OSBPL2-disrupted pigs recapitulate dual features of human hearing loss and hypercholesterolaemia

Oxysterol binding protein like 2(OSBPL2), an important regulator in cellular lipid metabolism and transport, was identified as a novel deafness-causal gene in our previous work. To resemble the phenotypic features of OSBPL2 mutation in animal models and elucidate the potential genotypephenotype associations, the OSBPL2-disrupted Bama miniature(BM) pig model was constructed using CRISPR/Cas9-mediated gene editing, somatic cell nuclear transfer(SCNT) and embryo transplantation approaches, and then subjected to phenotypic characterization of auditory function and serum lipid profiles. The OSBPL2-disrupted pigs displayed progressive hearing loss(HL) with degeneration/apoptosis of cochlea hair cells(HCs) and morphological abnormalities in HC stereocilia, as well as hypercholesterolaemia. High-fat diet(HFD) feeding aggravated the development of HL and led to more severe hypercholesterolaemia. The dual phenotypes of progressive HL and hypercholesterolaemia resembled in OSBPL2-disrupted pigs confirmed the implication of OSBPL2 mutation in nonsydromic hearing loss(NSHL) and contributed to the potential linkage between auditory dysfunction and dyslipidaemia/hypercholesterolaemia.     

Effects of different sucrose concentrations on vitrified porcine preantral follicles: Qualitative and quantitative analysis

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M–0.25 M – 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended.     

Fat accumulation in differentiated brown adipocytes is linked with expression of Hox genes

Homeobox (Hox) genes are involved in body plan of embryo along the anterior–posterior axis. Presence of several Hox genes in white adipose tissue (WAT) and brown adipose tissue (BAT) is indicative of involvement of Hox genes in adipogenesis. We propose that differentiation inducing agents viz. isobutyl-methyl-xanthine (IBMX), indomethacin, dexamethasone (DEX), triiodothyronine (T3) and insulin may regulate differentiation in brown adipose tissue through Hox genes. In vitro culture of brown fat stromalvascular fraction (SVF) in presence or absence of differentiation inducing agents was used for establishing relationship between fat accumulation in differentiated adipocytes and expression of Hox genes. Relative expression of Pref1, UCP1 and Hox genes was determined in different stages of adipogenesis. Presence or absence of IBMX, indomethacin and DEX during differentiation of proliferated pre-adipocytes resulted in marked differences in expression of Hox genes and lipid accumulation. In presence of these inducing agents, lipid accumulation as well as expression of HoxA1, HoxA5, HoxC4 & HoxC8 markedly enhanced. Irrespective of presence or absence of T3, insulin down regulates HoxA10. T3 results in over expression of HoxA5, HoxC4 and HoxC8 genes, whereas insulin up regulates expression of only HoxC8. Findings suggest that accumulation of fat in differentiated adipocytes is linked with expression of Hox genes.     

The oxidant-antioxidant equilibrium,activities of selected lysosomal enzymes and activity of acute phase protein in peripheral blood of 18-year-old football players after aerobic cycle ergometer test combined with ice-water immersion or recovery at room temperature

The goal of the study was to evaluate the effect of an aerobic exercise bout followed by ice-water immersion or recovery at room temperature on the redox state, activities of selected lysosomal enzymes and activity of α1-antitrypsin (AAT) in the blood of healthy sportsmen. Eleven amateur football players aged 18 were randomly assigned to two similar 30-min aerobic cycle ergometer tests followed by a recovery at room temperature (20 °C; Experiment 1) or ice-water immersion (3 °C, 5 min; Experiment 2). Peripheral blood was collected three times during both study experiments: before (baseline), as well as 20 and 40 min after the recovery or immersion. The concentrations of thiobarbituric acid reactive substances in blood plasma (plTBARS) and erythrocytes (erTBARS) were measured. The erythrocytic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were also determined. In the blood serum, the activities of acid phosphatase (AcP), arylsulphatase (ASA), cathepsin D (CTS D) and AAT were evaluated. The activities of AcP, ASA, CTS D and AAT changed similarly during both experiments. The GPx activity decreased 40 min after the exercise/recovery compared to the baseline activity and was lower than 40 min after the exercise/immersion. The exercise followed by the recovery or immersion had no significant effect on the serum lysosomal and AAT activities in the studied men. The exercise/recovery reduced the hydrogen peroxide concentration in the men's erythrocytes, however the exercise/immersion demonstrated the opposite effect.     

