A 12 kb multi-allelic copy number variation encompassing a GC gene enhancer is associated with mastitis resistance in dairy cattle

Clinical mastitis (CM) is an inflammatory disease occurring in the mammary glands of lactating cows. CM is under genetic control, and a prominent CM resistance QTL located on chromosome 6 was reported in various dairy cattle breeds. Nevertheless, the biological mechanism underpinning this QTL has been lacking. Herein, we mapped, fine-mapped, and discovered the putative causal variant underlying this CM resistance QTL in the Dutch dairy cattle population. We identified a ~12 kb multi-allelic copy number variant (CNV), that is in perfect linkage disequilibrium with a lead SNP, as a promising candidate variant. By implementing a fine-mapping and through expression QTL mapping, we showed that the group-specific component gene (GC), a gene encoding a vitamin D binding protein, is an excellent candidate causal gene for the QTL. The multiplicated alleles are associated with increased GC expression and low CM resistance. Ample evidence from functional genomics data supports the presence of an enhancer within this CNV, which would exert cis-regulatory effect on GC. We observed that strong positive selection swept the region near the CNV, and haplotypes associated with the multiplicated allele were strongly selected for. Moreover, the multiplicated allele showed pleiotropic effects for increased milk yield and reduced fertility, hinting that a shared underlying biology for these effects may revolve around the vitamin D pathway. These findings together suggest a putative causal variant of a CM resistance QTL, where a cis-regulatory element located within a CNV can alter gene expression and affect multiple economically important traits.     

Going "open" with Mesoscopy: a new dimension on multi-view imaging

OpenSPIM and OpenSpinMicroscopy emerged as open access platforms for Light Sheet and Optical Projection Imaging, often called as optical mesoscopy techniques. Both projects can be easily reproduced using comprehensive online instructions that should foster the implementation and further development of optical imaging techniques with sample rotation control. This additional dimension in an open system offers the possibility to make multi-view microscopy easily modified and will complement the emerging commercial solutions. Furthermore, it is deeply based on other open platforms such as MicroManager and Arduino, enabling development of tailored setups for very specific biological questions. In our perspective, the open access principle of OpenSPIM and OpenSpinMicroscopy is a game-changer, helping the concepts of light sheet and optical projection tomography (OPT) to enter the mainstream of biological imaging.     

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.     

Epithelial cadherin is present in bovine oviduct epithelial cells and gametes,and is involved in fertilization-related events

Fertilization is a calcium-dependent process that involves sequential cell–cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein β-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and β-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine–induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. β-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell–cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm–OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.     

Historical perspectives and recent research on superovulation in cattle

Superovulation protocols have evolved greatly over the past 40 to 50 years. The development of commercial pituitary extracts and prostaglandins in the 1970s, and partially purified pituitary extracts and progesterone-releasing devices in the 1980s and 1990s have provided for the development of many of the protocols that we use today. Furthermore, the knowledge of follicular wave dynamics through the use of real-time ultrasonography and the development of the means by which follicular wave emergence can be controlled have provided new practical approaches. Although some embryo transfer practitioners still initiate superstimulatory treatments during mid-cycle in donor cows, the elective control of follicular wave emergence and ovulation has had a great effect on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. The most common treatment for the synchronization of follicular wave emergence for many years has been estradiol and progestins. In countries where estradiol cannot be used, practitioners have turned to alternative treatments for the synchronization of follicle wave emergence, such as mechanical follicle ablation or the administration of GnRH to induce ovulation. An approach that has shown promise is to initiate FSH treatments at the time of the emergence of the new follicular wave after GnRH-induced ovulation of an induced persistent follicle. Alternatively, it has been suggested recently that it might be possible to ignore follicular wave status, and by extending the treatment protocol, induce small antral follicles to grow and superovulate. Recently, the mixing of FSH with sustained release polymers or the development of long-acting recombinant FSH products have permitted superstimulation with a single or alternatively, two gonadotropin treatments 48 hours apart, reducing the need for animal handling during superstimulation. Although the number of transferable embryos per donor cow superstimulated has not increased, the protocols that are used today have increased the numbers of transferable embryos recovered per unit time and have facilitated the application of on-farm embryo transfer programs. They are practical, easy to administer by farm personnel, and more importantly, they eliminate the need for detecting estrus.     

Electron microscopy morphology of the mitochondrial network in gliomas and their vascular microenvironment

Gliomas still represent a serious and discouraging brain tumor; despite of the diversity of therapeutic modalities, the prognosis for patients is still poor. Understanding the structural and functional characteristics of the vascular microenvironment in gliomas is essential for the design of future therapeutic strategies. This review describes and analyzes the electron microscopy morphology of the mitochondrial network in human gliomas and their vascular microenvironment. Heterogenous mitochondrial network alterations in glioma cells and in microvascular environment are implicated directly and indirectly in the processes linked to hypoxia-tolerant and hypoxia-sensitive cells phenotype, effects of the hypoxia-inducible factor-1α, increased expression of several glycolytic protein isoforms as well as fatty acid synthase, and survivin. The prevalent existence of partial and total cristolysis observed suggests that oxidative phosphorylation is severely compromised. A mixed therapy emerged as the most appropriate.     

The intracellular distal tail of the Na+/H+ exchanger NHE1 is intrinsically disordered: implications for NHE1 trafficking

Intrinsic disorder is important for protein regulation, yet its role in regulation of ion transport proteins is essentially uninvestigated. The ubiquitous plasma membrane carrier protein Na(+)/H(+) Exchanger isoform 1 (NHE1) plays pivotal roles in cellular pH and volume homeostasis, and its dysfunction is implicated in several clinically important diseases. This study shows, for the first time for any carrier protein, that the distal part of the C-terminal intracellular tail (the cdt, residues V686-Q815) from human (h) NHE1 is intrinsically disordered. Further, we experimentally demonstrated the presence of a similar region of intrinsic disorder (ID) in NHE1 from the teleost fish Pleuronectes americanus (paNHE1), and bioinformatic analysis suggested ID to be conserved in the NHE1 family. The sequential variation in structure propensity as determined by NMR, but not the amplitude, was largely conserved between the h- and paNHE1cdt. This suggests that both proteins contain molecular recognition features (MoRFs), i.e., local, transiently formed structures within an ID region. The functional relevance of the most conserved MoRF was investigated by introducing a point mutation that significantly disrupted the putative binding feature. When this mutant NHE1 was expressed in full length NHE1 in AP1 cells, it exhibited impaired trafficking to the plasma membrane. This study demonstrated that the distal regulatory domain of NHE1 is intrinsically disordered yet contains conserved regions of transient structure. We suggest that normal NHE1 function depends on a protein recognition element within the ID region that may be linked to NHE1 trafficking via an acidic ER export motif.