Subfertility decreases the efficiency of the cattle industry because artificial insemination employs spermatozoa from a single bull to inseminate thousands of cows. Variation in bull fertility has been demonstrated even among those animals exhibiting normal sperm numbers, motility, and morphology. Despite advances in research, molecular and cellular mechanisms underlying the causes of low fertility in some bulls have not been fully elucidated. In this study, we investigated the metabolic profile of bull spermatozoa using non-targeted metabolomics. Statistical analysis and bioinformatic tools were employed to evaluate the metabolic profiles high and low fertility groups. Metabolic pathways associated with the sperm metabolome were also reported.A total of 22 distinct metabolites were detected in spermatozoa from bulls with high fertility (HF) or low fertility (LF) phenotype. The major metabolite classes of bovine sperm were organic acids/derivatives and fatty acids/conjugates. We demonstrated that the abundance ratios of five sperm metabolites were statistically different between HF and LF groups including gamma-aminobutyric acid (GABA), carbamate, benzoic acid, lactic acid, and palmitic acid. Metabolites with different abundances in HF and LF bulls had also VIP scores of greater than 1.5 and AUC- ROC curves of more than 80%. In addition, four metabolic pathways associated with differential metabolites namely alanine, aspartate and glutamate metabolism, β-alanine metabolism, glycolysis or gluconeogenesis, and pyruvate metabolism were also explored.This is the first study aimed at ascertaining the metabolome of spermatozoa from bulls with different fertility phenotype using gas chromatography-mass spectrometry. We identified five metabolites in the two groups of sires and such molecules can be used, in the future, as key indicators of bull fertility. Fertility is the key to success for sustainability and economics of the livestock system in both beef and dairy cattle industries . In cattThe complex nature of events involved in fertilization is affected by the fluctuating concentrations of macromolecules found in the spermatozoon itself , 12, semIn recent years, studies using approaches in metabolomics have revealed potential fertility biomarkers in the seminal plasma of humans , 28, staTwenty-two metabolites were structurally identified in the bull spermatozoa, regardless of fertility phenotype of the animals. As the full scan spectra of the metabolites showed consistency in all samples, the retention time, target ion, two quantitative ions, and chemical structure of the derivative product of each metabolite were used for the metabolite identification Table . The NISThe metabolites identified in samples of bovine spermatozoa were categorized into eight major chemical classes, as determined by hierarchical clustering analysis: organic acids/derivatives, fatty acids and conjugates, inorganic acids and derivatives, carboxylic acids and derivatives, amino acids, peptides/analogues, keto acids and derivatives, steroids and derivatives, and carbohydrates/carbohydrate conjugates Table .E-04 ± 1E-03; 3.4E-03/0.016), oxalic acid , urea , benzoic acid , carbonate  as well as carbamate  and . The second largest group of sperm metabolites covered a remarkable diversity of fatty acids (22.73%), including nonanoic acid , azelaic acid , oleic acid , oleanitrile , and palmitic acid . In addition, there were three inorganic compounds, such as phosphoric acid, , borate , and phosphine , making 13.64% of all metabolites detected by GC-MS in the bull spermatozoa. Carboxylic acids and derivatives (9.1%) including acetic acid  and acetate  were also identified. Amino acids such as gamma-aminobutyric acid  and L-serine  comprised 9.1% of the bull sperm metabolite profile. Carbohydrates/carbohydrate conjugates (4.54%) such as glycerol  as well as steroids and derivatives (4.54%) such as cholesterol  were also detected. Furthermore, a member of keto acids and derivative class (4.5%), 2-ketobutyric acid , was identified in bull spermatozoa , carbamate (P = 0.000001), and glycerol (P = 0.00000002) , and the abundance ratio of carbamate was higher than those of glycerol (P = 0.003). In addition, the relative abundance within the least predominant metabolites had also displayed significant differences. Bull spermatozoa had a greater abundance ratio of benzoic acid as compared with L-serine (P = 0.0001), carbonate (P = 0.0008), and 2-ketobutyric acid (P = 0.000009), as shown in Fig. Oleic acid, phosphoric acid, phosphine, carbamate, and glycerol were the most abundant metabolites in bull spermatozoa Fig. a. In con02) Fig. a. Moreov P = 0.00. In addi2 = 0.428 and Q2 = 0.874, respectively.Partial Least Square-Discriminant Analysis (PLS-DA) analysis was assessed to determine the contribution of metabolites for the separation of high fertility (HF) and low fertility (LF) groups. PLS-DA two-dimensional score plots of sperm metabolites demonstrated that HF and LF phenotypes of bulls were separated from each other in two distinct clusters with a small overlap Fig. . The firThe Variable Importance in Projection (VIP) score based on the PLS-DA model represents the potential of the metabolite as a biomarker Fig.  and thosThe correlation matrix shows positive (red) and negative (blue) associations between the abundance ratios of the metabolites in HF and LF bulls Fig. . Thus, pP = 0.005), GABA , benzoic acid , and lactic acid  curve (AUC) of the sperm metabolites ranged from 0.52 to 0.92. Metabolites with an AUC > 80% were carbamate , β-alanine metabolism (P = 0.045), glycolysis or gluconeogenesis (P = 0.05), and pyruvate metabolism (P = 0.05), as shown in Fig. Metabolic pathway analysis was performed to evaluate the most relevant pathways associated with differential metabolites in the sperm of HF and LF bulls. The differential metabolites encompass four biochemical pathways, which may reveal the metabolic mechanisms within spermatozoa that might affect fertility. The metabolic pathways were alanine, aspartate and glutamate metabolism  is the most abundant monounsaturated fatty acids of the plasma membrane of ejaculated stallion , boar 4, and ram2) with an NH2 group of primary and secondary amines [2 increases [Our GC-MS-based analyses indicated that carbamate is the fourth most abundant metabolite of the bovine spermatozoa with the second highest VIP score. Carbamate was first reported in seminal plasma from healthy and asthenozoospermic men . Howevery amines , 61 whenncreases . The forncreases . Althougncreases . TherefoA/GABAB receptors were previously identified in human spermatozoa [A receptor [The principal inhibitory neurotransmitter in the adult brain, GABA, was found with greater abundance in spermatozoa from HF sires and it had the highest VIP score. The key enzyme in the synthesis of the GABA, glutamate decarboxylase, and GABArmatozoa . GABA harmatozoa , as wellrmatozoa . GABA inrmatozoa , 68, ratrmatozoa , and ramrmatozoa  and acrormatozoa . In the receptor . Moreovereceptor  and levereceptor . The abureceptor  and asthreceptor  as well receptor . A recenreceptor , suggest+) through the lactate dehydrogenase-mediated conversion of pyruvate to lactate [+ in the electron transport chain generates the ATP molecules by oxidative phosphorylation [Lactate is an important energy source for bull, human, stallion, and boar spermatozoa , 46. The lactate , 76. Therylation . Thus, wrylation , 46. In rylation , humans rylation , mice [8rylation , and goarylation . Thereforylation .Palmitic acid (C16:0), another metabolite found in bull spermatozoa, had the fifth highest VIP score associated with LF animals. This is consistent with previous studies showing increased levels of palmitic acid in infertile men , 83, 84 The importance of lipid metabolism for the production of energy for spermatozoa has been discussed in previous studies . Our anaThe present study reported for the first time the presence of azelaic acid  in bull spermatozoa. Azelaic acid is a nine-carbon saturated aliphatic dicarboxylic acid, and it has been reported in testes of rats . This meWe also evaluated the metabolic pathways of certain molecules and their potential contributions to male fertility. There were four significant pathways associated with differential sperm metabolites including alanine, aspartate and glutamate metabolism, β-alanine metabolism, glycolysis or gluconeogenesis, and pyruvate metabolism. Alanine, aspartate, and glutamate are linked to amino acid metabolism. As amino acids play key roles in multiple cellular processes, they influence the metabolic activity of the spermatozoa . The GABAlthough the sample size represents a limitation of the present study, the sensitivity of the GC-MS approach, together with the bioinformatic tools, enabled us to construct a metabolomics analytical model of sperm from bulls with different fertility phenotypes. Metabolites with different abundances in bulls of high and low fertility  are potential biomarkers of bull fertility.The metabolomic signatures of bull spermatozoa advance our current understanding of the multifactorial and complex processes related to the physiology of male fertility. The present study uncovered vital pieces of information about sperm metabolites for diagnosing male fertility. In addition, because of the strong similarities in physiology and genetics between cattle and other mammals, including humans and endangered mammals, the knowledge generated in the present investigation can be applied to enhance reproductive biotechnology of other species.n = 5) and LF (n = 5) bulls. Statistical and bioinformatics tools were also used to identify potential biomarkers of bull fertility.Metabolomic analysis of bull spermatozoa with two distinct and reliable fertility phenotypic scores was performed by GC-MS. Univariate and multivariate statistical models were employed to identify key differences between the two groups, HF . All animals were raised under the same management conditions and received the same nutrition. Ten ejaculates, one per bull, were collected using an artificial vagina and spermatozoa were separated from seminal plasma by centrifugation . Then, the pellet containing spermatozoa was washed twice  with cold phosphate-buffered saline (PBS) and further aliquoted (100 μl) into a new 2 ml Cryotube® . Following the second centrifugation, spermatozoa were snap-frozen in liquid nitrogen and transported to Mississippi State University (MSU). At MSU, snap-frozen spermatozoa were stored at − 80 °C until preparation for GC-MS analyses. The metabolomic profiles of the seminal plasma obtained from the same bull semen samples have been reported in our previous publication .2HPO4, and 2 mM KH2PO4; pH 7.4) was purchased from Thermo Fisher Scientific . Amber glass vials (2 ml) with 300 μl fixed insert were obtained from Agilent Technologies Inc. .GC-MS grade chemicals and solvents were all purchased from Sigma Aldrich in the highest purity available. Phosphate-buffered solution  were thawed in a water bath at 37 °C for 30 s. The thawed spermatozoa were suspended in a mixture of 8 ml of methanol and 1 ml of ultrapure water, followed by addition of 150 μl of heptadecanoic acid (1 mg/ml in methanol) as the internal standard. In addition, sperm suspension was subjected to five freeze/thaw cycles. Each cycle consisted of freezing sperm cells in liquid nitrogen vapor for 30 min. and subsequent thawing at room temperature