kit for Interleukin 1 Beta (IL1b) and ELISA Kit for Interleukin 6 (IL6), Uscn Life Science Inc, USA) according to the manufacturer’s manual.P ˂0.05.Data were statistically analyzed using SPSS  and expressed as median (min-max). The data were tested for normal distribution using the Kolmogoronov-Smirnov and Shapiro Wilks Test. The level of significance was set at U Test for 2-group comparison to determine intergroup differences.Kruskal Wallis H test was used to compare between controls and different disease groups followed by Post-Hoc testing using Mann-Whitney Spearman rank correlation coefficients were calculated between clinical parameters, cytologic data, PCT and interleukin concentrations. Correlations were classified as irrelevant (rs = 0–0.25), weak (rs = 0.25–0.5), moderate (rs = 0.5–0.75) and strong (rs > 0.75).According to the results of clinical scoring and BALF cytology, the 71 horses  presented for participation in this study were classified as follows: 15 horses were classified as free of respiratory disease , 21 as RAO (group II), 16 as IAD (group III), 12 as chronic interstitial pneumopathy (group IV), 4 horses suffered from acute respiratory infections and 3 could not be clearly assigned to groups I-IV. Therefore, the later 7 were excluded, leaving 64 horses for statistical analysis.3/μl), hematocrit (35 ± 5.1%), total protein (6.7 ± 0.5 g/dl) and ionized calcium (1.64 ± 0.10 mmol/l) were within normal limits. There was no difference between in ionized calcium between controls (1.58 ± 0.11 mmol/l) and horses affected by respiratory disease in groups II-IV (1.65 ± 0.09 mmol/l).In all 64 horses, controls and horses affected by respiratory disease, hematology was unremarkable. Leucocytes (8.1 ± 2.2 * 10n = 7) were higher than in group I . The median concentrations in group II (RAO) was even 482.23 ng/ml (n = 4).In 17 horses , PCT was measured in plasma using a specific ELISA for equine PCT . OverallAmerican Food and Drug Administration for Validation of Bioanalytic Methods (2013) the variation coefficients in medical ELISAs need to be below 15%, therefore the working range for equine BALF was defined as 25–250 ng/ml, as shown in Table According to the guidelines of the ethods 203 the varP = 0.033) as well as groups I and III .In 64 horses, PCT was measured in undiluted BALF supernatant using a specific ELISA for equine PCT  after adP = 0.060). The highest correlation was found between PCT in plasma and the respiratory rate . Negative correlations were found between PCT in plasma and the arterial PaO2 . A weak correlation was found between PCT concentration in BALF and airway obstruction  as well as overall clinical score . Neither PCT in plasma nor in BALF supernatant correlated to the percentage of neutrophils in BALF cytology, but PCT in plasma with the eosinophils .A weak correlation was found between PCT concentrations in plasma and BALF supernatant  or between controls and single disease groups (P = 0.365). Results are shown in detail in Table In 64 horses, interleukin concentrations were measured in undiluted BALF samples using a specific ELISA for equine IL-1ß and IL-6. No significant increases were found for IL-1ß or IL-6 between group I (controls) and groups II-IV in general P = 0.12 or betwIn the late 1990s, it has been shown that procalcitonin is a sensitive marker for systemic inflammatory processes caused by bacterial infection, but not of other pathogenesis –28. WhilEquine PCT was characterized in the early 2000s . HypocalFor the quantification of ePCT in plasma and BALF a validated sandwich ELISA using human anti-PCT Antibodies and equine recombinant PCT (erPCT) as standard was used . Rieger In human medicine, PCT concentrations are routinely measured systemically from blood samples. A good correlation has been found between PCT in serum and BALF , 41. StuA week positive correlation was found between PCT and clinical score. In human asthma, a correlation between PCT and the severity of disease has also been described . Pro-infFor IL-1ß, no significant differences were found between healthy controls and horses affected by chronic respiratory disease in general and single disease groups, while in former studies increased transcription and protein concentrations have been found in severe RAO –50. SimiFor IL-6, no significant differences were found between healthy controls and horses affected by chronic respiratory disease either. The highest median concentration was found in group II (RAO in exacerbation). Former studies have led to contrasting results. While some authors found no significant increases in IL-6 were found in RAO exacerbation , 55, 56,A weak point of this study was group definition, as samples were obtained from clinic patients. Therefore disease status was not as uniform as in experimental studies, where samples were obtained after defined disease induction. On the other hand, biomarkers are used in a clinical setting, therefore it seems logical to evaluate their usefulness in the same situation. Although IAD and RAO in remission were planned as two distinct groups, it was not possible to differentiate clearly between these two. Anamnestic information of recurrent respiratory distress was often unreliable and the majority of owners did not agree to a natural challenge test. Descriptions of equine CIP are rare in the literature and an international consensus statement is missing, so definition of this group was based on a quite old clinical case series including only 12 horses . ThoraciIn conclusion, correlations between PCT concentrations in plasma and BALF supernatant were found and also with clinical data, but PCT in BALF does not seem to be a superior marker compared to established clinical markers, in particular in cases of low-grade inflammation. Following the definition by Couëtil , a lower


Gennadas differ from each other largely only in male (petasma) and female (thelycum) copulatory characters, which were restudied in scanning electron microscopy and used as a basis for phylogenetic analyses. Twenty-six petasma characters and 49 thelycum characters were identified. All 16 recognized species of Gennadas and Aristaeomorpha foliacea (outgroup) were included as terminals. Four robust monophyletic clades were retrieved, described and diagnosed as new species groups. The thelycum characters had greater impact on tree topology and supported deeper nodes than did the petasma characters. We hypothesize that features of the thelycum evolved first followed by aspects of the petasma. Relatively more conservative characters include parts of the sternites of the thelycum and of the petasma, while the scuti and protuberances on the thelycum and the shape and subdivisions of the petasma lobes are evolutionarily plastic. We identified two groups of copulatory characters, which are likely coupled functionally and interlinked evolutionarily: (i) the external part of the petasma and the posterior part of the thelycum and (ii) the internal part of the petasma and anterior part of the thelycum. We reconstruct possible mating position during copulation for each of the new species groups presented here. We also present an updated key to genera of Benthesicymidae and key to species of Gennadas.Species within  The fGennadas, the most abundant and diverse genus of Benthesicymidae, which encompasses 16 of the 40 species of the family (40%). Most species of Gennadas are abundant and widely distributed in all oceans and therefore were discovered and described already in the late nineteenth century or in the beginning of twentieth century. Only one species, associated with the seamounts of the distant Nazca and Sala-y-Gomez Ridges, was described later . Burkenka, 1990  and Kenshe genus . For exazi, 1960  is basedGennadas, the genus offers a number of phylogenetic challenges due to the general similar morphology of many species. Indeed, regarding the external morphology, the genus is morphologically more uniform than the rest of the Benthesicymidae, which may be explained by the fact that they occupy very similar ecological niches in the marine habitats: all species are mesopelagic migrants occurring between 500 and 1500 m in the daytime and between 200 and 500 m at night in the Atlantic [In addition to the general biodiversity and taxonomic interest related to Atlantic , PacificAtlantic  and IndiAtlantic ,10 OceanGennadas and are therefore promising for phylogenetic reconstruction as was shown for other Dendrobranchiata [However, genital structures  show an outstanding diversity in anchiata –14. Despanchiata ,5,10.Gennadas. We also restudy and/or revise all available copulatory structures in search of suitable phylogenetic characters on which a new classification can be based. As in previous studies on the classification of other pelagic shrimps, we examine genital structures by the use of scanning electron microscopy (SEM). Based on the phylogeny, we test statistically the possibility of coevolution between male and female genital structures, test their contribution to the phylogeny and discuss questions such as: which genital structures arose first, female or male? Which genital structures are evolutionarily conservative and which are more plastic? Based on both phylogenetic and morphological considerations, we also propose hypotheses for how the complex female and male genital structures may operate and interact with each other during spermatophore deposition.In this study, we provide an inventory of the global fauna of 2.2.1.Gennadas; individuals were then selected for further study by SEM.The material used for this study is primarily