Effect of resveratrol on vitrified in vitro produced bovine embryos: Recovering the initial quality

Although vitrification is the current routine method for human embryo cryopreservation, it may cause detrimental effects. The aim of this study was to evaluate the effect of supplementing in vitro culture (IVC) media and/or vitrification solutions (VS) with Resveratrol on the presence of apoptotic markers, reactive oxygen species (ROS) level, glutathione (GSH) content and relative gene abundance. Abattoir-derived oocytes were matured and fertilized in vitro according to a standard procedure. Zygotes were cultured in IVC medium supplemented with or without 0.5 μM Resveratrol (C^R, C⁻ respectively). On day 7, blastocysts were vitrified using the minimum volume vitrification method supplementing VS with (C⁻V^R, C^RV^R) or without (C^–V^-, C^RV⁻) 0.5 μM Resveratrol. After warming, embryonic quality parameters were evaluated. Survival rates were significantly lower in C^RV^R group compared with C^RV⁻ group, but no differences in hatching rate were observed between groups. Vitrification/warming process did not alter total cell number or the presence of apoptotic or dead cells, but C^RV⁻ and C^RV^R groups presented a significant increase in dead cells (P < 0.05 by ANOVA). Resveratrol supplementation in VS (C⁻V^R) restored GSH content (P < 0.05) to the level found in the C^R group. Vitrification/warming process significantly increased the expression of FOXO3A, PNPLA2, BCL2L1 and BAX genes (P < 0.05). Resveratrol addition to IVC medium or VS partially compensated this increase for FOXO3A and PNPLA2 (P < 0.05) but not for BCL2L1 and BAX. In conclusion, supplementation of IVC media or VS with 0.5 μM resveratrol may help embryos to partially restore the initial quality they had before the cryopreservation process.     

Immunological response induced by cryoablation against murine H22 hepatoma cell line in vivo

ObjectivesTo describe immunological consequences induced by cryoablation against H22 cells in vivo.MethodsAdult BALB/c mice underwent subcutaneous implantation of H22 cells. All of them were assigned into three groups randomly: group A (false surgery), group B (cryoablation) and group C (cryoablation plus Freund's adjuvant). Animals were sacrificed 1, 2 and 3 weeks after treatment. Serum IFN-γ and IL-4, Th1/Th2 in spleens and cytotoxicity were detected.ResultsCompared with that of group A, (1) INF-γ of group B was higher, but IL-4 was lower; cryoablation plus Freund's adjuvant enhanced these effects. (2) Th1/Th2 rose significantly in both group B and group C. (3) Strong cytolytic activity against H22 cells of group B and group C was found on day 7, 14 and 21.ConclusionsOur study showed a marked shift toward Th1 and IFN-γ expression after cryoablation, with an immuno-stimulatory effect against murine H22 hepatoma Cell.     

Phenotypic and genotypic intra-diversity among Anatolian durum wheat “Kunduru” landraces

Kunduru is an important Anatolian landrace having peculiar traits that are appreciated by farmers and breeders. 33 accessions known as Kunduru collected by ICARDA from six geographical provinces of Turkey, were used to study the phenotypic and genotypic intra-diversity. Kunduru landraces exhibited high intra-diversity for most of the studied morphological traits. GPC (12.10–14.90%), vitreousness (75–100%), TKW (31.80–56.70 g), YP (4.70–8.00 ppm), b*-value (14.30–19.50), ash content (1.60–2.0%) and gluten strength (14–60 ml) showed marked variations. Gliadin and glutenin banding patterns showed high polymorphism. 65 alleles were detected with 14 SSR markers, giving a mean of 6.77 alleles per locus. The average PIC value was 0.44 and ranged from 0.11 to 0.70. The average genetic distance between pairs of landraces was 0.47 and ranged between 0.11 and 0.72. This study showed that Kunduru landraces maintains high allelic variation. PCoA indicated that eco-geographical variables have a significant effect on SSR diversity as well as morphological traits. Many of the landraces studied are in danger of disappearing from the local farmers' fields; this study demonstrates the importance of maintaining and conserving this precious genetic resources.     