for 30 min. Following freeze/thaw cycles, the cell suspension was sonicated in an ultrasonic bath at 25 °C for 30 min. at 120 W and 40 kHz , followed by ultracentrifugation  using an OptimaTM L-90 k and Type 70 Ti Rotor . The supernatant was filtered through a 0.2 μm nylon membrane  and the filtrate was evaporated under a stream of high-purity nitrogen gas  at 40 °C. An aliquot of methanol (1 ml) was added to dissolve metabolites. The metabolite extracts were subsequently transferred into a 2 ml amber vial and evaporated to dryness again with high-purity nitrogen gas at 40 °C.Sperm metabolites were isolated from bull spermatozoa as previously described by Paiva et al. , with moThe dried extracts were resuspended in 50 μl of methoxyamine hydrochloride (20 mg/ml in pyridine) vortexed vigorously for 1 min, and further heated in a water bath at 70 °C for 1 h. The samples were then derivatized by adding 100 μl N, O-Bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS) and heated in a water bath at 70 °C for 1 h. The final derivatives were transferred into a new 2 ml amber glass vial with a 300 μl fixed insert for GC-MS analysis. A quality-control sample for the experiment was prepared by pooling equal volumes of sperm extract samples to ensure that the detection of metabolites was consistent.Screening of the untargeted sperm metabolites was performed using GC-MS as previously described by Velho et al. , with mowww.hmdb.ca/) [http://www.metaboanalyst.ca). MetaboAnalyst is a comprehensive web-based tool designed to help users easily perform metabolomic data analysis, visualization, and functional interpretation [t-test) was used to determine if differences in metabolite abundances in spermatozoa of HF and LF sires were significantly different.Sperm metabolites were identified by their retention time as well as one target and two quantitative ions, in comparison with mass spectra of authentic standards and mass spectra in the NIST mass spectral library. Abundances of the target ions of metabolites were divided by the abundance of target ion of the internal standard (heptadecanoic acid), and the ratios were used for statistical analysis. Identified compounds were categorized based on their chemical classifications using Human Metabolome Database version 3.6  , 110. Stretation . Sum andMultivariate analysis was applied to provide additional information for the interpretation of the data. The PLS-DA defined the separation metabolome of sperm from HF and LF bulls. Potential biomarkers were identified according to the significance of their contributions to variable classification in the PLS-DA model, which was determined by the VIP score. The VIP score summarizes the contribution that a variable makes to the model, and it is calculated as the weighted sum of the squared correlations between the original variable and the PLS-DA components. The weights correspond to the percentage variation explained by the PLS-DA component in the model. The number of terms in the sum depends on the number of PLS-DA components found to be significant in distinguishing the classes. In the present study, we considered metabolites with VIP > 1.5 as potential biomarkers associated with bull fertility. The ROC analysis was applied to examine the specificity and sensitivity of the biomarkers. The area under the ROC curve was calculated to assess the effectiveness of the potential biomarkers. A guide for assessing the performance of metabolites as a biomarker based on its AUC is as follows: AUC of 0.9 to 1.0 = excellent, 0.8 to 0.9 = good, 0.7 to 0.8 = fair, 0.6 to 0.7 = poor, and 0.5 to 0.6 = fail . PearsonBos taurus’ library. The default ‘hypergeometric test’ and ‘Relative Betweenness Centrality’ for pathway enrichment and pathway topological analyses, respectively, were selected. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway was also employed. All matched pathways were visualized by plotting the −log(p) values from pathway enrichment analysis on Y-axis and pathway impact values from pathway topology analysis on X-axis [p-value and the node radius was associated with their pathway impact values. Metabolic pathways with p-value < 0.05 and false discover rate values of 0.7 were screened as pathways of interest.Differential metabolites were also evaluated by using metabolic pathway analysis (MetPA) , 114. Fo


Temnothorax elmenshawyi sp. n., a new ant species from the Asir Mountains of the southwestern region of the Kingdom of Saudi Arabia, is described based on the worker caste. The new species is a member of the T. exilis species group and is distinguished from the other species included in this group by the impressed metanotal groove, the short, acute and broadly-based propodeal spines, the finely punctate posterior half of cephalic surface, and absence of a median clypeal carina. Despite extensive collecting by the authors at the type locality, only two specimens are available for description, suggesting that this species may be rare and likely endemic to the Asir Mountains. The species description is complemented by still images of volume renderings of a 3D model and a 3D rotation video of the holotype based on x-ray microtomography (micro-CT), allowing remote in-depth examination of the specimen. The virtual micro-CT data is provided as cybertype dataset and freely available online 10.5061/dryad.4gg39k6, as well as 3D surface model . An updated identification key to the Arabian species is presented. Temnothorax Mayr1 is one of the largest genera in the Family Formicidae with 450 valid species and subspecies known worldwide from all zoogeographic regions5. Species of this genus inhabit a wide range of habitats from deserts to rainforests6. The taxonomic history of the genus is relatively long and complex including the synonymy with Leptothorax Mayr, 18557 and subsequent removal from synonymy10. The complete nomenclatorial history of the genus is available in Bolton3. Recently, Prebus5 reviewed the evolution, biography, and natural history of the genus postulating a Nearctic origin during the Eocene-Oligocene with a shift to arboreal nesting habits during the Oligocene.The ant genus Temnothorax is diagnosed for the Arabian Peninsula by the following characters for the worker caste11: Antennae 12-segmented with a 3-segmented club; mandibles armed with five teeth; palp formula 5, 3; clypeus broadly inserted posteriorly between frontal lobes; anterior clypeal margin convex in full-face view, not forming an apron over mandibular surface in profile; promesonotal suture absent; propodeum bidentate. Members of the T. exilis species group can be characterized by the following characters12: colour usually dark brown or black; promesonotal suture distinct; propodeal spines short and acute; petiolar node in profile triangular; body sculpture feeble.The genus 14), molecular phylogenetics (e.g.16), morphometrics17, or integrative approaches combining different lines of evidence18. Recently, interactive and three-dimensional (3D) imagery, such as x-ray microtomography (micro-CT), is gaining popularity and momentum within arthropod systematics. Micro-CT is a cutting-edge imaging technology that generates high-resolution, virtual, and interactive 3D reconstructions of whole specimens or particular body parts, thus allowing a maximum of morphological accuracy and fidelity21. Furthermore, a crucial benefit of applying micro-CT for insect taxonomy is the use of openly available cybertype datasets linked to the original, physical type material23.The field of insect taxonomy has advanced at great pace within the last decades through the implementation of new tools and methods, such as DNA barcoding , and T. megalops . Sharaf et al.24 described T. arabicus from the Asir Mountains, Kingdom of Saudi Arabia (KSA), based on the worker caste and reviewed the available regional records for the genus. In the present work, another new species of the Temnothorax is described from the Asir Mountains (KSA) based on the worker caste.Sharaf Temnothorax arabicus Sharaf & Akbar, 2017.Temnothorax elmenshawyi Sharaf, Wachkoo, Hita Garcia sp. n.Temnothorax liviae Temnothorax megalops Holotype worker. Saudi Arabia: Asir Province, Abha, Raydah, 18.201583°N, 42.408933°E, 2578 m., 31.vii.2015, Al Dhafer et al., deposited in the King Saud University Museum of Arthropods (CASENT0922350), College of Food and Agriculture Sciences, King Saud University, Riyadh, KSA. Paratype worker. One worker with same data as the holotype, also deposited in the King Saud University Museum of Arthropods (CASENT0790240)  Figs , 2A–H.FiHolotype measurements (paratype in brackets). EL 0.20 (0.17); FRS 0.15 (0.12); HL 0.75 (0.75); HW 0.62 (0.57); IOD 0.52 (0.52); MD 0.25 (0.20); PPH 0.20 (0.17); PPL 0.15 (0.17); PPW 0.20 (0.22); PTH 0.22 (0.22); PTL 0.27 (0.22); PTW; 0.15 (0.12) PW 0.45 (0.40); SL 0.55 (0.45); SPST 0.22 (0.15); WL 0.92 (0.87). Indices. CI 83 (76); DPeI 56 (55); DPpI 133 (129); LPpI 75 (100); OI 32 (30); PeNI 33 (30); PPI 133 (100); PpNI 44 (55); PSLI 29 (20); SI 73 (60).elmenshawyi_CASENT0790240_video.mp4, see Suppl. material XXX), still images of surface volume rendering, and 3D surface (in PLY format) of the physical paratype (CASENT0790240) in addition to montage photos illustrating head in full-face view, profile and dorsal views of the body. The data is deposited at Dryad23 (10.5061/dryad.4gg39k6) and can be freely accessed as virtual representation of the type. In addition to the cybertype data at Dryad, we also provide a freely accessible 3D surface model of the holotype at Sketchfab (https://skfb.ly/6HYRz).Cybertype. Volumetric raw data (in DICOM format), 3D rotation video  clearly not reaching posterior margin of head by about length of second funicular segment in full-face view; eyes moderately large  with about 16 ommatidia in the longest row. Mesosoma. Promesonotal suture indistinct; promesonotum flat in profile; metanotal grove distinct; propodeal spines short, acute and broadly based (PSLI 20–29). Petiole. In profile without a peduncle; the anterior face forms a shallow concavity anteriorly; anterior face of petiolar node forms a right angle with posterior face; subpetiolar process reduced to a small denticle. Postpetiole. In profile globular (LPpI 75–100) and relatively lower than the height of the petiole; in dorsal view trapezoidal broadest anteriorly, 1.2–1.3 broader than long (DPpI 129–133). Sculpture. Mandibles longitudinally rugulose; clypeus and cephalic surface behind posterior levels of eyes to posterior margin of head mostly unsculptured medially and shiny, laterally with sparse punctate ground sculpture; cephalic surface starting from posterior margins of clypeus to posterior level of eyes faintly longitudinally irregularly rugulose; dorsal surface of mesosoma densely and finely punctate; lateral sides of mesosoma densely punctate; area between mesopleura and metapleura with distinct longitudinal rugae; promesonotum and mesonotum smooth in dorsal view; propodeum irregularly rugulose; petiole and postpetiole densely and finely punctate; gaster smooth and shining. Pilosity. Anterior clypeal margin with six protrusive setae, two short lateral and four central longer ones; clypeus and cephalic surface with appressed scattered pubescence; posterior margin of head with four pairs of erect setae; antennae with abundant appressed pubescence; promesonotum with seven pairs of blunt stiff, short erect setae; mesonotum and propodeum each with two pairs of setae; propodeal spines with one pair of setae; petiole with three pairs of longer setae directed posteriad; postpetiole with five pairs of setae; gaster with scattered blunt setae. Colour. Uniformly black, tibiae and tarsi pale brown.Queens and Males: Unknown.Etymology. This new species is named in the honor of the late Egyptian Qur’an reader Mohamed Siddiq El-Menshawy (1920–1969).Habitat. The type locality (Raydah)  remaining on the Arabian Peninsula.ah) Fig.  is locatBiology and Ecology. Nothing is known of the biology and ecology of the new species.T. liviaeEyes exceptionally large (OI 46) Fig. ………………………Eyes well developed but significantly smaller (OI 32–37) Fig. ……..22.T. elmenshawyiBody colour very dark brown to blackish; cephalic dorsum and sides of head behind posterior eye level mostly unsculptured and shiny with sparse punctate ground sculpture Fig.  ……………………Body colour yellowish; most of head with distinct rugose/rugulose sculpture Fig. ………………………3.T. arabicusFrontal carinae well-developed; propodeal spines long and acute; petiole with two pairs of erect setae Fig. ………………………T. megalops.Frontal carinae feeble; propodeal spines short and blunt; petiole with a single pair of erect setae Fig. ………………………T. exilis species group as defined by Cagniant & Espadaler12 in the inventory of Moroccan Temnothorax, and will not key successfully to any of the Moroccan or African species4. It has to be pointed out though, that the T. exilis species group is not a monophyletic group based on a recent global phylogeny of the genus5, which was already suspected by Cagniant & Espadaler12. Without any other existing higher-level classification above species level available for the genus, we prefer to place T. elmenshawyi in the T. exilis group, at least temporarily.This new species belongs to the Temnothorax elmenshawyi superficially resembles T. exilis (Emery) from Italy but can be readily distinguished by the deeply impressed metanotal groove, the finely punctate posterior half of the cephalic surface, and absence of median clypeal carina. Workers of T. exilis have an indistinct metanotal groove, acute propodeal spines, a reticulate-rugulose posterior half of cephalic surface, and a median clypeal carina. With regards to the Arabian Temnothorax fauna, T. elmenshawyi cannot be confused with any of the Arabian species. It can be immediately separated by the uniformly very dark brown to blackish colour (vs. yellowish in the other three species), moderately large eyes (vs. extremely large eyes in T. liviae), and the mostly unsculptured posterior half of the head behind the level of the eyes (vs. conspicuously rugulose/rugose in the other three species).T. elmenshawyi may also occur in that country. It is hoped that future surveys of ants of this region will confirm this distribution, and result in the discovery of additional specimens, especially the male and queen castes.More than 220 pitfall traps were placed in the Asir Mountains during two years of sampling and only two specimens of the new species were trapped. Undoubtedly, this may reflect the scarcity of this species and indicate a high probability of it being a regional endemic. Due to the extension of the Asir Mountains into Yemen, 21, the use of micro-CT has great advantages for taxonomy. The non-destructive x-ray scan allows the generation of a virtual avatar or cybertype of the physical type material, providing a detailed and almost complete virtual reconstruction of the morphology, thus permitting a high degree of interactive examination for users without access to the physical specimen. Since standard ant taxonomy is still mostly based on the study and comparison of external morphology, virtual study of cybertype datasets can obviate the need to obtain loans from natural history museums, which are often difficult to organize, or to travel to the actual museum collection, which are usually costly and time-intensive. This is of special importance for developing countries where loans are almost never shipped to and travel budgets rather limited. The cybertype provided in this study is the first such virtual 3D dataset for an insect from the Arabian Peninsula or for the ant genus Temnothorax.As discussed in previous studies26.The specimens were measured with Leica M205C stereomicroscope at magnifications of up to 160x. Measurements are expressed in millimeters and followingEL Eye length: maximum diameter of compound eye measured in oblique lateral view.FRS Distance of the frontal carinae immediately caudal of posterior intersection points between frontal carinae and lamellae dorsal of torulus.HL Head length: maximum distance from mid-point of anterior clypeal margin to mid-point of posterior margin of head, measured in full-face view.HW Head width: width of head directly behind eyes, measured in full-face view.IOcD Inter-ocellar distance: minimum distance between posterior-most pair of ocelli.IOD Inter-ocular distance: minimum distance between compound eyes, measured in full-face view.MD Malar distance: minimum distance between anterior margin of compound eye and base of mandible.PH Petiole height: maximum height of petiole, measured from apex of node to ventral edge of petiole, parallel to anterior margin of petiole.PL Petiole length: maximum length of petiole node measured in dorsal view from anterior notch close to propodeum to articulation with postpetiole.PPH Postpetiole height: maximum height of postpetiole measured in lateral view from highest (median) point of node to ventral outline.PPL Postpetiole length: maximum length of postpetiole node measured in dorsal view, excluding helcium.PPW Postpetiole width: maximum width of postpetiole node measured in dorsal view.PTW Petiole width: maximum width of petiole node measured in dorsal view.PW Pronotal width: maximum width of pronotum measured in dorsal view.SL Scape length: maximum scape length excluding basal condyle and neck.SPST Distance between center of propodeal stigma and spine tip. The stigma center refers to midpoint defined by outer cuticular ring but not to center of stigma opening, which may be positioned eccentrically.WL Weber’s length: diagonal length of mesosoma in lateral view from postero-ventral margin of propodeal lobe to anterior-most point of pronotal slope, excluding the neck.Indices.CI Cephalic index: HW/HL × 100DPeI Dorsal petiole index: PTW/PTL × 100DPpI Dorsal postpetiole index: PPW/PPL × 100LPpI Lateral postpetiole index: PPL/PPH × 100OI Ocular index: EL/HW × 100PeNI Petiolar node index: PTW/PW × 100PPI Postpetiole index: PPW/PTW × 100PpNI Postpetiolar node index: PPW/PW × 100PSLI Propodeal spine index: SPST/HL × 100SI Scape index: SL/HL × 100et al.21. For further details on micro-CT scanning and post-processing workflow pipeline, we refer to Hita Garcia et al.23.The micro-CT scan was performed using a Zeiss Xradia 510 Versa 3D X-ray microscope operated with the Zeiss Scout-and-Scan Control System software (version 11.1.6411.17883). 3D reconstruction of the resulting scan raw data was done with the Zeiss Scout-and- Scan Control System Reconstructor (version 11.1.6411.17883) and saved in DICOM file format. Volume renderings, surface mesh generations, and virtual examinations were performed with Amira software (version 6.3.0). Post-processing of mesh data in order to generate a clean surface was done with Meshlab (version 1.3.3). The methodology for the virtual examination of 3D surface models, generation of 3D rotation videos, and virtual dissections follow Hita Garcia


Microbiological criteria for diagnosing nontuberculous mycobacterial pulmonary disease (NTM-PD) include positive culture results from at least two separately expectorated sputum specimens or one bronchial washing or lavage. However, the clinical similarities and differences between patients diagnosed by these two methods remain unclear. We compared clinical features and prognoses of patients with NTM-PD diagnosed from both specimen types.ClinicalTrials.gov identifier: NCT01616745). Baseline demographics, symptoms, radiographic findings, disease progression, and treatment responses were summarized and compared between patients diagnosed from sputum specimens and patients diagnosed from bronchoscopic specimens.We analysed data from patients who participated in the Seoul National University Hospital NTM-PD cohort  were diagnosed from two separately expectorated sputum specimens, and 68 (19.6%) were diagnosed from bronchoscopic specimens. Patients diagnosed from sputum specimens had more frequent and severe cough, sputum, postnasal drip, and high St. George’s Respiratory Questionnaire scores. However, the extent and severity of the radiographic lesions, disease progression, and treatment responses were similar for both groups. Further analysis based on the following three groups  suggested that the scanty sputum/bronchoscopy group appeared to have the worst prognosis in terms of both time to progression and time to culture conversion.Although some symptoms and quality of life were worse in patients with NTM-PD diagnosed from sputum specimens, their prognoses were similar to those of patients diagnosed by bronchoscopic specimen. We recommend bronchoscopic sampling for patients in whom NTM-PD is suspected clinically or radiographically, especially those who have no or scanty sputum.The online version of this article (10.1186/s12879-019-4078-0) contains supplementary material, which is available to authorized users. The incidence and prevalence of nontuberculous mycobacterial (NTM) pulmonary disease (PD) is increasing worldwide –8. In SoDiagnosing NTM-PD relies on combining clinical and microbiological criteria , 12. CliHowever, it remains unclear whether the clinical features differ between NTM patients diagnosed by the two methods . DiagnosClinicalTrials.gov identifier: NCT01616745), which commenced on 1 July 2011, were included in this analysis [Patients aged ≥20 years with NTM-PD who participated in the ongoing prospective cohort, Seoul National University Hospital NTM cohort  per the modified Medical Research Council dyspnoea scale . Other c1), forced vital capacity (FVC), FEV1/FVC ratio, and diffusing capacity for carbon monoxide. Computed tomography (CT) scans of the chest were obtained upon diagnosis. Radiographic subtype was classified as nodular bronchiectatic form or upper lobe cavitary form. When the subtype was unclassifiable, it was allocated into either subtype based on the dominant feature for further analysis in this study. In addition, radiographic NTM-PD severity was assessed by two board-certified radiologists, who were blinded to the patients’ clinical data, using a previously introduced scoring system [Baseline serum laboratory tests were performed, and pulmonary function was tested for forced expiratory volume in 1 second  or isolation of NTM from a bronchoscopic specimen (bronchoscopy group). When NTM were isolated from both sputum and bronchoscopic specimens, patients were classified by the test that yielded NTM sooner. For the sputum group, the date of the second positive sputum culture was considered the date of diagnosis. For the bronchoscopy group, the date of the bronchoscopy that yielded a positive NTM culture was considered the date of diagnosis.Disease progression was defined as physician recommendation for treatment initiation based on clinical and radiographic deterioration, as used previously by us and other researchers , 24, 25.Mycobacterium avium complex (MAC)-PD was treated with a