from Danish and Russian expeditions exploring the pelagic zone of the world oceans. Most material used for the study is stored in the crustacean collection of the Natural History Museum of Denmark (NHMD) . The work involved sorting and identification of the about 200 samples of Gennadas petasma, which consists of two symmetrical halves, are represented in Prior to treatment for SEM, relevant parts  of selected specimens were dissected in order to expose important structures for further study. The material was dehydrated in a graded ethanol series, critical point dried, mounted and coated with a mixture of platinum and palladium following standard procedures . The SEM2.2.Aristaeomorpha foliacea  [Gennadas and Bentheogennema were included as terminal taxa.Since the taxonomic validity of the genera of Benthesicymidae are uncertain and the phylogenetic relationships between them unknown, an outgroup was chosen outside the family. As molecular data have shown, Benthesicymidae is sister to Aristeidae  and we co, 1827) , type foGennadas, we made three analyses: (i) one with only female characters included , (ii) another with only male characters included  and (iii) a final with all characters included . Data were handled and analysed under maximum-parsimony settings using a combination of programs: WINCLADA/NONA, NDE (Nexus Data Editor) and TNT [We used 49 thelycum-related and 26 petasma-related characters . The data matrix is presented in electronic supplementary material, appendix S3. To estimate the contribution of the female and the male genital structures to the morphology-based phylogeny of  and TNT ,19.All characters were unordered (non-additive) and equally weighted. Trees were generated in TNT using the ‘implicit enumeration’ options. Relative stability of clades was assessed by standard bootstrapping (sample with replacement) with 10 000 pseudoreplicates and by Bremer support . We considered the clades statistically significant if they were supported both by Bremer values greater than or equal to 3 and bootstrap values greater than or equal to 80.2.3.To assess possible coevolution of characters, all morphological characters were divided into nine groups associated with the following structures : (i) sixth thoracic sternite in females (characters 1–9), (ii) anterior part of the seventh thoracic sternite in females (characters 10–25), (iii) posterior part of the seventh thoracic sternite in females (characters 26–39), (iv) eighth thoracic sternite in females (characters 40–48), (v) pars interna (PI) of the petasma  and further analysed via ANOSIM and non-metric MDS analysis and hierarchical clustering .p < 0.05.Calculations and analyses were carried out with the use of Excel, STATISTICA and PAST v. 3.04 . Correla3.3.1.Gennadas is formed by the sixth (S6), seventh (S7) and eight (S8) thoracic sternites. The seventh sternite is split into an anterior (AS7) and a posterior (PS7) parts in all Gennadas species, except G. elegans (a). The shape of each sternite is very characteristic in the various species and a number of distinct types could be recognized as seen in the following.Gennadas, except G. elegans, in which S6 is enlarged and has a complex relief (a).S6 is small and subtriangular in all x relief a.a,b,e,f), trapezoid with posterior incision , bilobed with notched elevation , bat-like with lateral edges not reaching coxae , as two separate lateral ear-like structures , or W-shaped with lateral edges reaching coxae .AS7 may be simple and unspecialized, without prominent relief a,b,e,f, incision b,e,f, bilevation c,d, bat-ng coxae g,h, as tructures c,d, or Wng coxae g,h.a,b,e,f), subtriangular (b–f), trapezoid, with anterior incision , as a long chitinized strip laterally produced beyond coxae .PS7 may be simple and unspecialized, without prominent relief a,b,e,f, iangular b–f, trapincision g,h, as and coxae c,d,g,h.e), may have medial ridges , medial grooves  or antelateral spinose extensions (figures h and 5a).S8 are nearly ortho-/pentagonal or trapezoid, sometimes soft and not chitinized e, may hal ridges e,f, medi grooves g,h or an figures h and 5a.The thelycum in  elegans a. The sha) or anteriorly produced from AS7 (figures b and 5h). In G. incertus (figure 5f), a very unusual structure is produced from the central part of S8 and extending in all directions as a mushroom cap over S8.Each of sternites may have a scutum of various size, shape, direction and armature. The scutum-related characters are very characteristic for each species figures  and 5. Ifigure 5 figures b and 5h.Gennadas  have strong specialized setae of various size and shape. They may include medial setae on S6 , sublateral and lateral setae  on PS7.In addition to scuti, species of ae on S6 b,e,d, sual setae c,d on PSGennadas are usually present (closed thelycum), although they may be developed to different extent. The female orifices may be guarded by scuti, which are either posteriorly extended from S6  and/or anteriorly extended from AS7 . In four species, orifices are bordered by compound posterior and anterior projections of S6 and AS7, respectively . In G. tinayrei, the receptacles involve also fifth thoracic sternite (d), which have a long posteriorly directed spinose process.The seminal receptacles in ectively c,d,g,h. sternite d, which G. crassus Tirmizi, 1960 [G. gilchristi. Taking into account the very elaborate thelyca in all other Gennadas, which are used as a fingerprint for identification of species, we consider the former species a junior synonym of the latter.The description of zi, 1960  was base3.2.G. elegans, in which PM and LA are absent (a).The petasma is consisting of PI, PM, PE and LA in all species except e absent a.G. elegans (a), the distal part of PI is transformed into what we propose is a specialized grasping structure, which is characteristic for each species and may include protuberances and invaginations of various sizes and shapes (figures 2b–h and 3a–h). The grasping structure is well chitinized and rigid in most species, soft and folded in two species . Sometimes, the grasping structure bears circinnuli greatly enlarged and/or transformed into hooks and/or spines .PI is covered with one to five rows of circinnuli along the medial edge. In all species except  elegans a, the di species a,b. Somee,f) or terminal hook (d). LA may be as a club, which is nearly orthogonal to the main lamella (h) or flat and laterally extended, overlapping PI in the lateral direction .LA is extending from that side of the petasma, which is directed towards the abdominal sternites. In some species, LA is very characteristic and has a greatly expanded apron-like apex e,f or tenal hook d. LA may lamella h or flatirection c,d.e,f) or wide and long  lobe. PM may consist of two independent lobules, which are either joining basally  or widely separated (figure 3f).PM may be short and rounded  or consisting of two long independent lobules (figures b and 3a). PE may have a terminal notch dividing apex either into pointed  or rounded  branches. PE may have an additional, small, rounded (d), pointed  or toothed  lobule. PE may have a few terminal tiny teeth (d) or be serrate (d).PE may be entire figures a and 3b  figures b and 3a. pointed e,f or ro rounded g,h branc rounded d, pointe pointed e,f or to toothed c,d lobulny teeth d or be s serrate d.3.3.a) with only thelycum-related characters retrieved a single most parsimonious (MP) tree, with a score of 50 . The trees showed that G. elegans  was separate from the core Gennadas, which further branched into four clades (a): (i) G. talismani + G. valens, (ii) G. scutatus + G. propinquus + G. burkenroadi + G. brevirostris, (iii) G. incertus + G. capensis + G. kempi + G. bouvieri and (iv) G. tinayrei + G. gilchristi + G. barbari +G. sordidus + G. parvus. The last three clades were further divided into six terminal clades, consisting of two species each. All these clades received high statistical support.Analysis 1 a with onr clades a: (i) G.b) with only petasma-related characters retrieved a single MP tree, with a score of 27 . The trees showed a basal branching similar to that of Analysis 1 (G. elegans outside the remaining Gennadas), while further branching was greatly less resolved. Only terminal clades  were revealed, but only three were robust: G. kempi + G. bouvieri, G. talismani + G. valens and G. sordidus + G. parvus.Analysis 2 b with onc) with all characters included retrieved a single MP tree, with a score of 80 . The trees had a topology very similar to that of Analysis 1. All clades received better support (c). As in Analysis 1, the branching can be described as occurring at three levels: (i) basal branching (G. elegans and the core Gennadas), (ii) medial-level branching into four species groups and (iii) terminal branching with each terminal clade consisting of two species.Analysis 3 c with al support c. As in 3.4.Gennadas  was supported by synapomorphies mostly involving general morphological characters: presence of specialized setae, shape/division of thoracic sternites in the thelycum and presence/absence of lobes in the petasma (c). The medial-level branching was supported by synapomorphies mostly relating to the morphology of the medial part of the thelycum (AS7 and PS7); G. valens species group was additionally supported by characters relating to S8 and the petasma (c). The terminal clades (each consisting of two species) were supported by the characters related to detailed morphology: degree of extension and chitinization of sternites, presence and shape of specialized groups of setae, shape and development of scuti