Sternal gland structures in males of bean flower thrips,Megalurothrips sjostedti,and Poinsettia thrips,Echinothrips americanus,in comparison with those of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them.We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III–VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III–VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV–VII. Pore plate and pore plate area were distributed similarly on sternites III–VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.     

The ACBP gene family in Rhodnius prolixus: Expression,characterization and function of RpACBP-1

The acyl-CoA-binding proteins (ACBP) constitute a family of conserved proteins that bind acyl-CoA with high affinity and protect it from hydrolysis. Thus, ACBPs may have essential roles in basal cellular lipid metabolism. The genome of the insect Rhodnius prolixus encodes five ACBP genes similar to those described for other insect species. The qPCR analysis revealed that these genes have characteristic expression profiles in insect organs, suggesting that they have specific roles in insect physiology. Recombinant RpACBP-1 was able to bind acyl-CoA in an in vitro gel-shift assay. Moreover, heterologous RpACBP-1 expression in acb1Δ mutant yeast rescued the multi-lobed vacuole phenotype, indicating that RpACBP-1 acts as a bona fide acyl-CoA-binding protein. RpACBP-1 knockdown using RNAi caused triacylglycerol accumulation in the insect posterior midgut and a reduction in the number of deposited eggs. The amount of stored triacylglycerol was reduced in flight muscle, and the incorporation of fatty acids in cholesteryl esters was increased in the fat body. These results showed that RpACBP-1 participates in several lipid metabolism steps in R. prolixus.     

Structural features,evolutionary relationships,and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta

C-type lectins (CTLs) are a large family of Ca²⁺-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca²⁺-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes,fibH, fibL and fhx,in the silk gland of the silkworm,Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.     

Spatial and temporal distribution of Patched-related protein in the Drosophila embryo

Patched-related (Ptr) encodes a protein with 12 potential transmembrane domains and a sterol-sensing domain that is closely related in predicted topology and domain organization to Patched, the canonical receptor of the Hedgehog pathway. Here we describe the production of an antibody specific for Drosophila Ptr and analyse its spatial and temporal distribution in the embryo. We find that at early developmental stages Ptr is predominantly localized at cell periphery but later on it becomes strongly and almost exclusively expressed in hemocytes. Interestingly Ptr null mutant embryos died without hatching. Our findings suggest that Ptr plays an essential function in Drosophila development, perhaps as a new receptor of embryonic hemocytes.     

Decomposition of sugarcane bagasse with lignocellulose-derived thermotolerant and thermoresistant Penicillia and Aspergilli

To promote the decomposition of sugarcane bagasse (SCB) for conversion into value-added products and to reduce waste, the capability of fungal mixes (FMs) to degrade SCB was examined. A total of 169 isolates from SCB and non-SCB were categorized as thermotolerant and thermoresistant. Thirty-six fungal candidates were screened for the presence of polyphenol oxidase, endoglucanase (EDN) and xylanase (XLN) activities, and EDN and XLN activities were quantitated. Five identified isolates (Aspergillus flavus AG10; Aspergillus niger AG68 & NB23; and Penicillium citrinum AG93 & AG140) were selected as the best enzyme producers, and 15 moderately to highly xylolytic, cellulolytic and ligninolytic isolates were added to construct FMs. Using a Taguchi design, the top ten reducing sugar-producing FMs (no. 12 showed the maximum amount of reducing sugar, at 2.11 mg g⁻¹, followed by no. 7, 15, 2, 16, 11, 13, 6, 4, & 8) were selected as potential agents for decomposition durations of 1, 2 and 3 months. The maximum decrease in SCB materials compared with the control was generated by FM 6 (9.08% cellulose reduction); FM 13 (21.03% hemicellulose reduction); and FM 16 (9.21% lignin reduction). These results indicate the potential use of SCB as a substrate for synergistic FMs. These FMs could be applied to the large-scale composting of SCB and other related agricultural residues, thus improving the biological pretreatment of lignocellulose.     

Optimization of the cryopreservation of biological resources,Toxoplasma gondii tachyzoites,using flow cytometry

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10⁶ tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me₂SO without incubation. A cooling rate of ∼1 °C/min to −80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.