macrolide, ethambutol, and rifampicin using a thrice weekly or daily schedule, and an injectable aminoglycoside was considered in patients with a severe disease extent. M. abscessus-PD was initially treated with a combination of azithromycin, aminoglycoside, and imipenem (or cefoxitin) for the first 3 weeks, and aminoglycoside administration (3 to 5 times per week) was continued for at least 3 more months with azithromycin during the continuation phase. Clofazimine has been used for both MAC-PD and M. abscessus-PD patients who were refractory to treatment or resistant to clarithromycin. When treating patients with mixed NTM infections, we targeted the most frequently isolated NTM species.NTM-PD was treated as per recent guidelines , 12. ClaCulture conversion was defined as at least three consecutive negative results from sputum mycobacterial cultures after initiating treatment and adopted as a parameter for treatment response . The colP-values less than 0.05 were further included in a multivariate model to calculate the adjusted HR of each variable. All statistical analyses were performed using Stata, version 13.0 .Categorical variables were summarized as numbers with percentages and compared using a chi-square test or Fisher’s exact test. Continuous variables are presented as the median with interquartile range (IQR) and analysed using the Mann-Whitney U test. The Wilcoxon signed-rank test was also used to evaluate changes in NTM-PD radiographic severity scores. To estimate the probability of disease progression and culture conversion after treatment, Kaplan-Meier survival analyses with a log-rank test were used. To estimate hazard ratios (HRs) with 95% confidence intervals (CIs) for each potential variable affecting the study outcomes, we utilized a Cox proportional hazards model. Covariates with n = 11), had withdrawn consent (n = 10), started treatment before diagnosis (n = 3), or had poor compliance (n = 1) were excluded. Finally, 347 patients with NTM-PD were included in the analysis. Of these, 279 (80.4%) were diagnosed from two consecutive sputum specimens obtained at a median interval of 3.2 months , whereas 68 (19.6%) were diagnosed by examination of single bronchoscopic specimens. The reasons for undergoing bronchoscopic sampling were as follows: could not expectorate or had too little sputum (47 patients) or suspected NTM-PD but negative acid-fast staining or mycobacterial culture (21 patients) on ≥2 sputum specimens.On the date of extraction of initial data (11 April 2017), 372 patients were enrolled in the cohort. Of these, patients who did not fulfil the diagnostic criteria for NTM-PD (P = 0.046). History of tuberculosis treatment was more common in the sputum group than in the bronchoscopy group . Nodular bronchiectatic form was the main radiographic subtype in both groups: 90.0 and 94.1% in the sputum and bronchoscopy groups, respectively (P = 0.288). NTM species were similarly distributed in both groups. MAC was the most common (n = 243), and M. abscessus the second most common (n = 63). M. abscessus consists of 32 strains of M. abscessus subsp. abscessus and 31 of M. abscessus subsp. massiliense. Twelve of 32 strains of M. abscessus subsp. abscessus (37.5%) and 26 strains of 31 M. abscessus subsp. massiliense (83.9%) were clarithromycin-susceptible. The rate of smear positivity did not differ according to the type of specimen: 16.9 and 22.1% were positive in the sputum and bronchoscopy group, respectively (P = 0.314)  than did the bronchoscopy group  1 was lower in the sputum group than in the bronchoscopy group, although this was not statistically significant . Other laboratory test details revealed no significant differences between the two groups [see Additional file FEVP = 0.002). Likewise, 140 patients (50.2%) in the sputum group had sputum “almost every day”, compared with only 14 (20.6%) in the bronchoscopy group (P < 0.001). Patients in the sputum group had more severe respiratory symptoms and worse quality of life overall than those in the bronchoscopy group, showing higher St. George’s Respiratory Questionnaire scores . The frequencies of other symptoms, including dyspnoea, haemoptysis, fever, weight loss, anxiety, and depression were similar between the groups  had coughing “almost every day”, compared with 11 (16.2%) in the bronchoscopy group (7.2% had P = 0.907).Patients were followed for a median of 2.9 years  after their NTM-PD diagnosis: median 3.0 years  for the sputum group vs. 2.1 years  for the bronchoscopy group. Among the 347 patients, 133 (38.3%) experienced disease progression. In the sputum group, disease progression manifested as clinical deterioration in 17 patients (15.3%), radiographic deterioration in 54 (48.7%), and both in 40 (36.0%). The ratios were similar in the bronchoscopy group, namely 4 (18.2%), 11 (50.0%), and 7 (31.8%) patients, respectively, in the above three categories (P = 0.772)  and M. abscessus-PD (log-rank P = 0.455) patients. Younger age  and the upper lobe cavitary disease form  were identified as independent factors for earlier disease progression. However, diagnostic method (sputum vs. bronchoscopy) was not a predictor of earlier disease progression; the HR of the bronchoscopy group was 0.82 with a 95% CI 0.48–1.38 compared with the sputum group (Table Time to progression for NTM-PD was similar between the sputum and bronchoscopy groups (log-rank 72) Fig. a. In theP < 0.001 by paired t-test). These scores ranged from median 8  to 10  for the sputum group (P < 0.001) and from median 8  to 10  for the bronchoscopy group (P = 0.002).The baseline radiographic severity score showed a median of 8 , which was elevated to median 10  with disease progression  refused, and 78 (58.6%) remained on treatment at the time of data retrieval. The median treatment duration was 1.4 years  in all patients who initiated treatment, with medians of 1.5 years  for the sputum group and 1.2 years  for the bronchoscopy group.P = 0.869)  and M. abscessus-PD (log-rank P = 0.267).Time to culture conversion was compared between the 13 patients in the bronchoscopy group whose subsequent sputum cultures yielded NTM and who therefore started treatment and the 109 patients in the sputum group who also started treatment. Culture conversion of sputum was achieved in 7 of 13 patients in the bronchoscopy group (53.9%) within a median of 1.8 months  and in 71 of 109 patients in the sputum group (65.1%) within a median of 1.8 months . Time to culture conversion did not differ significantly between the sputum and bronchoscopy groups   . Although some symptoms and quality of life were worse in patients diagnosed from sputum, times to disease progression and culture conversion with treatment were similar between both groups.The similar prognoses despite worse symptoms found in this study for patients diagnosed from sputum requires further explanation. Sputum group patients more frequently complained of symptoms related to sputum production or expectoration . However, other symptoms not directly associated with sputum yield  were similar between both groups. Results of other tests, including pulmonary function and radiographic severity, were also similar. Finally, disease progression and culture conversion with treatment were similar between both groups. These findings suggest that the specimen collection method reflects the sputum accessibility, rather than the overall disease severity. Our results support the microbiologic criteria for NTM-PD suggested by the current ATS/IDSA and BTS guidelines , 12.It is interesting to note the differences between the sputum culture negative/bronchoscopy and scanty sputum/bronchoscopy groups. Our analysis suggested that the scanty sputum/bronchoscopy group appeared to have the worst prognosis in terms of both time to progression and time to culture conversion; however, this difference was not statistically significant. This finding suggests that bronchoscopy should be performed more actively in patients with possible NTM-PD who are not producing sputum.In our institution, bronchoscopic sampling, rather than sputum induction, was performed for patients who could not expectorate sputum for the diagnosis of NTM-PD. Although the induction of sputum is considered as one of the sampling methods for patients with suspicion of NTM-PD as well as pulmonary tuberculosis, the superiority of sputum induction over routine sputum examination is questionable . In addiThe strength of this study was that the analyses were based on the prospective cohort, which adopted not only interviews and questionnaires, but objective measures including pulmonary function tests and chest CT scans. Pulmonary function and CT scores taken at baseline evaluation confirmed that both groups were similar in disease severity. Furthermore, disease progression, determined by the on-duty physician during follow-up, was also confirmed by worsening lesions on the CT, which was scored by board-certified radiologists who were blinded to the patients’ clinical data.In this study, only culture conversion was adopted as the treatment response, which could be a limitation. Because follow-up durations were relatively short after initiating treatment, other treatment outcomes such as ‘microbiological cure’ or ‘treatment failure’ could not be compared for this study. Long-term observation of these patients could elucidate treatment responses more clearly. Another limitation of this study is that rates of both disease progression and culture conversion may have been affected by the different follow-up durations in different groups. Follow-up durations varied according to the time of enrolment.In conclusion, disease progression and treatment response were similar between patients with NTM-PD diagnosed from sputum specimens and those diagnosed from bronchoscopic specimens, despite some symptoms and quality of life being worse in patients diagnosed from sputum. We recommend bronchoscopic sampling for patients in whom NTM-PD is suspected clinically or radiographically, especially those who have no or scanty sputum.Additional file 1:Table S1. Laboratory findings for patients enrolled in the nontuberculous pulmonary disease cohort. (DOCX 21 kb)