on the thelycum and lobi on the petasma (c).The core  petasma c. The me petasma c. The te petasma c.3.5.d). The use of all available evolutionary events (nodes on phylogenetic tree—figure 6c) showed the following correlations:(1) between posterior part of the thelycum (S8) and external part of the petasma (PE) and(2) between anterior part of the thelycum (S6 and AS7) and interior part of the petasma .ANOSIM multidimensional scaling and hierarchical clustering showed that morphology of different parts of copulatory structures have coevolved d. The usOne part of the thelycum (PS7) was not grouped with any of the other copulatory structures.4.4.1.4.1.1.Gennadas burkenroadi was described as Bentheogennema burkenroadi Krygier and Wasmer, 1975 [Gennadas and Bentheogennema in having a single pair of lateral spines on the telson  and podobranchs on the second maxilliped posteriad to the third pereopod . The authors considered podobranchs as a ‘primitive characteristic, a more important generic trait than the number of pairs of spines on the telson’ [Bentheogennema. We consider both traits above as equally important in the generic taxonomy of Decapoda and therefore refer to two additional evidences to resolve the position of B. burkenroadi. We examined type material of B. burkenroadi and found the thelycum and petasma of this species to be greatly elaborate and structurally similar to those seen in the core species of Gennadas (figures d and 5d), and significantly different from those in Bentheogennema. Our phylogenetic analysis showed that this species is deeply nested into the Gennadas tree and belongs to G. brevirostris species group. Hence, B. burkenroadi belongs to Gennadas which makes some changes in the diagnosis of Gennadas necessary: figures d and 5d,Gennadas Bate, 1881.Emended diagnosis: Integument membranous; rostrum not reaching eye cornea, armed with one apical and one dorsal tooth, setose in between; carapace with distinct cervical and postcervical sulci reaching dorsal midline, antennal angle rounded, branchiostegal angle square, hepatic and branchiostegal carinae weak; abdomen with first to fifth somites dorsally rounded and sixth somite dorsally carinate; telson posteriorly truncate, with a single pair of movable posterolateral spines. Eyestalks with long tubercle; first maxilliped with exopod not segmented distally; fourth and fifth pereopods with dactyl slender, entire. Petasma as wide lamina with developed elongate PE; appendix masculina bilamellate, with inner lobe spinose; thelycum elaborated, formed by sixth to eighth thoracic segments.4.1.2.Gennadas with independent spermathecal orifices  the distolateral lobe of the petasma is entire, not subdivided; whereas in all species with orifices contained within a common atrium , the distolateral lobe is bifurcated’. On the basis of these findings, he divided Gennadas into two groups, which were followed hereafter. However, this subdivision of Gennadas could not be further substantiated by our work. First of all, the information revealed by SEM showed that in most Gennadas, the thelycum is more or less closed and the orifices are covered by the shields either anteriorly or posteriorly. Open thelyca were found only in G. incertus and G. propinquus , in which the PE  of the petasma was either divided  or entire . Second, there are numerous exceptions from Burkenroad's rule, for example G. barbari , and G.Gennadas consists of two major phylogenetic lineages: G. elegans and the rest of Gennadas. Contrary to the core Gennadas but like other Benthesicymidae (Bentheogennema and Benthesicymus), G. elegans has a simple petasma without LA, PM and grasping structure on PI. However, the morphology of the thelycum in G. elegans is elaborate and more similar to the core Gennadas than to the other genera. Our phylogenetic analysis did not support grouping this species with the core genus. Gennadas elegans probably should be placed in a separate genus Amalopenaeus  in ordeGennadas further branches into four very robust clades, each consisting of two to five species. To better represent the phylogenetic information in the classification, we subdivide Gennadas into four species groups and treat G. elegans as separate from these.Phylogenetic analysis has shown that the core G. elegansDiagnosis: (figures a and 4a) figures a and 4aThelycum: specialized setae absent; sixth sternite with posteriorly directed scutum in the anterior part and with W-shaped posterior prominence; seventh sternite undivided, as a simple oval plate.Petasma: PI without distal grasping structure, LA and PM absent, PE leaf-like. Both lobes of appendix masculina spinose.G. valens species groupDiagnosis: Thelycum: specialized setae present; sixth sternite simple subtriangular, without scutum; seventh sternite divided into two shields, anterior shield bat-like, lateral edges not reaching coxae, posterior shield trapezoid, with anterior incision, lateral edges not reaching coxae, anteriorly armed with a row of strong spines; eighth sternite with median groove in the posterior part, without scutum.Petasma: PI with distal grasping structure, LA long and distally extended, directed nearly orthogonal to main lamina, PM apically cleft into smaller interior and larger exterior lobules, PE with rounded apical notch as bit of key. Outer lobe of appendix masculina unarmed.G. talismani and G. valens.Species included: G. brevirostris species groupDiagnosis: Thelycum: specialized setae present; sixth sternite simple subtriangular; seventh sternite divided into two shields, posterior shield as long chitinized strip, laterally produced beyond coxae, with two groups of lateral setae in addition to two groups of sublateral setae.Petasma: PI with distal grasping structure, LA, PM and PE present. Outer lobe of appendix masculina unarmed.G. brevirostris, G. burkenroadi, G. propinquus and G. scutatus.Species included: G. bouvieri species groupDiagnosis: Thelycum: specialized setae present; sixth sternite simple subtriangular; seventh sternite divided into two parts, both simple and unspecialized, without prominent relief, posterior part as soft plain narrow strip.Petasma: PI with distal grasping structure, LA, PM and PE present. Outer lobe of appendix masculina unarmed.G. bouvieri, G. capensis, G. incertus and G. kempi.Species included: with five species: G. parvus species groupDiagnosis: (figures 2b–f and 4b–f)Thelycum: specialized setae present; sixth sternite simple subtriangular; seventh sternite divided into two shields, anterior shield bilobed, with medial depression, lateral edges not reaching coxae, posterior shield subtriangular, with beak-like anterior elevation, lateral edges not reaching coxae.Petasma: PI with distal grasping structure, LA, PM and PE present. Outer lobe of appendix masculina unarmed.G. barbari, G. gilchristi, G. parvus, G. sordidus and G. tinayrei.Species included: with five species: 4.1.3.Gennadas Spence Bate, 1881Telson armed with a single pair of terminal spines  — — Telson armed with lateral spines in addition to terminal spines  — 2Bentheogennema Burkenroad, 1936Fifth abdominal somite without dorsal carina  — — Fifth abdominal somite with dorsal carina on the posterior part at least  — 3Dactyli of fourth and fifth pereopods greatly elongate and subsegmentedBenthonectes Smith, 1885  — — Dactyli of fourth and fifth pereopods ordinary, unisegmented  — 4Rostrum dorsally unarmed or bearing a single rudimentary dorsal tooth. Third abdominal segment with well-developed dorsal carina. Petasma: PE greatly overreaching PIAltelatipes Crosnier & Vereshchaka, 2008  — — Rostrum dorsally armed with one or more well-developed dorsal teeth. Third abdominal segment without well-developed dorsal carina. Petasma: PE not overreaching PIBenthesicymus Spence Bate, 1881  — Key to Genera of BenthesicymidaeGennadasa); both lobes of appendix masculina spinose. Thelycum: S6 with posteriorly directed scutum in the anterior part and with W-shaped posterior prominence; S7 undivided, as a simple oval plate (a)Petasma: PI without distal grasping structure; LA and PM absent a; both lal plate aG. elegans   — — Petasma: PI with distal grasping structure; LA and PM present; outer lobe of appendix masculina unarmed. Thelycum: S6 simple subtriangular; S7 divided, not as a simple oval plate  — 2g,h). Thelycum: PS7 trapezoid, with anterior incision, lateral edges not reaching coxae, anteriorly armed with a row of strong spines; S8 with median groove in the posterior part Petasma: LA club-like; PE with a rounded apical notch as a bit of a key g,h. Thelior part g,hG. valens species group)  — 3 . Thelycum: S8 anteriorly convex, unarmed (g)— Gennadas valens Petasma: PM with lobules subequal in width g. Thelyc unarmed g— Gennadh). Thelycum: S8 anteriorly bilobe, with two spinose projections (h)— Gennadas talismani Bouvier, 1906— Petasma: PM with lobules unequal in width h. Thelycjections h— Gennadc,d,g,h)Thelycum: PS7 as a narrow chitinized strip, produced laterally beyond coxae, with two groups of lateral setae in addition to two groups of sublateral setae c,d,g,hG. brevirostris species group)  — 5 . Thelycum: AS7 as two separate lateral ear-like structures; PS7 as a W-shaped strip; S8 with short anteriorly spinose scutum — 6Petasma: LA laterally expanded, overlapping PI in the lateral direction; PE narrow c,d. Thele scutum c,d— 6g,h). Thelycum: AS7 W-like, with lateral edges reaching coxae; PS7 as a nearly linear strip; scutum of S8, if present, not short and spinose — 7— Petasma: LA not overlapping PI in the lateral direction; PE wide g,h. Thel spinose g,h— 7c). Thelycum: S6 with