A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

Development of a microtitre plate indirect ELISA for measuring cortisol in teleosts, and evaluation of stress responses in rainbow trout and gilthead sea bream

A microtitre plate indirect enzyme‐linked immunoassay (ELISA) was developed for measuring plasma cortisol levels in rainbow trout Oncorhynchus mykiss, gilthead sea bream Sparus auratus sea bass Dicentrarchus labrax and Senegalese sole Solea senegalensis. Covalink microplates pretreated with disuccinimidyl suberate were coated with bovine serum albumin (BSA) conjugated to cortisol‐3‐carboxymethyl oxime. After blocking with BSA, competition was started by addition of plasma samples and anti‐cortisol antibody raised in rabbit. Goat anti‐rabbit IgG conjugated‐peroxidase was added as second antibody and then incubated with orthophenylenediamine as substrate. Reaction was stopped with 0·1 M HCl and absorbance was read at 450 nm in an automatic plate reader. The standard curve was linear from the lower limit of sensitivity of the assay (c. 0·3 ng ml^?1) to c. 3000 ng ml^?1. Dose‐response inhibition curves using serially diluted plasma samples of four species consistently showed parallelism with the standard curve using cortisol. The ELISA satisfied the strictest criteria of specificity (cross‐reactivity of anti‐cortisol antibody with testosterone, progesterone and 17ß‐oestradiol was negligible, cross‐reactivity with cortisone, corticosterone and 11‐deoxycortisol, was 1·5, 1 and 0·1%, respectively), reproducibility (interassay CV <6%), precision (intra‐assay CV <4%), and accuracy (average recovery >98%). Plasma cortisol concentration in rested fishes was in the range of 5–30 ng ml^?1. To physiologically validate the technique, changes in plasma cortisol concentrations were also measured in plasma of rainbow trout and gilthead sea bream following an acute 15 min chasing or 3 min air‐exposure stress, respectively. In both species plasma concentrations of cortisol, glucose and lactate rose significantly with respect to controls, showing concentrations similar to those reported previously for these species under similar stress conditions. Furthermore, gilthead sea bream chronically stressed by maintaining for 14 days under increased stocking density conditions also showed increased concentrations of plasma cortisol and glucose. These results validate the indirect ELISA technique developed for use in the evaluation of plasma cortisol concentration of at least four fish species.     

Hot spots in apolipoprotein A-II misfolding and amyloidosis in mice and men

ApoA-II is the second-major protein of high-density lipoproteins. C-terminal extension in human apoA-II or point substitutions in murine apoA-II cause amyloidosis. The molecular mechanism of apolipoprotein misfolding, from the native predominantly α-helical conformation to cross-β-sheet in amyloid, is unknown. We used 12 sequence-based prediction algorithms to identify two ten-residue segments in apoA-II that probably initiate β-aggregation. Previous studies of apoA-II fragments experimentally verify this prediction. Together, experimental and bioinformatics studies explain why the C-terminal extension in human apoA-II causes amyloidosis and why, unlike murine apoA-II, human apoA-II normally does not cause amyloidosis despite its unusually high sequence propensity for β-aggregation.     

Isolation and characterization of polymorphic microsatellite loci from RAPD product in half-smooth tongue sole (Cynoglossus semilaevis) and a test of cross-species amplification

Eleven polymorphic microsatellite loci have been isolated and characterized from random amplified polymorphic DNA product in half-smooth tongue sole, Cynoglossus semilaevis. Twenty-one microsatellites were selected for designing microsatellite primers, of which 11 gave working primer pairs. They had between three and 12 alleles. Observed and expected heterozygosities varied from 0.53 to 0.93, and from 0.52 to 0.80, respectively. Five additional fish species assessed for cross-species amplification revealed between one and three positive amplifications and between zero and three polymorphic loci per species.     

Comparison of Solea solea macroparasites between two nursery-continental shelf systems in the Bay of Biscay and the Portuguese coast

Digenean metacercariae of 0 year group common sole Solea solea ( n = 70) were more abundant in the embayed nursery of the Pertuis Charentais than in the Tagus estuary nursery. Macroparasite assemblages of adult sole ( n = 119) displayed only one species in common between the Bay of Biscay and the Portuguese coast continental shelves. These data highlighted the potential use of macroparasites as biological tags in various aspects of common sole ecology.     

Positive effects of plantar vibration training for the treatment of diabetic peripheral neuropathy: A pilot study

Purpose: To evaluate the effectiveness of 8-week low-frequency plantar vibration training on patients with sensorimotor diabetic peripheral neuropathy (DPN).

Participants: Twelve patients took part in the investigation.

Results: An increase of the nerve conductive velocity of soral and peroneal nerves of feet, increased postural stability, and disappearance of the pain and tingling were observed.

Conclusion: The obtained results provide evidence for beneficial effects of 8-week plantar vibration training in patients with DPN.     

扶正活血祛瘀法治疗慢性足底溃疡的临床疗效观察

目的观察扶正活血祛瘀法治疗慢性足底溃疡的临床疗效。方法采用四妙勇安汤加减内服,红萝卜加马齿苋煎液浸泡外洗溃疡处,外敷呋喃西林纱布保护伤口的综合治疗方法。结果36例中治愈27例,占75．0％;好转6例,占16．7％;无效3例,占8．3％．姑论以扶正活血祛痰法内服为主,结合红萝卜加马齿苋煎液浸泡外洗与呋喃西林纱布外敷治疗慢性足底溃疡有较好的临床疗效．     

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

三唑磷降解菌的筛选及其降解途径研究

从三唑磷生产厂周围的土壤中用土壤富集的方法筛选分离出一株三唑磷降解菌Klebsiella sp.,它能以三唑磷为唯一碳源、唯一氮源、唯一磷源生长同时实现对三唑磷的降解,三唑磷作为唯一氮源时的降解速度最快,是实现三唑磷降解的最佳营养方案。在三唑磷为唯一氮源时,1000 mg/L的三唑磷浓度对菌体降解无抑制作用。此降解菌首先通过水解作用实现对TAP 的降解,之后把水解产物进一步降解为无毒的无机物质。降解菌的这些降解特性表明了它用于生物降解消除三唑磷污染的巨大潜力。