SCNN1A, SCNN1B, or SCNN1G genes that encode epithelial sodium channel subunits.Liddle syndrome is a monogenic disease with autosomal dominant inheritance. Basic characteristics of this disease are hypertension, reduced concentration of aldosterone and renin activity, as well as increased excretion of potassium leading to low level of potassium in serum and metabolic alkalosis. The cause of Liddle syndrome is missense or frameshift mutations in SCNN1G, which encodes the γ subunit of ENaC in vertebrates. The father and younger sister also harbor this heterozygous deletion. Treatment with amiloride of proband and his sister did not normalize the blood pressure, but normalized level of plasma renin activity.We describe a family with Liddle syndrome from Russia. 15-year-old proband has arterial hypertension, hypokalemia, hyporeninemia, metabolic alkalosis, but aldosterone level is within the normal range. At 12 years of age, arterial hypertension was noticed for the first time. We identified novel frameshift mutation c.1769delG (p.Gly590Alafs) in Our results expand the mutational spectrum of Liddle syndrome and provide further proof that the conserved PY motif is crucial to control of ENaC activity. Genetic analysis has implications for the management of hypertension, specific treatment with amiloride and counselling in families with Liddle syndrome. The key clinical characteristics of this syndrome are early onset salt-sensitive hypertension with low level of K+ through the apical membrane from lumen into the epithelial cell [+ ion balance in the extracellular fluid (ECF) and in the kidney, these channels are a significant part of ECF volume and blood pressure (BP) regulation [Mutations (missense or frameshift) in the genes of epithelial sodium channel (ENaC) subunits cause the Liddle syndrome. These channels belong to the ENaC/DEG (degenerin) family of proton-gated cation channels. ENaC mediates transport of Naial cell . Since Egulation .SCNN1A), β (SCNN1B), and γ (SCNN1G). All subunits have similar structure: cytoplasmic N-terminus, extracellular loop, two short hydrophobic segments (transmembrane domains 1 and 2) and cytoplasmic C-terminus. The N- and C-termini are turned to the cytosolic surface, whereas the extracellular loop is turned to the extracellular surface [ENaC channels are present in the apical portion of epithelial cells of distal nephron, distal colon, lung and ducts of exocrine glands. The channel consists of three subunits encoded by three genes: α , cortisol, dehydroepiandrosterone, aldosterone , adrenaline, noradrenaline, dopamine, and serotonin was normal. Urine level of metanephrines was normal. Serum level of renin activity was reduced to 0,1 ng/mL/h . Treatment with Lisinopril  and Amlodipine  was ineffective.He was admitted to Veltischev Research and Clinical Institute for Pediatrics of the Pirogov Russian National Research Medical University  at 14, 15 and 16 years.On admission at 14 years he had high BP (160/120 mmHg), reduced level of potassium  and renin activity . Ultrasound showed no abnormality of kidney. Treatment with Amlodipine and Bisoprolol (beta-blocker) did not normalize hypertension.During a year, the patient received Irbesartan, Amlodipine, Bisoprolol and Moxonidine. But arterial hypertension persisted.3 std. 31 mmol/L. Other biochemical parameters  as well as the aldosterone level , thyroid gland hormonal profile and cardiac indicators were within the normal range. Urine analysis was without any significant changes.On admission at 15 years his weight was 70 kg, height was 175 cm. Intellectual development corresponded to his age. Maximum BP was 159/109 mmHg. Heart rate while lying was 66 bpm, heart rate while standing was 76 bpm. The heart was not enlarged; heart sounds were clear and rhythmic. Biochemical analysis showed reduced level of potassium , reduced renin activity , elevated level of lactate dehydrogenase  and creatine kinase . The acid-base balance is shifted towards metabolic alkalosis: pH 7,45, BE (Base Excess) + 7,4 mmol/L, HCOEchocardiography showed concentric left ventricular hypertrophy, tricuspid and mitral valve regurgitation. Global systolic and diastolic myocardial functions were not abnormal. Daily monitoring of BP showed stable systolic and diastolic arterial hypertension during night and daytime hours. Abdominal ultrasound revealed no significant changes.Due to the presence of hypertension, hypokalemia, hyporeninemia, and metabolic alkalosis monogenic form of arterial hypertension was expected in patient. Differential diagnosis was conducted between two conditions: Liddle syndrome and syndrome of apparent mineralocorticoid excess.3 std. 22 mmol/L). Urine analysis was without any significant changes. Echocardiography showed symmetrical left ventricular hypertrophy.The younger sister  of proband had normal BP (120/80 mmHg) with a periodic increase to 160/100 mmHg. From 12 years, she has been complaining of headache and nausea in the morning. She had a reduced serum level of aldosterone , reduced renin activity  and normal level of potassium . The level of sodium (141 mmol/L) and cortisol  were within the normal range. There were no signs of metabolic alkalosis in the sister’s blood  also had arterial hypertension (BP 170/120 mmHg with periodic increase to 210/140 mmHg) since the age of 18. Ultrasound showed kidney cysts. It is impossible to describe detailed clinical symptoms of the father due to his refusal to undergo an examination and treatment.Genomic DNA was extracted from peripheral blood samples of proband, his mother, father and younger sister.Sequencing of clinically relevant genes  was done by Genotek Ltd. The Genotek’s Ethics Committee (08/2018) approved all studies. Parents gave written informed consent to research and publication of photos, clinical and sequencing data of themselves, proband and his sister.NEBNext Ultra DNA Library Prep Kit for Illumina  was used as reagent for preparation of DNA libraries. Target enrichment was performed with SureSelect XT2  system. Next samples were sequenced on Illumina HiSeq 2500 (paired-end 100 bp reads). To trim 3′-nucleotides, we used Cutadapt . Reads wAll of the exons of the SCNN1A, SCNN1B and SCNN1G genes were sequenced. An additional table file shows other genes and exons that have been sequenced [see Additional file SCNN1G pathogenic variant was identified in proband and his relatives using exome sequencing  and then was verified by Sanger sequencing.After exome sequencing proband and his sister were commenced on amiloride (with hydrochlorothiazide) and low-salt diet.A dose of 5 mg of amiloride /50 mg of hydrochlorothiazide caused severe side effects  in proband. The dose of the drug was reduced to 2,5 mg of amiloride /25 mg of hydrochlorothiazide. After 6-months therapy, BP of proband did not normalize with a maximum increase to 160/110. Also, elevated level of homocysteine was revealed . Other biochemical parameters as well as level of aldosterone and renin activity were within the normal range. However, ultrasound revealed diffuse changes in the renal parenchyma.A dose of 5 mg of amiloride /50 mg of hydrochlorothiazide caused an increase in creatine levels up to 85 μmol/L in sister of proband. The dose of the drug was reduced to 2,5 mg of amiloride /25 mg of hydrochlorothiazide. After 6-months therapy, BP of the sister tended to normalize with a maximum increase to 141/98. Other biochemical parameters as well as level of aldosterone and renin activity were within the normal range. However, ultrasound revealed an increase in volume and thickening of the renal parenchyma.SCNN1G (NM_001039.3) by NGS analysis and confirmed by Sanger sequencing. We identified this mutation in samples of proband, his father and sister. There was no mutation of the SCNN1G in mother  of the DSG2 gene: c.458A > G, p.Asn153Ser (NM_001943.3). According to the ACMG recommendation, this mutation is considered to be likely pathogenic. Mutation in this gene are regarded to cause arrhythmogenic (right ventricular) dysplasia (OMIM 610193). No other pathogenic variants and SNVs were found.We also found that proband, his mother and sister have heterozygous missense mutation in The c.1769delG mutation is deletion in exon 13 that shifted a reading frame. Mutalyzer  predictsSCNN1G can lead to Liddle syndrome. To date, 6 different mutations of SCNN1G have been reported in 8 families from Finland, China and Japan [Heterozygous mutations in the nd Japan –32 (Tabl624PPPKY628). This segment is engaged in negative regulation of the channel and its overactivation [Most of described mutations remove a proline rich segment  in the carboxyl cytoplasmic tail of γ subunit  in the gene encoding the γ subunit of the epithelial sodium channel.Additional file 1. Genes and exons that have been sequenced in all patients. The file is a list of genes that were sequenced during the analysis, with indication of the number of sequenced exons for each gene.


In 2016, two thirds of diagnosed human immunodeficiency virus (HIV) infections in the United States were attributed to male-to-male sexual contact , CDC anaAll states and U.S. dependent areas report cases of HIV infection and associated patient demographic and clinical information to NHSS. CDC analyzed data reported through December 2017 from the U.S. states and DC, statistically adjusted for missing risk factor information  tabulated by age group and race/ethnicity and by age group and region of residence at diagnosis. The APC is calculated by using a generalized log linear model. Prevalence trends among MSM living with diagnosed HIV infection were measured using APCs tabulated by age group and last known jurisdiction of residence at year-end during 2008–2015. Changes were considered statistically significant if the APC’s 95% confidence interval (CI) excluded zero.Among 236,150 MSM with HIV infection diagnosed during 2008–2016, a total of 106,258 (45%) were aged 13–29 years, 100,857 (43%) were aged 30–49 years, and 29,034 (12%) were aged ≥50 years . During During 2008–2015, the number of MSM living with diagnosed HIV infection increased 4.5% per year, including a 7.7% annual increase among MSM aged 13–29 years, from 40,991 in 2008 to 69,505 in 2015 . Among MThe number of MSM aged ≥50 years living with HIV infection increased in all jurisdictions, ranging from an estimated average of 7.8% in Alaska to 16.0% per year in Idaho. Among MSM aged ≥50 years, the number of persons living with HIV infection increased 10.8% per year, from 108,544 in 2008 to 223,210 in 2015. In 12 jurisdictions, at least half of MSM living with diagnosed HIV infection were aged ≥50 years. Seven of these states were in the West , four were in the Northeast  and one was in the Midwest (South Dakota). Nine of 10 states with the highest percentages of MSM living with diagnosed HIV infection aged 13–29 years were in the South.During 2008–2016, the annual number of diagnoses of HIV infection among MSM increased 3% per year among persons aged 13–29 years, decreased 4% per year among those aged 30–49 years and was stable among those aged ≥50 years. The number of diagnoses among MSM aged 13–29 years was four times that among MSM ≥50 years.Racial/ethnic disparities in the occurrence of annual diagnoses of HIV infection persisted, particularly among younger MSM. Compared with non-Hispanic whites, blacks and Hispanics/Latinos accounted for a disproportionate number of cases. Among MSM aged 13–29 years, American Indians/Alaska Natives, Asians, and residents of the South experienced the steepest increases in trends in annual diagnoses of HIV infection compared with other racial/ethnic groups and other U.S. regions; however, the numbers of annual diagnoses of HIV infection among American Indian/Alaska Native and Asian MSM were small.During 2008–2015, the number of MSM aged ≥50 years living with diagnosed HIV infection increased by 11% per year, and at year-end 2015, this group accounted for the largest age group of MSM living with diagnosed HIV infection, presumably as a result of increased survival associated with widespread use of antiretroviral therapy  infections were attributed to male-to-male sexual contact.During 2008–2016, the number of HIV diagnoses increased 3% annually among men who have sex with men (MSM) aged 13–29 years. The number of HIV diagnoses among MSM aged 13–29 years was four times that of MSM aged ≥50 years. Racial/ethnic inequities in HIV persisted, particularly among younger black/African American and Hispanic/Latino MSM.MSM may be tested at least annually and, if positive, linked to and retained in HIV medical care. Those testing negative might benefit from prevention services, including preexposure prophylaxis. Strengthened efforts can reduce racial/ethnic inequities.