scutum directed posteriorly; S8 with scutum concave, armed with uninterrupted row of spines (c)Petasma: LA without hooks; PM divided; PE unarmed c. Thelycf spines cGennadas brevirostris Bouvier, 1905  — d). Thelycum: S6 with scutum directed anteriorly; S8 with scutum truncate, armed with an interrupted row of spines (d)— Petasma: LA with an apical hook, PM entire; PE serrate d. Thelycf spines dGennadas burkenroadi   — g). Thelycum: S8 without scutum (g)Petasma: PE entire g. Thelyct scutum gGennadas propinquus Rathbun, 1906  — h). Thelycum: S8 with a long unarmed scutum (h)— Petasma: PE apically cleft h. Thelycd scutum hGennadas scutatus Bouvier, 1906  — a,b,e,f)Thelycum: PS7 plain, without prominent relief, as an unchitinized narrow strip a,b,e,fG. bouvieri species group)  — 9 (b–f)— Thelycum: PS7 with prominent relief, subtriangular b–fG. parvus species group)  — 12 . Thelycum: AS7 chitinized, subrectangular, with a pair of lateral locks overlapping scutum — 10Petasma: LA soft; PM nearly reaching the end of PI a,b. Thelg scutum a,b— 10e,f). Thelycum: AS7 unchitinized, as plain strip, without lateral locks overlapping scutum — 11— Petasma: LA rigid; PM not reaching the end of PI e,f. Thelg scutum e,f— 11a). Thelycum: S6 with large posteriorly extended scutum in the posterior part (overlapping AS7 and PS7); S8 with a pair of anterolateral spinose extensions (a) — Gennadas bouvieri Kemp, 1909Petasma: PM and PE bifid a. Thelyctensions a — Gennab). Thelycum: S6 without scutum; S8 without spinose extensions (b)— Gennadas kempi Stebbing, 1914— Petasma: PM and PE entire b. Thelyctensions b— Gennade). Thelycum: S6 with scutum directed posteriorly; S8 without shield (e)Petasma: grasping structure greatly enlarged, inflated; PM entire; PE shallowly notched e. Thelyct shield eGennadas capensis Calman, 1925  — f).— Petasma: grasping structure small, not inflated; both PM and PE deeply cleft f.f)Thelycum: S6 with scutum directed anteriorly; S8 with a large oval shield fGennadas incertus   — e,f). Thelycum: beak-like structures on S6 and AS7 forming forceps present; S8 without shield Petasma: PT with a greatly expanded apron-like apical structure e,f. Thelt shield e,f  — 13b–d). Thelycum: no beak-like structures on S6 and AS7 not forming forceps; S8 with anterior shield (b–d)— Petasma: PT without greatly expanded apron-like apical structure b–d. Thelr shield b–d  — 14e). Thelycum: AS7 with a pair of spinose lateral protuberances at base of fourth pereopods (e)Petasma: LA with apron-like apical structure apically concave e. Thelycereopods eGennadas parvus Spence Bate, 1881  — f). Thelycum: AS7 without spinose lateral protuberances at base of fourth pereopods (f)— Petasma: LA with apron-like apical structure apically convex f. Thelycereopods fGennadas sordidus Kemp, 1910  — b). Thelycum: AS7 without shield; S8 with anteriorly setose shield (b)— Gennadas barbari Vereshchaka, 1990Petasma: PE deeply cleft b. Thelyce shield b— Gennadc,d). Thelycum: AS7 with anteriorly directed bilobed shield; shield on S8 not setose — 15— Petasma: PE shallowly notched c,d. Thelt setose c,d— 15c). Thelycum: no setose medial processus extending posteriorly from the fifth thoracic segment; scutum of S8 anteriorly concave (c)Petasma: PM divided; PE unarmed c. Thelyc concave cGennadas gilchristi Calman, 1925  — d). Thelycum: fifth thoracic segment with setose medial processus extending posteriorly; scutum of S8 anteriorly convex (d)— Petasma: PM entire; PE with lateral lobule serrate d. Thelycy convex dGennadas tinayrei Bouvier, 1906  — Key to Species of 4.2.The phylogenetic tree shows that the medium-level clades (the species groups) are primarily supported by thelycum-related characters, while petasma-related characters are more important for the support of the terminal clades. The greater importance of the thelycum for the main branches of the tree is also illustrated when comparing the thelycum-based and the petasma-based MP trees: the female tree is much more resolved than the male tree and has the same topology as the ‘total evidence’ tree. It is remarkable that the terminal clades (species pairs) are similar in all trees. However, the clade support is weaker in the male tree, where some species group-level clades fail to appear. We here hypothesize that the thelycum-based characters were first to appear in evolution followed by the petasma-based characters. Among these possibly co-evolving characters are the structure of the sixth and seventh thoracic sternites and the presence of specialized groups of setae on thelycum.Other thelycum-related characters are much less conservative. Even such remarkable species-diagnostic characters as scuti, occurring in different parts of the thelycum (from S6 to S8) and being very characteristic in shape, size and armature, seem evolved later in evolution, at the species level. In most species, they are not fully homologous, which is indicated by the fact that they occur on different somites.The petasma is generally an evolutionary more plastic character than the thelycum. Only presence of the major lobes  determining the rough topology of the petasma is conservative. Such characters as further division and shape of the lobes, the presence of additional lobules and their serration, etc. are species-specific and have appeared late.Gennadas were the first to evolve elaborate sexual structures externally (topology of sternites) later followed by males, in which a very specific petasma is present in each species. The lobules of the petasma, which are very specific in size and shape, probably evolved to fit species-specific parts of the thelycum (scuti and microrelief).We thus suggest a ‘lock and key’ hypothesis and tentatively conclude that females of 4.3.An analysis of the coevolution between central aspects of the sexual structures showed a couple of statistically significant correlations: (i) between the external part of the petasma and the posterior part of the thelycum and (ii) between the internal and medial parts of the petasma and the anterior part of the thelycum.in situ observations of copulation in Dendrobranchiata are very rare for coastal species and entirely absent for pelagic ones. Bauer [Sicyonia dorsalis. He found that the position of the male was at right angles below the female during copulations and that males were able to inseminate only the spermatheca on one side per successful copulation. He proposed that the petasma is not a sperm injection device; instead, this organ is used to hook onto the female thelycum, adjusting the proper position during copulation.The explanation for the coevolution between certain sexual structures in females and males must be that these structures interact functionally during copulation. However, s. Bauer  was amonGennadas, we reconstruct the details of spermatophore deposition based on which parts of the petasma and thelycum fit to each other. Gennadas. It is clear that in all cases, PE of the petasma always fits to S8 of the thelycum, and that the grasping structure of PI of the petasma fits to the seminal receptacles of the thelycum. The grasping structure, which is synapomorphic for the core Gennadas, may serve for carrying the spermatophore and fixing it to the receptacles. It is noteworthy that the nearly terminal position of the grasping structure on the petasma facilitates receiving the spermatophores from the genital apertures, which face the dorsal (anterior) side of the petasma.Assuming that a comparable mechanism exists in pelagic G. incertus (a) has an unusual fork-like PE of the petasma, which may be passed under the cap of the mushroom-like structure on S8 of thelycum . The apron-like apex of LA in the petasma of G. parvus is suitable for hanging onto the coxa of the third pereopod (b). The mitten-like PM of the petasma of G. scutatus and G. talismani may be adapted to pass under the scutum of the former and anterior setose projections of S8 of the latter .In some species, the petasma and the thelycum both have very peculiar structures, which provide further evidence for how female and male structures fit with each other during copulation. For example, incertus a has an pereopod b. The mie latter c,d.4.4.Gennadas of Benthesicymidae is characterized by a simpler (but still elaborate) petasma and by the most elaborate thelycum among the eucarids. The thelyca of Gennadas species play a larger role in stable copulation and sperm transfer than in other eucarids.Recent analyses have suggested that the elaborate male petasmata are important for successful colonization of the pelagic zone by shrimp-like eucarids . In the Gennadas is among the most widely distributed pelagic genera. Indeed, four species (25%) are panoceanic: G. bouvieri, G. capensis, G. scutatus and G. tinayrei. Four species live in the Atlantic , four species live in the Indo-Pacific  and two species live in the Indo-West Pacific (G. gilchristi and G. kempi). There are only two species with regional distribution: G. burkenroadi from the northeast Pacific and G. barbari from the southeast Pacific. The latter is benthopelagic and associated with seamounts of the Nazca and Sala-y-Gomez Ridges, which may explain its restricted distribution [ribution ,10.Gennadas and the absence of significant individual variations in these structures both favour sexual isolation between species, which are otherwise ecologically similar in the mesopelagic habitat.The greatly elaborate copulatory structures of


Large area, fullerene‐free organic solar cells with improved stability and efficiency of up to 1% are fabricated by the roll‐coating process on indium tin oxide free and flexible substrates, under ambient conditions. The solar cell is one of the best candidates for clean energy and sustainable development. Especially, the bulk heterojunction (BHJ) organic solar cells (OSCs) are promising because of some advantages, such as simple preparation, low cost, light weight, and large area flexible fabrication. This has been the focus of research in the past decade.61BM and PC71BM) are widely used as electron acceptors in OSCs because of ultrafast photoinduced charge transfer at donor/acceptor (D/A) interfaces,VOC), weak absorption in the visible region, relatively high cost,Nowadays, fullerene derivatives 2),2) on ITO/glass substrates, with vacuum evaporated electrodes and nitrogen protection.Although FF‐OSCs potentially have a bright future, compared with fullerene based OSCs, they are still far away from industrial manufacture. Fullerene‐based OSCs can be fabricated on large area , flexible substrates, ITO‐free, vacuum‐free, and preparation under ambient conditions. Moreover, relative to PC71BM‐based control devices, the FF‐OSCs exhibited much better stability.In this work, we report FF‐OSCs based on a small molecule nonfullerene acceptor fabricated by the roll‐coating process. The structure of inverted FF‐OSCs is shown in Figure−1 in total. The ink was roll‐coated on four stripes on a flexible PET substrate, precoated with ZnO on high conductive PEDOT:PSS PH1000 cathodic stripes   Figure c. The coC Figure d. FinallC Figure e were thJ–V curves of FF‐OSCs with different D/A weight ratio, wet thickness of active layer, and thermal annealing time are shown in Figures S1, S2, and S3 of the Supporting Information, respectively. Table S1, Supporting Information, summarizes the average and best device data of FF‐OSCs under different device conditions. The optimized device conditions were D/A weight ratio of 1:1.25, wet thickness of active layer of 9 μm, and thermal annealing at 120 °C for 1 min. FigureJ–V and external quantum efficiency (EQE) spectra of FF‐OSCs with different processing solvent (chloroform (CF); o‐xylene; chlorobenzene (CB), and dichlorobenzene (DCB)) and thermal annealing under the optimized device conditions. Figure TableVOC, short circuit current density (JSC) and fill factor (FF). The VOC of devices processed with CF, DCB, and CB solvents was similar and much higher than that of o‐xylene processed devices. The FF of devices processed with CF and CB was higher than those of o‐xylene and DCB processed devices. The devices processed with CB had higher JSC and PCE; the average VOC, JSC, FF, and PCE of these devices was 0.7748 V, 2.847 mA cm−2, 37.22%, and 0.821%, respectively. Thermal annealing led to performance improvement in FF‐OSCs. The solar cells exhibited the best performance after thermal annealing at 120 °C for 1 min; average VOC increased from 0.7748 to 0.7983 V, average JSC increased from 2.847 to 3.086 mA cm−2, average FF increased from 37.22% to 40.11%, and average PCE increased from 0.821% to 0.988%. The best PCE of these devices was 1.019%. As shown in Figure JSC. To evaluate the accuracy of the photovoltaic results, the JSC values were calculated from integration of the EQE spectra with the AM 1.5G reference spectrum. The calculated JSC was similar to J–V measurement .Figure71BM (processing by DCB solvent and 5% DIO) devices in air under continuous AM1.5G illumination for 180 h. The PCE of PC71BM‐based device decreased more quickly than that of nonfullerene acceptor‐based device. The polymer/nonfullerene device showed much better stability (maintained 85% PCE after 180 h) relative to polymer/fullerene device (maintained 50% PCE after 180 h). Figure71BM films before and after 180 h stability test. The appearance of PBDTTT‐C‐T/DC‐IDT2T film did not change, while some black dots (≈10 μm) were clearly observed in PBDTTT‐C‐T/PC71BM film after 180 h stability test, which decreased the PCE. Figure71BM films before and after 180 h stability test. The measured current values for each point can be translated into a scale of different color hues and represented as a map showing areas of high and low current response to the incident light.71BM films after the 180 h stability test became darker , flexible substrates, ITO‐free, vacuum‐free, and ambient conditions. The nonfullerene‐based device showed much better stability than the fullerene‐based control device. Our preliminary results demonstrate that nonfullerene small molecule acceptors could be promising for industrial manufacture of high‐performance OSCs.In summary, we report FF‐OSCs based on polymer donor PBDTTT‐C‐T and nonfullerene small molecule acceptor DC‐IDT2T fabricated by the roll‐coating process. The solar cells fabricated using processing solvent CB and thermal annealing exhibited the best performance due to the uniform and smooth surface of the blended film. The best PCE of 1.019% was achieved with large device area  should be addressed to the authors.SupplementaryClick here for additional data file.


The aim of this study is to examine whether advanced magnetic resonance imaging (MRI) techniques can detect early brain injury caused by intrauterine inflammation and inappropriate initial respiratory support in preterm lambs.Neuropathology caused by intrauterine inflammation is exacerbated by mechanical ventilation at birth and is detectable with advanced MRI techniques.n = 6) or protective  ventilation strategy. MRI of the brain was conducted 90 min after preterm delivery, using structural, diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS) techniques. A colour map threshold technique was utilised to compare distributions of low diffusivity voxels in the brains of LPS-exposed lambs with those not exposed to LPS .Pregnant ewes received intra-amniotic lipopolysaccharide (LPS) 7 days prior to delivery at ~125 days of gestation (85% of gestation), whereupon lambs were delivered and randomised to receive an injurious (LPS + INJ, N-acetylaspartate, were not different between LPS-exposed groups. Lambs exposed to LPS had lower diffusivity within the white matter regions assessed than non-LPS-treated control lambs.No overt cerebral injury was identified on structural MRI images of any lamb. However, on DTI, axial diffusivity, radial diffusivity, and mean diffusivity values were lower and significantly more heterogeneous in specific brain regions of lambs in the LPS + INJ group compared to the LPS + PROT group. Colour mapping revealed lower diffusivity in the thalamus, periventricular white matter, internal capsule, and frontal white matter in the LPS + INJ group compared to LPS + PROT group. The MRS peak area ratios of lactate, relative to those for the metabolites creatine, choline, and DTI colour map threshold techniques detected early brain injury in preterm lambs exposed to intrauterine inflammation and detected differences between injurious and protective ventilation strategies. DTI mapping approaches are potentially useful for early detection of subtle brain injury in premature infants. Infants born preterm are at a high risk of brain injury, with the rate of neurodevelopmental disability exceeding 50%, and cerebral palsy rates at ~10% . FurtherIntrauterine inflammation, clinically defined as chorioamnionitis, is a common antecedent of preterm birth , 9 and aMRI is commonly used in neonatology as a diagnostic and prognostic tool and is usually conducted several days to weeks after birth , 21. GivWe have previously demonstrated the utility of DTI in detecting early brain injury using a region of interest (ROI) analysis . An alteT) ventilation after chronic intrauterine inflammation exacerbates brain injury compared to protective ventilation and is detectable using DTI on a clinical MR scanner.The objective of this study was to develop new techniques to detect early brain injury using diffusion-weighted MRI that have the potential to improve future diagnostic strategies for identifying abnormalities in white matter. In particular, we aimed to examine whether non-invasive MRI techniques can differentially detect brain injury caused by injurious or protective ventilation in preterm lambs exposed to intrauterine inflammation, factors known to cause early brain injury. Although we have demonstrated that both protective (PROT) and injurious (INJ) ventilation strategies increased brain inflammation and oxidative stress, and more so in the INJ group , it has The experimental protocol was approved by Monash Medical Centre animal ethics committee at Monash University and conducted in accordance with guidelines established by the National Health and Medical Research Council (NH&MRC) of Australia.Escherichia coli 055; Sigma-Aldrich, Australia) were administered to one amniotic sac of twin-bearing ewes  at 118 ± 2 days of gestation (mean ± SD). Lambs were delivered by caesarean section 7 days later, dried, and a transcutaneous oxygen saturation probe was placed around a forelimb . The lambs were intubated and then randomised to receive either a protective ventilation strategy  or injurious ventilation strategy , as described previously (T ventilation (targeting 12–15 mL/kg) using a neonatal positive-pressure ventilator . After 15 min, LPS + INJ lambs received surfactant , followed by tidal ventilation targeting VT of 7 mL/kg. LPS + PROT lambs received prophylactic surfactant, a single 30-s sustained inflation at peak inspiration pressure (PIP) 35 cmH2O , followed by tidal ventilation (7 mL/kg) for 3 min prior to umbilical cord clamping . Samples of arterial blood were collected every 5 min for the initial 15 min and at 15-min intervals thereafter