Cuphea ignea is one of the herbal resources belonging to Lythraceae family. Some species of this family have been used traditionally in South and Central America’s folk medicine for treating stomach disorders. Therefore, the present study was performed to evaluate the gastropreventive effect of aqueous ethanolic extract of C. ignea aerial parts on ethanol-induced gastric ulcer.C. ignea aerial parts extract at doses of 250 and 500 mg/kg body weight and ranitidine (a reference drug) at a dose of 30 mg/kg body weight were orally administrated daily for 7 days before ulcer induction. One hour after ethanol administration blood samples were collected and then stomachs of sacrificed rats were subjected to biochemical, macroscopic and microscopic studies.Gastric ulcers were induced in Sprague Dawley rats using one oral dose of absolute ethanol (1.5 mL/rat). The C. ignea extract significantly attenuated gastric ulcer as revealed by significant reduction in the gastric ulcer index and volume of gastric juice while significantly increased preventive percentage, gastric pH value and pepsin activity. Pre-treatment of C. ignea extract markedly improved the serum level of TNF-α, the gastric MPO activity and NO content. Furthermore, C. ignea pre-treatment significantly increased the gastric levels of enzymatic and non- enzymatic antioxidants namely CAT, SOD, GSH-Px, and GSH with concomitant reduction in MDA level compared with those in the ethanol group. These results were further supported by histopathological findings which revealed the curing effect of C. ignea on the hemorrhagic shock induced by ethanol toxicity.Oral administration of C. ignea extract showed a potential gastroprotective effect on ethanol-induced gastric ulcer, and its effect may be mediated through suppression of oxidative stress and gastric inflammation. Helicobactor pylori [Gastric ulcer is a benign lesion with multiple etiologies, associated with an imbalance between gastric protective factors and aggressive physical, chemical or psychological factors on the mucosal epithelium . These ar pylori . Among tr pylori . Thus, tr pylori .In spite of the domination of synthetic drugs in managing most of human diseases including gastric ulcer, extensive proportion worldwide now directed to traditional medicine . This maCuphea, a new world genus, is considered the largest genera of Lythraceae family [Plants of Lythraceae family are regarded as a valuable source of exclusive natural products for developing medications against various diseases . Cuphea,e family . Plants e family . Some spe family , 14.Cuphea ignea, cigar plant, is a flowering species in genus Cuphea. It is a tropical, densely branched evergreen subshrub produces tubular, bright red to orange flowers resemble a lit cigar, hence its name. C. ignea is native to Mexico and the West Indies; however, in recent years its popularity is on rise everywhere [erywhere .C. ignea except Bate-Smith [C. ignea plant. Recently, we isolated from this extract a coumarin with a rare structure, namely, 7-hydroxy 3-methoxy coumarin 5-O-β-glucopyranoside, which offered potent antioxidant activities in vitro [C. ignea in vivo. Therefore, the present study was undertaken to evaluate phytochemical constituents of the aqueous ethanolic extract of C. ignea aerial parts and to estimate its gastroprotective effect of C. ignea extract against ethanol induced gastric ulcer in rats.So far there are no studies regarding the phytochemistry of te-Smith  who studin vitro . To dateC. ignea aerial parts were collected from 30 k north Cairo. Authentication of the plant was carried out by Prof. Dr. Salwa Kawashty at the NRC. A voucher specimen was deposited at the herbarium of the NRC (voucher number C 182).The collected C. ignea plant was dried in the shadow, crushed and exhaustively extracted with 70% (v/v) aqueous EtOH under reflux. The obtained eluent was dried under vacuum at 55–60 °C then dissolved in EtOH. Then, the extract was stored for future use.Fresh samples of Phytochemical analysis of the aqueous ethanolic extract was carried out as described by Sofowora .2 = 0.998), which represented results as gallic acid equivalents.Folin-Ciocalteu method  was usedC. ignea plant was determined according to Žilić et al. [3 solution was added and the volume was adjusted to 2.5 mL using distilled water. After 7 min, 1.5 mL of 1 M NaOH was added, followed by centrifugation (5000 g/10 min). Absorbance of the supernatant was measured at 510 nm against the solvent blank. Total flavonoids were estimated using a catachine calibration curve and the equation: y = 0.003x - 0.004 (R2 = 0.998).Total flavonoid content in the aqueous ethanolic extract of ć et al.  using alThe quantitative scavenging of 2,2-diphenyl-1-picrylhydrazyl radical was determined following the Brand et al. method . The extC. ignea extract on the reduction of ferric cyanide into ferrous cyanide was evaluated according to Yen and Duh [3) was measured at 700 nm.The effect of  and Duh . A seriaC. ignea extract on acute toxicity using OECD 425 guidelines [C. ignea extract. Group 6 was given an equivalent volume of distilled water. Animals were evaluated clinically and toxicologically for 3 days after receiving the extract, while death rates were monitored for 14 days.In order to detect the maximal safe dose, Sprague Dawley rat model was used to investigate the effect of idelines . Thirty n = 6) as follows: Group 1 ; Group 2 (ethanol ulcerated rats); Group 3 ; Group 4 (rats administered only 250 mg/kg of extract); Group 5 (ethanol ulcerated rats pretreated with 250 mg/kg of extract); Group 6 (rats were administered only 500 mg/kg of extract); Group 7 (ethanol ulcerated rats pretreated with 500 mg/kg of extract). Ranitidine and extract treatments were given orally once/day for 7 days before ulcer induction. Groups 4 and 6 were used to evaluate the negative/toxicological effects of the extract. Groups 2, 3, 5 and 7 were supplied only with water for 24 h before ulcer induction. A single gavage of absolute ethanol (1.5 mL/rat) was used to induce gastric ulcer after 24 h fasting according to Liu et al. [The experiments were performed on adult female Sprague-Dawley rats (150-200 g) obtained from the animal house colony of the National Research Centre, Dokki, Giza, Egypt. The animals were kept in polypropylene cages with wood shaving under standardized animal house conditions , fed with standard pellet and allowed free access to water. Distilled water was used for the oral administration of standard drug and plant extracts in all in vivo assays. The animal experiments were conducted according to the international regulations of the usage and welfare of laboratory animals and were approved by the Ethics Committee of the National Research Centre, Cairo, Egypt. Rats were randomized into seven groups , where clear serum was separated and stored at −20 °C until analysis. In parallel, animal stomachs were rapidly taken away, opened along the greater curvature, where their contents were collected for volume and pH determination. The gastric secretion was stored for estimating pepsin activity. Gastric tissue specimens were thereafter rinsed gently with phosphate buffer saline (PBS) to remove any blood clots and then examined macroscopically to calculate gastric ulcer index . SecondlFor each group, ulcer index was determined using the following equation:The percentage of ulcer preventive index was then calculated as follows:Pepsin activity was determined using stop-point assay of denatured hemoglobin hydrolysis .Serum aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) activities were determined using commercial kits . Serum urea and creatinine level were assayed as kidney function tests using kits also provided by Spectrum Diagnostics Company, (Egypt). Serum necrosis factor-alpha (TNF-α) was investigated by the enzyme-linked immunosorbent assay using Koma Biotec Inc. kits, Korea. The operational processes were measured in accordance with the kit instructions.Myeloperoxidase, a marker of neutrophil infiltration, was assayed using a modified method of Bradley et al. . In brieNitric oxide (NO) concentration was assayed by measuring nitrite formed from NO oxidation , based o2O2 disappearance at 240 nm was used to detect catalase (CAT) activity according to Aebi [2O2/min/25°C at pH 7.0.The initial rate of H to Aebi . Enzyme Superoxide dismutase (SOD) activity estimated according to method of Minami and Yoshikawa , where pGlutathione peroxidase (GSH-Px) activity was determined according to method of Necheles et al.  at 412 n3)n, then centrifuged for at least 2 min at 4000 rpm to eliminate proteins in order to avoid interferences of protein R-SH groups.The method of Beutler et al.  was usedMalondialdehyde (MDA), resulting from lipid peroxidation, was assayed  based onTissue specimens were taken mainly from the glandular part of stomach of the all groups and fixed in 10% neutral buffer formalin overnight. Routine tissue processing was carried out according to Suvarna et al. . Tissue P < 0.05.All results were expressed as means ± SD. The data were evaluated with SPSS 19.0 . The statistical significance of differences for each parameter among the groups was evaluated by one-way ANOVA, followed by LSD test. The significance level was set at C. ignea plant revealed the presence of phenolics, flavonoids, tannins, alkaloids, carbohydrates, glycosides, triterpenens and unsaturated sterols. The quantitative phytochemical investigation of total phenolic and flavonoid contents in the extract was shown in Table The qualitative phytochemical screening of aqueous ethanolic extract of C. ignea extract at seven different concentrations ranging from 0.5 to 500 μg/mL  used in comparison with ascorbic acid as standard reference. C. ignea extract exhibited nearly similar RSA as ascorbic acid at a concentration of 50 μg/mL, the extract completely inhibited the scavenging radical absorbance and give a potent radical scavenging activity with 98% at concentration of 100 μg/mL, indicating a remarkable antioxidant capacity.Radical scavenging assay is used to determine the in vitro antioxidant activity of plant extracts. Figure C. ignea extract had concentration-dependent reducing power. Also, it had an appreciable reducing power when compared to standard quercetin.The reducing power of phenolic compounds serves as good indicator of its antioxidant activity. Figure C. ignea extract revealed insignificant changes in the liver and kidney functional tests  were observed in rats during the period of 72 h study up to the maximum dose (5 g/kg). 250 and 500 mg/kg doses of the extract were selected for the antiulcer studies. One week treatment with 250 or 500 mg/kg of C. ignea gavaging prior to ethanol administration showed improved protection against ulceration degree. As shown in Fig. C. ignea extract significantly reduced gastric ulcer index. The improvement in gastric ulcer index was more pronounced in C. ignea pretreated group than in ranitidine group. The preventive index recorded 90.49 and 88.89% for 250 and 500 mg/kg of C. ignea, respectively.Oral administration of absolute ethanol induced gross lesions in the gastric lumen of rats with markedly high ulceration index. C. ignea extract (250 and 500 mg/kg) produced significant increase in pH value by 17.73 and 29.91% associated with significant decrease in gastric volume by 38.78 and 27.73%, respectively as compared to ulcerated rats , SOD 54.46%), GSH-Px (18.06%), GSH (57.13%), contents, and typically showed significant increase in MDA level (1.77 fold) as compared to normal group. On the other hand, pre-treatment with 4.46%, GSC. ignea extract at both doses showed no toxic or deteriorating effects on the gastric mucosa of the treated rats as; Normal non congested gastric mucosa with normal thickening were appeared . Some other areas of gastric mucosa exhibited severe coaggulative necrosis . The ranitidine treated group revealed that; the extravasation of RBCs in the core of gastric villi among the intervillus spaces became negligible. More over the desquamation of the mucous cells of the gastric mucosa and complete separation in the pyloric glands are still pronounced even with the ranitidine treatment  and restoring the depleted GSH levels together with reducing MDA levels. This antioxidant effect of the C. ignea extract could be attributed to its strong free radical scavenging activity due to the presence of a significant amount of, the powerful antioxidants, flavonoids and phenolic compounds. This is consistent with Mei et al. [C. ignea extract had strong antioxidant effect, which is comparable to that of ranitidine. Ahmadi et al. [Laine et al.  stated ti et al.  who estai et al.  previousC. ignea aerial parts at both doses attenuated ethanol-induced gastric ulcer through its antioxidant and anti-inflammatory effects. This gastroprotective efficiency of C. ignea aerial parts extract could be possibly attributed to the presence of wealthy phytoconstituents as total polyphenols, flavonoids and tannins. Therefore, C. ignea could be used as a promising anti-ulcer agent in the treatment of gastric ulcers due to its comparable anti-ulcer effect to that of ranitidine. However, further researches should be taken to further explore the underlying mechanisms of action.The results of the present study demonstrated that the aqueous ethanolic extract of