for measurement of the partial pressure of arterial carbon dioxide (PaCO2), oxygen (PaO2), oxygen saturation (SaO2), and pH . Ventilation and oxygen delivery were altered to target PaCO2 of 45–55 mmHg and an oxygen saturation of 90–95%. Ewes were killed humanely (sodium pentobarbitone 100 mg/kg i.v.) immediately after delivery of the lambs, while still under general anaesthesia. Lambs were killed humanely (sodium pentobarbitone 100 mg/kg i.v.) after the MRI.After delivery, umbilical arterial and venous catheters were inserted for measurement of blood pressure, arterial blood gas sampling, and invasive real-time monitoring of heart rate (HR) . Anaesthesia was maintained 2 were continuously monitored to guide anaesthesia maintenance, and blood gas parameters were recorded at 15-min intervals to monitor lamb well-being and guide mechanical ventilation. Scans were performed on a 3T MR scanner  at Monash Biomedical Imaging, Monash University , with a 15-channel radio frequency (RF) coil for both RF transmission and reception. The MRI protocol comprised structural imaging sequences , susceptibility-weighted imaging (SWI), and a single-voxel MRS . HR and SpOoxel MRS . The totA 3D MPRAGE sequence was applied to achieve T1-weighted images, 102.5 mm thick, for whole brain coverage. The repetition time (TR) was 1,440 ms, with echo time (TE) of 3.92 ms, an inversion time of 900 ms, and a flip angle of 9°. The data matrix was 256 × 256 × 128, collected from a field of view (FOV) of 200 mm × 200 mm, giving a voxel size of 0.78 mm × 0.78 mm × 0.8 mm.T2-weighted images with a 3D SPACE sequence, 44 mm thick, were also obtained to provide whole brain coverage . The matrix size was 384 × 384 × 88, collected from a FOV of 192 mm × 192 mm, giving a voxel size = 0.5 mm × 0.5 mm × 0.5 mm.A standard 3D gradient echo sequence was used to acquire SWI for the whole brain with a slab thickness of 72 mm . The matrix size was 288 × 216 × 60, collected from a FOV of 176 mm × 132 mm, giving voxel size = 0.6 mm × 0.6 mm × 1.2 mm. The second phase encoding direction for 1.2-mm voxel was acquired in a parallel direction to the main magnetic field to optimise the susceptibility effect.b0. Diffusion encoding gradients were acquired in 30 directions with a b value of 1,000 s/mm2. The diffusion-weighted imaging was performed twice, and five b = 0 volumes were acquired.Diffusion tensor imaging with a spin-echo echo planar imaging sequence in the axial plane was obtained with 50 contiguous 1.2-mm slices . The data matrix was 128 × 128, collected from a FOV of 154 mm × 154 mm, giving a voxel size of 1.2 mm × 1.2 mm × 1.2 mm; for five A single-voxel spin-echo sequence was used to acquire MRS, localised on the supratentorial deep grey matter and central white matter  with a voxel size of 15 mm × 15 mm × 20 mm.b0 image was used as the reference for the coregistration.MRI examination, image processing and analysis, and interpretation of the structural MR images were performed completely blind. Once ROI was drawn, the group allocation of each lamb was revealed for the final statistical analysis. The colour mapping approach was then completed, but not performed in a blinded fashion. Images acquired from T1, T2, and SWI scans were used to determine the presence of gross brain injury including infarcts and haemorrhages. DTI images were converted into NIFTI format, and then a brain mask was manually placed over the image using the FMRIB Software Library . DTI daROI were defined manually on high-resolution T2 images and then exported to the FA, AD, MD, and RD maps to calculate the region mean values for each lamb. ROIs were placed in specific regions including the thalamus (Th), periventricular white matter (PVWM), internal capsule (IC), frontal white matter (FWM), and the cerebellum (CB) vermis, targeting the stalk and midline white matter Figure . The SheN-acetylaspartate (NAA) , creatine , and choline  were calculated. In addition, we calculated peak area ratios for NAA/Cho, NAA/Cr, and Cho/Cr.MRS data were processed using TARQUIN software to implement spectral fitting in the time domain . Peak arDTI data from the ROIs appeared to show more heterogeneity in the injured lamb brains. To further examine this variability, we utilised a colour map threshold technique to determine where the low diffusivity voxels were distributed in the DTI volumes. ImageJ  was used to create a histogram of the distribution of the MR image pixel intensities at each position. For the purposes of comparison between animals, the histograms from all lambs in the two groups were normalised. The total pixel intensity distribution (histograms) for each lamb brain was compared between the minimum and maximum intensities within the brain for 256 bins in all DTI images. Histograms of the diffusivities for the whole brains of individual lambs in the two groups exposed to LPS were determined . The LPS + INJ group was compared to the LPS + PROT group to assess between-group differences in DTI and MRS parameters. The mean and SEM of voxel intensities within the predefined ROIs were calculated, and independent t-tests were used to compare treatment groups with the significance level set at P < 0.05. Data from the ROIs are presented as mean (box 5-95% CI of mean) with maximum − minimum error bars. Further, we calculated the SD for the ROIs in each lamb’s brain to test within-animal variability and to test the heterogeneity of DTI diffusivities for differences between groups using MATLAB  software.Physiological data were compared using a two-way repeated measure ANOVA and Holm–Sidak LPS was successfully injected into the amniotic sac as confirmed by electrolyte analysis of amniotic fluid aspirated at the time of injection and the presence of thickened fetal membranes characteristic of this experimental intervention observed at delivery .Mean gestational age (125 ± 2 days), body weights , and sex distributions of the lambs  were not different between groups.P < 0.001), resulting in a significantly higher VT during the initial 15 min than the LPS + PROT group . Oxygen saturation (SaO2) did not differ significantly between groups after the first 5 min of ventilation .Carotid blood flow was variable within groups and between groups, but overall there was no significant differences observed  Figure  as well While mean AD, RD, and MD values were lower in the LPS + INJ lambs compared to the LPS + PROT lambs in many of the ROIs Figure , no statColour map techniques were used to describe the tensor orientation in relation to the white matter tract direction . HistogrIn the low diffusivity maps obtained from the lambs exposed to LPS during the perinatal period, the red shading (threshold range) was more visible in the Th of AD and RD maps Figure  and 7. IAs a control reference, we applied the colour map technique to lambs that underwent the same ventilator and MRI protocols but were not exposed to LPS (PROT and INJ control lambs); the physiology and other MRI data have been reported previously . Lambs fPreterm neonates exposed to intrauterine inflammation are at an increased risk of ventilation-induced brain inflammation and injury . In thisAdvanced MRI methods such as DTI and MRS provide useful information about structural connectivity and altered brain chemistry, allowing improved detection of newborn brain injury over conventional MRI –33. Howeex vivo MRI analyses that were strongly correlated with histopathological evidence of injury  between PROT and INJ groups following LPS administration. These ratios are indicative of brain metabolism and correlate with neonatal brain injury  and subsThere are some limitations to this study. We did not correlate our quantitative DTI findings with histological assessment to conclusively demonstrate that these alterations truly represent microstructural changes exacerbated by multiple risk factors such as injurious ventilation and chronic inflammation. Further, conducting longer term studies is required to investigate whether the early injury markers manifest as disability later in life. Moreover, we concede that the greatest limitation of the application of this imaging and analysis approach to very preterm and very low-birth-weight neonates in the NICU is that it is difficult to image ventilated babies in an MR scanner within hours of birth, and this would be discouraged by the general policy of minimal handling of such neonates. However, if we can develop robust strategies that can accurately determine brain injury, then the potential benefit of early identification and treatment would likely outweigh the minimal handling policies of such units. The use of new MRI-compatible cribs might make such early diagnosis easier to implement.In conclusion, the use of colour mapping demonstrated that injurious ventilation after LPS was associated with low AD, RD, and MD in the cerebral white matter of preterm lambs, which was not detected using standard MRI techniques. Colour mapping also demonstrated greater alterations in water diffusivity, and therefore neural function, in LPS lambs compared to controls. The colour map technique might be a sensitive way to detect early white matter injury and may lead to improved detection of preterm brain injury within a suitable time frame for clinical intervention.All authors made substantial contributions to the conception or design of the work, or the acquisition, analysis, or interpretation of data for the work; revised drafts; and approved the final version to be published.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare that they do not have any conflicts of interests.