MPN diagnosis affects survival of individuals as compared with matched populations. Concerning PMF, survival is currently stratified on the basis of the International Prognostic Scoring System (IPSS)2 and its variants or on the most recent mutation-based MIPSS-703. In post-PV MF and post-ET MF, namely secondary myelofibrosis (SMF), the MYSEC PM (Myelofibrosis Secondary to PV and ET-Prognostic Model), has been recently developed to assess survival4. Epidemiological data have revealed an advantage for women in surviving a diagnosis of cancer compared with men5. Sex hormones might play a role in the hematopoiesis and in the pathogenesis of hematologic malignancies6. Concerning MPNs, gender differences have been observed in terms of disease distribution , JAK2V617F allele burden 7, and numbers of homozygous mutant colonies 8.Myeloproliferative neoplasms (MPN) include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) and are established clonal disorders 1. Molecular and genetic tests were performed as previously described10. The study was approved by the ethical committee of each institution and conducted in accordance with the Declaration of Helsinki. Statistical analyses considered clinical and laboratory data collected at the time of progression to SMF. Wilcoxon rank sum and Pearson’s chi-squared tests were performed to report differences between the groups, whereas Kaplan–Meier estimators, log-rank tests, and Cox regression models were used for time-to-event analysis.In this study, we assessed the prognostic impact of gender in the study population of the multicenter MYSEC project including 684 SMF patients with driver mutational status available. Diagnosis of SMF was performed between 1981 and 2015 and were locally reviewed according to the International Working Group on Myeloproliferative Neoplasm Research and Treatment criteria (IWG-MRT 2008)p = 0.027) and that age at the time of SMF transformation was similar between the genders . This is in favor of a slower progression of the diseases in females, as documented by a longer time to progression in SMF .The first observation we found is that diagnosis of ET and PV occurred at younger age in females versus males . In SMF, female sex was correlated with higher platelet count (p = 0.041), smaller palpable spleen (p = 0.016), and lower frequency of circulating blasts ≥ 1% (p = 0.008). We found also a correlation with lower hemoglobin levels (p = 0.036), but this was evident only in younger women as a possible expression of a pre-menopausal phase. Within the PET MF cohort, female sex was significantly associated with smaller palpable spleen (p = 0.024) and lower frequency of circulating blasts ≥1% (p = 0.027). Conversely, we did not find gender differences within PPV MF cohort.Table JAK2 in 257 (78%), CALR in 47 (14%), MPL in 17 (5%), and triple negative in seven females . Within females with PET MF, the distribution was as follows: 96 JAK2 (57%), 47 CALR (28%), 17 MPL (10%), 7 TN (4%). Mutation frequency was similar in the two genders (p = 0.57). Furthermore, female gender frequency per genotype was 46% in JAK2-pos PPV MF, 53% in JAK2-pos PET MF, 46% in CALR-pos, 57% in MPL-pos, and 37% in TN cases without significant differences with respect to male gender (p = 0.9). Concerning cytogenetic profile, abnormalities have been described in 58 females and 58 males . Of interest, the rate of complex karyotype  was lower in females versus males .Driver mutational status within SMF females was p = 0.32). In addition, the rate of fatal thrombosis was superimposable. Concerning blast phase (BP), 52 (8%) transformations occurred with an incidence of 1.8/100 patient-years and 2/100 patient-years in the female and male groups, respectively (p = 0.88).During a median follow-up of 3 years , 67 (10%) thrombosis occurred with an incidence of 3.1/100 patient-years and 2.4/100 patient-years in the female and male groups, respectively without significant differences  with a Cox regression model, was uninfluential (p = 0.3). Of note, female gender retained a survival advantage, albeit with a weak statistical significance, even when added as a covariate to the MYSEC-PM risk strata and type of diagnosis (PPV MF and PET MF) . Taking into account earlier disease onset in females, a multivariable analysis including age at diagnosis and gender found that female sex still remains significant .Death occurred in 168 (25%) patients. Overall survival from diagnosis of SMF was better in female  than in male patients , with a hazard ratio (HR) of 0.68  and a longer life expectancy than male counterpart. Survival difference between men and women in the general population is well recognized, and in cancer subset shorter survival might be linked to host factors, such as activity of sex steroid hormone pathway, occupational exposures, and lifestyle factors, as already described11. This is indicative of a lower genomic instability in females during disease progression. Looking for gene expression between genders in MPNs, a differential gene expression in female patients as compared with male patients has been reported with 235 genes differentially regulated in women, vs. 571 genes differentially regulated in men12. In our analysis, we found that women have a lower rate of complex karyotype  as a result of lesser genomic instability. The correlation between complex karyotype and worse survival has been recently documented in SMF10. This could add explanations to the different survivals that we found between the genders.From a biological viewpoint, MPNs patients exhibit decreased telomere length, more evident in men than in women13 demonstrated that older women with ET live longer than their male counterparts and that gender might supersede thrombosis history as a risk variable for overall survival. An analysis on 1109 PMF showed that mild anemia, defined as hemoglobin between 10 g/dl and sex-adjusted lower limit of normal, independently predicted shortened survival in men, but not in women14.The impact of gender on clinical phenotype has been recently investigated in ET and PMF, but never in SMF. Tefferi et al.15, this can eventually overbalance the overall drug results.The magnitude of difference in benefit we found in term of survival between genders  seems clinically relevant and has some implications. The most practical is for the doctor/patient communication at the time of diagnosis of SMF. The second is for helping decision making in those areas where a case by case decision is suggested. So far, new therapeutic approaches in MPNs have not taken sex into consideration. However, focusing on subgroup analysis of the COMFORT trials, women receiving ruxolitinib have better survival than men In conclusion, our study in SMF indicates that female gender has a more indolent disease with a specific phenotype and better prognosis. This finding finally led to a more accurate definition of the natural history of the disease and drove the attention toward a careful interpretation of clinical trial results.


New-onset atrial fibrillation (NOAF) is commonly encountered in critically ill adults. Evidence evaluating the association between NOAF and patient-important outcomes in this population is conflicting. Furthermore, little is known regarding the association between NOAF and resource use or hospital costs.Retrospective analysis (2011–2016) of a prospectively collected registry from two Canadian hospitals of consecutive ICU patients aged ≥ 18 years. We excluded patients with a known history of AF prior to hospital admission. Any occurrence of atrial fibrillation (AF) was prospectively recorded by bedside nurses. The primary outcome was hospital mortality, and we used multivariable logistic regression to adjust for confounders. We used a generalized linear model to evaluate contributors to total cost.We included 15,014 patients, and 1541 (10.3%) had NOAF during their ICU admission. While NOAF was not associated with increased odds of hospital death among the entire cohort , an interaction was noted between NOAF and sepsis, and the presence of both was associated with higher odds of hospital mortality (aOR 1.28 [95% CI 1.09–1.36]) than either alone. Patients with NOAF had higher total costs (cost ratio [CR] 1.09 [95% CI 1.02–1.20]). Among patients with NOAF, treatment with a rhythm-control strategy was associated with higher costs (CR 1.24 [95% CI 1.07–1.40]).While NOAF was not associated with death or requiring discharge to long-term care among critically ill patients, it was associated with increased length of stay in ICU and increased total costs. Atrial fibrillation (AF) is the most common cardiac dysrhythmia, with a lifetime risk of 1 in 4 among older adults . In the The clinical importance of critical illness-associated NOAF is a matter of ongoing uncertainty . AF in aEqually important is the relationship between NOAF, and subsequent ICU resource use and costs , 12. SinWe obtained ethics approval for this study from the Ottawa Health Science Network Research Ethics Board (protocol 20160570-01H).We studied ICU patients from two hospitals within the Ottawa Hospital network . These hospitals have approximately 2500 combined ICU admissions per year. These are combined medical and non-cardiac surgical ICUs. We retrospectively examined prospectively collected data from the Ottawa Hospital Data Warehouse, a health administrative database used in previous studies –19. FromWe included all consecutive patients ≥ 18 years of age, admitted to one of the two ICUs between January 2011 and December 2016. Sample size was determined pragmatically, on the basis of available patients in the Data Warehouse. We also examined pre-specified subgroups of patients, including those with suspected infection, sepsis, and septic shock, as based on the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) . “SuspecWe obtained all data from the Ottawa Hospital Data Warehouse. We abstracted demographic data, comorbidities, Elixhauser Comorbidity Score , and MulWe determined patient costs using the case-costing system of the Ottawa Hospital Data Warehouse, as done previously , 23, 27.The primary outcome was hospital mortality. Secondary outcomes included discharge directly from hospital to long-term care , hospital readmission within 30 days of hospital discharge among survivors, ICU length of stay (LOS), hospital LOS, resource utilization , and total hospital costs.For each patient, the occurrence of any AF was prospectively recorded by bedside nurses for the purposes of quality assurance. The date and time of AF, as captured by the bedside nurse, was stored in the Data Warehouse. Patients identified through this method were then evaluated by a single investigator (SMF), to confirm the diagnosis. Since there is no consensus definition for NOAF , we follt test , Mann-Whitney test , and χ2  were performed to determine between-group differences. In keeping with existing guidelines, we did not perform pairwise comparisons of baseline characteristics [P value of ≤ 0.05 was considered statistically significant.We performed all statistical analyses using R version 3.3.3) and IBM SPSS version 24.0). We present data as mean values, with standard deviation (SD), or medians, with interquartile range (IQR), where appropriate. Student’s eristics . To adjueristics . As per .3.3 and .0. We preristics , we usederistics . We evaleristics . If a steristics , 32. We A total of 17,173 patients were admitted to the participating ICUs from 2011 to 2016  had NOAF while in the ICU. Baseline characteristics of patient with and without NOAF are shown in Table n = 4837, Table n = 1944, 40.2% of “suspected infection” population), and septic shock  are included in Additional files P < 0.001) and median total hospital LOS . Among patients with NOAF, factors associated with increased risk of hospital mortality included increasing age, increased MODS score, history of CHF (as identified in the Data Warehouse), and sustained AF  or non-invasive ventilation . Vasoactive medication use was higher among patients with NOAF . In terms of treatment strategy for NOAF, 747 (48.5%) patients received antiarrhythmic medical therapy , while 644 (41.8%) received therapy with a beta-blocker, calcium channel blocker, or digoxin. A total of 128 patients (8.3%) received a combination of the above therapies.Comparisons of resources used between patients with and without AF are shown in Table P < 0.001), as were mean total ICU costs . Total cost per survivor for patients with NOAF was $81,120, as compared to $59,710 for patients without NOAF. Presence of NOAF in our ICU cohort was a significant predictor of total hospital costs  was associated with higher cost, but decrease in mortality. While this study was not designed to test the efficacy of therapeutic agents for NOAF, the little evidence that exists on this topic seems to suggest no beneficial effect of any particular agent , 4. ThisWe used a large multicenter database of patients with prospective identification of NOAF, and provide novel data related to hospital mortality, resource utilization, and cost. We also closely followed recommendations for control of confounding in observational studies . HoweverWhile NOAF was not associated with hospital mortality among all critically ill patients, it was associated with mortality in subgroups of patients with suspected infection, sepsis, and septic shock. Among patients with NOAF, sustained AF was associated with higher risk of hospital mortality. Finally, patients with NOAF had higher costs than patients without NOAF, and NOAF was a predictor of increased total costs among all ICU patients.Additional file 1 : Table S1. Multivariable Logistic Regression Model for hospital mortality for entire study cohort . Multivariable Logistic Regression Model for hospital mortality for entire study cohort .Additional file 2 : Table S2. Multivariable Logistic Regression Model for hospital mortality for patients with sepsis (n = 1944). Multivariable Logistic Regression Model for hospital mortality for patients with sepsis (n = 1944).Additional file 3 : Table S3. Multivariable Logistic Regression Model for hospital mortality for patients with septic shock (n = 1286). Multivariable Logistic Regression Model for hospital mortality for patients with septic shock (n = 1286).Additional file 4 : Table S4. Multivariable Logistic Regression Model for hospital mortality for entire study cohort , including interaction terms. Multivariable Logistic Regression Model for hospital mortality for entire study cohort , including interaction terms.Additional file 5 : Table S5. Multivariable Logistic Regression Model for hospital mortality among patients with new-onset atrial fibrillation (n = 1541). Multivariable Logistic Regression Model for hospital mortality among patients with new-onset atrial fibrillation (n = 1541).Additional file 6 : Table S6. Generalized Linear Model with gamma distribution and log link for total cost for entire study cohort . Generalized Linear Model with gamma distribution and log link for total cost for entire study cohort .Additional file 7 : Table S7. Generalized Linear Model with gamma distribution and log link for total cost for patients with new-onset atrial fibrillation . Generalized Linear Model with gamma distribution and log link for total cost for patients with new-onset atrial fibrillation .


According to the WHO Multicentre Growth Reference Study Group recommendations, boys and girls have different growth trajectories after birth. Our aim was to develop gender-specific fetal growth curves in a low-risk population and to compare immediate birth outcomes.First, second, and third trimester fetal ultrasound examinations were conducted between 2002 and 2012. The data was selected using the following criteria: routine examinations in uncomplicated singleton pregnancies, Caucasian ethnicity, and confirmation of gestational age by a crown-rump length (CRL) measurement in the first trimester. Generalized Additive Model for Location, Scale and Shape (GAMLSS) was used to align the time frames of the longitudinal fetal measurements, corresponding with the methods of the postnatal growth curves of the WHO MGRS Group.p < 0.001) equating to a 3-day difference at 20–24 weeks. Boys were significantly heavier, longer, and had greater head circumference than girls (p < 0.001) at birth. The Apgar score at 1 min (p = 0.01) and arterial cord pH (p < 0.001) were lower in boys.A total of 27,680 complete scans were selected from the astraia© ultrasound database representing 12,368 pregnancies. Gender-specific fetal growth curves for biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL) were derived. The HC and BPD were significantly larger in boys compared to girls from 20 weeks of gestation onwards  contains supplementary material, which is available to authorized users. Ultrasound has been an indispensable tool for diagnosis in obstetrics and fetal growth assessment for at least 4 decades –3. CliniMany charts have been published on fetal growth using different methodologies from the early 1990s until early in this decade, after which new (dating) protocols emerged . Most noOur aim was to develop gender-specific longitudinal first, second, and third trimester normal growth reference curves within a low-risk Caucasian population with a robust WHO-endorsed longitudinal statistical methodology. Further, we aimed to test the validity of these curves by comparing the estimated fetal weights derived from these charts to actual birth weight, and determine whether there were gender differences in fetal growth trajectories and immediate birth outcomes.This was an observational longitudinal cohort study of first, second, and third trimester fetal biometry ultrasound examinations performed during 2002–2012 in the University Hospital Leuven. The study was approved by the ethics committee of the University Hospitals KU Leuven. The data was selected from the astraia© ultrasound database with the following criteria  was applied to construct the growth curves for all four fetal routine fetal biometry measurements: BPD, HC, AC, and FL, by the use of the R package software [10 EFW = 1.3350.0034 (AC) (FL) + 0.0316 (BPD) + 0.0457 (AC) + 0.1623 (FL)] was used [Outliers in BPD, HC, AC, or FL were removed from the data. Generalized Additive Models for Location, Scale and Shape , 6.6% of the women smoked. Gender-specific birth datasets could be ascertained in 76.1% of the cases and are outlined in Table p < 0.001) different for boys  as compared to girls . A low 1-min AS (≤ 5) was more common in boys (3.8%) as compared to girls (2.9%) (p = 0.01) as was a low 5-min AS (≤ 7) for boys (3.2%) compared to girls . The arterial umbilical cord pH was lower in boys compared to girls (p < 0.001). There was no difference in asphyxia, defined as a pH < 7.10, in boys (0.9%) compared to girls , and abnormal BE (< − 10 mEq/L) was the same for both sexes. There was no difference in preterm birth (< 37 weeks) for girls (5.7%) and boys , longer (p = 0.005), and had larger head circumferences (p = 0.006). The immediate outcome of AS and pH were also different in boys and girls, although not statistically different due to the smaller preterm group  difference for all percentiles in boys having higher BPD measurements  [2), non-smoking women, 18–35 years of age and recruited in selected institutes. This highly qualitative study  were measured in standardized circumstances in accordance with international guidelines . Longitu0 weeks) . In Belg0 weeks) . Also, t0 weeks) –19, 39. 0 weeks) . Finally0 weeks) , 19. Eth0 weeks) , 41. The0 weeks) . The stuSome limitations on constructing these charts have to be addressed. The study was performed in a university teaching hospital, a large tertiary referral center, not necessarily reflecting a routine setting. This center, on the other hand, also has a regional remit for routine obstetric care for low-risk pregnancies, but the included cases were not selected on maternal morbidity nor on parental characteristics. Some maternal characteristics  were not excluded in the selected cohort, deliberately to prevent “super-normalization” of the cohort. But artificial conception was excluded for intracytoplasmic sperm injection, since this is a level 1 ultrasound indication. Finally, it is expected that within this large time period, some women with subsequent pregnancies were included more than once for this cohort.Our fetal growth curves for the Caucasian population resemble predictive growth curves with the gender specified which can discern aberrant from normal fetal growth. The longitudinal aspect and large cohort, covering the full trimesters, have not been reported before in the Caucasian population. The neonatal data gave us the opportunity to customize for the fetal gender. There was a marked difference between fetal boys and girls in their growth trajectory for fetal head measurements and to a lesser extent the abdominal circumferences. Also for the estimated fetal weight, there was a difference. This gender differentiation is important in antenatal and perinatal care. Prenatal ultrasound is used not only to define fetal growth, but also gestational age. Both growth and fetal age are important in defining the time point of fetal viability and the optimization of the timing of obstetrical interventions, e.g., medical elective birth or administration of corticosteroids for fetal lung maturation in cases of threatened premature birth. Second trimester dating depends on fetal growth parameters and particular on the fetal head measurement. Our results suggest a gender-specific approach in counseling future parents on important issues when fetal viability starts and when is the best time point to start obstetrical interventions.The gender differences are further demonstrated by the immediate birth outcomes for males: different anthropometry , lower AS, and lower cord pH. The significant lower AS and umbilical cord pH in boys underline the fetal male vulnerability, although in the asphyxia group (pH < 7.10), there was no predominance by males, stating that boys do not have a higher risk of acidemia at birth in a routine population. Therefore, one can argue on the clinical importance of the pH findings  in our study.In summary, we present fetal growth curves with the latest statistical tools in a large, routine pregnant population with state-of-the-art ultrasound technology. The data covers the pregnancy period from 12 weeks onwards, and there were differences between boys and girls for the fetal head and fetal abdomen measurements and the estimated fetal weight. Also, the immediate neonatal outcome demonstrated gender differences favoring the girls. This could give caretakers the opportunity to take into account a gender-tailored approach in life decision care both at the margins of viability and post-term.Additional file 1:Ultrasound protocol guidelines .  pregnancies. (DOCX 23 kb)Additional file 3:Boys vs Girls combined scatterplots and curves. (DOCX 816 kb)Additional file 4:Boys vs Girls combined gridcurves and reference values. (DOCX 4051 kb)Additional file 5:Boys vs Girls combined centiles and real values. (DOCX 35 kb)Additional file 6:Table S2. Fetal charts characteristics, quality control. (DOCX 17 kb)