Saliva and plasma samples were collected prior to and after a standard evening meal at specific intervals. Plasma non-esterified fatty acid (NEFA) levels were significantly higher pre-meal in DL compared to BL (P < 0.01). Plasma glucose and insulin levels were significantly greater post-meal in the BL compared to DL session , respectively. Salivary melatonin levels were significantly higher in the DL compared to those in BL session (P = 0.005). BL at night was associated with significant increases in plasma glucose and insulin suggestive of glucose intolerance and insulin insensitivity. Raised pre-prandial NEFA levels may be due to changes in insulin sensitivity or the presence of melatonin and/or light at night. Plasma triglyceride (TAG) levels were the same in both sessions. These results may explain some of the health issues reported in shift workers; however, further studies are needed to elucidate the cause of these metabolic changes.Many animal studies have reported an association between melatonin suppression and the disturbance of metabolic responses; yet, few human studies have investigated bright light effects on metabolic and hormonal responses at night. This study investigated the impact of light on plasma hormones and metabolites prior to, and after, an evening meal in healthy participants. Seventeen healthy participants, 8 females (22.2 ± 2.59 years, mean ±  Artificial light exposure at night has become commonplace throughout the developed world , 2. LighA majority of the previous studies investigating the effect of light on hormone and metabolic responses have either been carried out on experimental animals  or underAll procedures received a favourable ethical opinion from the University of Surrey Ethics Committee (UEC/2013/93/FHMS) and were conducted in accordance with the Declaration of Helsinki (1975) as revised in 1983 and conformed to international ethical standards. Volunteer information was coded and held securely in compliance with the Data Protection Act, UK (1998). All participants gave written informed consent after full explanation of the purpose and nature of all procedures involved.s.d.) and 9 males (22.8 ± 3.5 years) were recruited from students and staff at the University of Surrey. The two genders were matched for age and body mass index (BMI)  . ParticiAll participants maintained a standard self-selected regular sleep–wake cycle  for at least 7 days before the in-laboratory sessions, as confirmed by actigraphy  and sleep diaries . 24-h prAll participants were randomised to a two-way cross-over design protocol; dim light and bright light. All study sessions were held at the Clinical Investigation Unit (CIU), which was equipped with overhead light control. During the dim light session, lighting levels were <5 lux and in the bright light session, lighting was >500 lux between 18:00 h and 06:00 h the next day . ParticiBlood samples were collected hourly from 18:00 h until the evening meal; then, every 15 min for the first hour after the meal, then at 30-min intervals until the end of the session. In total, 22 blood samples were collected in each session from each participant for analysis of insulin, glucose, triglyceride (TAG) and non-esterified fatty acids (NEFAs). Saliva samples were collected every 30 min from 18:00 h to 06:00 h the following day for analysis of melatonin levels.n = 221; DL 1.06 ± 0.06 lux, BL 305 ± 10.1 lux; mean ± s.e.m.) and vertical level towards the lights .Light intensity was measured at 2 different positions horizontal level (direction of gaze)  and vertical level . The light source provided in both studies are fluorescent light, and the spectral composition of the light is shown in Spectral composition of the light source was measured using a R203 power radiometer at horizontal  and NEFAs  were analysed by standard automated enzymatic spectrophotometric methods (ILAB600). The interassay coefficients of variation were less than 5% for glucose, NEFAs and TAGs. Plasma immunoreactive insulin was measured using radioimmunoassay (RIA) (Millipore). Salivary melatonin and urinary aMT6s were analysed using in-house RIAs , 26. TheAn index of fasting insulin resistance (HOMA-IR) and postprandial insulin resistance (HOMA-PP) were determined for the evening meal in both BL and DL sessions.HOMA-IR was calculated using the HOMA calculator based on HOMA model 2 developed by Jonathan Levy .HOMA-PP was calculated as the incremental area under the curve (IAUC) glucose (mmol/L min) × IAUC insulin (U/L min).This equation has been validated against the intravenous glucose tolerance .A power calculation was performed using PS software  with a power of 80% and a significance level 0.05 utilising NEFA data obtained from a previous pilot investigation. From this power calculation, ≥18 participants were required.Urine aMT6s data were subjected to cosinor analysis , to ascertain calculated peak time of aMT6s (acrophase).s.d.) and standard error of mean (s.e.m.) were calculated from individual data sets. All hormonal and metabolic data were subjected to three-factor repeated measures ANOVA  followed by Tukey’s honest significance post hoc test to locate individual differences, using Statistica Statsoft . The trapezoidal rule was used to determine the total area under curve (TAUC). All hormone and metabolite data were analysed using TAUC, followed by 2-tailed paired Student’s t-test. The significance level was set at P < 0.05.All data were checked for normality using D’Agostino Pearson omnibus normality test (Graphpad). The mean value plus the standard deviation  are shown in P = 0.02). No significant difference was seen in basal TAGs between DL and BL (P = 0.81). Basal melatonin levels were significantly greater in DL than those in BL (P < 0.001)  .Figure P = 0.03). Similarly, salivary melatonin was significantly higher in the DL session (P < 0.001)  .Figure P = 0.01, P = 0.008), respectively. Salivary melatonin was significantly greater in DL than that in BL sessions (P < 0.001). There were no differences in postprandial TAGs responses after BL and DL sessions . Post hoc tests showed significant differences at +180, +210 and +270 min. Similarly, plasma glucose showed a significant increase in BL compared to that in DL sessions (P = 0.02). Post hoc test showed significant differences at +180 and +210 min after the meal. In contrast, there was a significant pre-prandial increase of plasma NEFA in DL session (P = 0.005). Post hoc tests showed the difference was directly prior to the evening meal (T = 0). Plasma TAGs showed no significant difference between the DL and BL sessions. All 4 plasma parameters showed significant effects of time, whereas no significant effects of gender were observed.Graphical representations of the female, male and all participants during DL and BL sessions are shown in P < 0.001). Post hoc tests showed significant differences at pre-prandial and postprandial time points between −120 and +330 min. Both males and females show similar increase in salivary melatonin in DL compared to those in BL sessions, females showed higher levels of salivary melatonin than males although not significant . In contrast, a significant increase in TAUCs for plasma insulin and glucose were shown in BL compared to DL (P = 0.004 and P = 0.03), respectively. TAUC of plasma insulin was 129,119 ± 10,343 pmol/L min in DL and 109,875 ± 9817 pmol/L min BL, whereas TAUCs of plasma glucose were 3805.76 ± 60 mmol/L min in DL and 3985 ± 73 mmol/L min in BL. No significant difference was observed in plasma TAGs responses. TAUCs showed a significant suppression of salivary melatonin in BL compared to those in DL sessions (P < 0.0001), and TAUCs were 3171 ± 530 pg/mL min in BL session and 10,362 ± 1777 pg/mL min in DL sessions.TAUCs for, insulin, glucose, NEFAs, TAGs and melatonin were calculated and are shown in HOMA-PP and HOMA-IR are shown in Few studies have investigated the influence of light at night on plasma hormones and metabolites in healthy humans as a majority of the studies reported to have either been carried out on experimental animals  or underThe salivary melatonin profile was significantly reduced by bright light exposure at night. This was expected as the light intensity delivered at the angle of gaze was 305 lux, and previous human studies have shown that 200 lux was sufficient to suppress salivary melatonin by 50% in healthy participants . In thisPlasma glucose responses were significantly elevated three hours after the meal in the BL session, confirmed by TAUC analysis. Insulin levels were significantly elevated after the meal in BL compared to those in the DL sessions. Raised glucose and insulin responses suggest changes in glucose tolerance and insulin sensitivity. Calculation of HOMA-PP and HOMA-IR confirmed these findings although significance was not achieved. These results agree with rodent studies that showed decreased blood glucose and insulin in constant darkness , 15. FonIn contrast, a human study by Rubio-Sastre and coworkers gave exogenous melatonin prior to an oral glucose tolerance test (OGTT) in the morning and evening and resulted in raised insulin and glucose responses compared to placebo . This stT = 0) when melatonin levels were already high in the DL. This potentially suggests the possible stimulatory effect of melatonin on glucagon  at night on metabolic and hormonal responses in healthy young participants. Significantly higher glucose and insulin in the BL session suggests glucose intolerance and insulin insensitivity. Elevated NEFAs level in the DL session prior to the meal could either be due to the stimulatory effects of melatonin on glucagon or the inhibitory effects on insulin, resulting in higher HSL activity. Our recent results could be due to light or melatonin or a combination of the two. These results support the idea that nocturnal lifestyle, such as in night shift work, is likely to be one of the risk factors to health in modern society, including diabetes. Further studies are needed to determine whether melatonin causes the present metabolic changes or other processes are involved.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This research was supported by Abu Dhabi Health Service Company (SEHA) in United Arab Emirates.The authors’ responsibilities were as follows: M S A, B M and S M H designed research; M S A conducted research; M S A analysed data; M S A wrote the paper; M S A, B M and S M H had primary responsibility for final content. All authors read and approved the final manuscript.


PTCL) is a heterogeneous group of aggressive non‐Hodgkin lymphomas typically associated with poor prognosis. Most patients with PTCL receive chemotherapy as first‐line treatment, but many experience rapid relapse. For patients with relapsed/refractory PTCL, responses to treatment and long‐term outcomes tend to worsen with increasing lines of therapy. Romidepsin is a potent class I histone deacetylase inhibitor approved by the US Food and Drug Administration for the treatment of PTCL in patients who have received ≥1 prior therapy. A pivotal phase 2 trial of romidepsin in patients with relapsed/refractory PTCL demonstrated an objective response rate of 25% (33/130), including 15% with confirmed/unconfirmed complete response, and a median duration of response of 28 months. In the analysis presented herein, romidepsin was shown to have similar responses and long‐term outcomes in patients with 1, 2, and ≥3 prior lines of treatment, including in patients with disease refractory to the last prior therapy. Although adverse events increased with increasing lines of treatment, the rate of dose modifications and discontinuations due to adverse events was not significantly different. These data support the use of romidepsin as salvage treatment for PTCL irrespective of the number of prior therapies.Peripheral T‐cell lymphoma ( Peripheral T‐cell lymphoma (PTCL) is a relatively rare, heterogeneous group of mature T‐cell and natural killer (NK) cell disorders which accounted for 5–10% of the estimated 71,850 new cases of non‐Hodgkin lymphoma (NHL) diagnosed in the United States in 2015 N = 205) of patients with PTCL demonstrated that objective response rates (ORRs) and rates of complete response (CR), as well as long‐term outcomes , worsened with each successive line of treatment With the exception of ALK+ ALCL, for which National Comprehensive Cancer Network guidelines suggest first‐line treatment with CHOP  or CHOEP (CHOP+etoposide), there is no well‐defined algorithm for treating patients with PTCL The epigenetic modifying agent romidepsin is a structurally unique, potent, bicyclic class I selective histone deacetylase (HDAC) inhibitor γδ TCL, transformed mycosis fungoides, hepatosplenic TCL, ALK− ALCL, or ALK+ ALCL (restricted to patients with relapsed disease following autologous stem cell transplant). Diagnosis of PTCL for enrollment was histologically confirmed by a local pathologist and then reviewed by a central laboratory  for PTCL subtyping. Eligible patients had relapsed or refractory disease and had received ≥1 prior systemic therapy, measurable disease according to International Working Group criteria (IWG) The details for this phase 2, open‐label, single‐arm, international study  have been reported in detail elsewhere 2 on days 1, 8, and 15 of 28‐day cycles for up to 6 cycles. Patients who had a response  or stable disease (SD) could continue to receive treatment beyond 6 cycles at the discretion of the patient and the investigator. By protocol amendment, patients treated for ≥12 cycles could receive maintenance dosing of 2 rather than 3 doses per 28‐day cycle Romidepsin was administered intravenously over 4 hours at 14 mg/mStudy endpoints have been described in detail elsewhere All descriptive statistical analyses were performed using the SAS statistical software version 9.2 . Differences in response rates were compared using Fisher's exact test. Time to event data was summarized using Kaplan–Meier methods. Differences in DOR, PFS, and OS were compared using the log‐rank test.A total of 131 patients were enrolled; 130 with histopathologically confirmed PTCL were included and one patient diagnosed with diffuse large B‐cell lymphoma was excluded. For this analysis, patients were divided into three groups according to the number of prior systemic treatments . There were a comparable number of patients in each group , there were also no significant differences in response rates, DOR, and survival by number of prior lines of treatment (Table n = 21), the ORR was 24%, including 10% with CR/CRu; median DOR was 3.1 months , median OS was 8.3 months (range: 2.1 to NE), and median PFS was 2.1 months (range: 1.7–5.6). Most patients (18/21) who had received transplants, including 4 of 5 responding patients who had received transplants, had received ≥3 prior lines of treatment.Overall, the responses to romidepsin were similar irrespective of the number of prior lines of treatment. As a benchmark, patients who had received 1 prior systemic therapy  were similar across groups Fig. , rates on = 3).Rates of dose adjustment (romidepsin dose held or reduced) and discontinuation as a result of AEs were not significantly different by number of prior lines of treatment, although rates were numerically highest for patients who had received ≥3 prior lines of treatment Table . The ratN = 57) of heavily pretreated patients with relapsed or refractory PTCL treated with pralatrexate demonstrated that a pattern of gradually declining treatment efficacy with number of prior lines of treatment  was reversed with pralatrexate (median PFS of 134 days and 40% ORR) Given the aggressiveness of PTCL, the limited treatment options, and no accepted standard of care, studies that explore the optimal use of current therapies are important to define a robust treatment algorithm. Generally, there have been limited data published on the impact of line of treatment on the clinical efficacy of therapies. A retrospective analysis . In a recent analysis of a similarly designed National Cancer Institute study of romidepsin in patients with relapsed or refractory PTCL and CTCL, patients with PTCL who had received ≥2 prior treatments had a similar response rate and durability of responses to the overall population The median DOR for romidepsin in the pivotal study of PTCL was 28 months, with no significant difference in DOR across the three most common PTCL subtypes , and the longest response ongoing at 56 months in a patient with AITL The safety profile of romidepsin showed discrete changes with increasing number of lines of treatment. Although differences in the rates of dose modification and withdrawal from treatment due to AEs were not significant, incidences of specific AEs were numerically higher with increasing number of lines of treatment. As romidepsin was introduced in later lines, the incidence of grade ≥3 AEs  tended to increase; most notably, the incidence of thrombocytopenia , dyspnea , and drug‐related neutropenia  increased. Interestingly, a recent analysis of the AE profile of romidepsin in patients with relapsed or refractory PTCL  also showed that patients who received prior monoclonal antibody treatment  had significantly higher incidence of grade ≥3 infection (20% vs. 4%) and neutropenia (50% vs. 14%) versus those who did not receive antibody treatment FF: advisory board and consultancy: Celgene Corporation, Eisai, Seattle Genetics, Kyowa Kirin, Spectrum Pharmaceuticals; honoraria: Celgene Corporation, Spectrum Pharmaceuticals; research funding: Celgene Corporation. BP: advisory board and consultancy: Celgene Corporation; honoraria: Celgene Corporation. HMP: advisory board and consultancy: Celgene Corporation; honoraria: Celgene Corporation; research funding: Celgene Corporation. LS: advisory board and consultancy: Celgene Corporation; research funding: Gloucester. SH: advisory board and consultancy: Janssen Pharmaceuticals, Millennium Pharmaceuticals, Amgen, Inc., Bristol‐Myers Squibb, Celgene Corporation; honoraria: Janssen Pharmaceuticals, Millennium Pharmaceuticals, Amgen, Inc., Bristol‐Myers Squibb, Celgene Corporation; research funding: Celgene Corporation, Millennium Pharmaceuticals, Infinity Pharmaceuticals, Kyowa Kirin, Seattle Genetics, Spectrum Pharmaceuticals. BC, advisory board and consultancy: Celgene Corporation. DC has nothing to disclose.