Secreted kinase phosphorylates extracellular proteins that regulate biomineralization

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization, including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Fam20C is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.     

High throughput phenotyping for aphid resistance in large plant collections

ABSTRACT: BACKGROUND: Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. RESULTS: In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. CONCLUSIONS: This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.     

The pre-emptive effect of epidural ketamine on wound sensitivity in horses tested by using von Frey filaments

Objective To evaluate the pre‐emptive analgesic effect of pre‐incisional epidural ketamine. Study Design A blinded, randomized experimental study. Animals Sixteen mixed breed mares, 7.6 ± 2.8 years old, weighing 352 ± 32 kg. Methods In a pilot study, an incision was made on one lateral thigh using a lidocaine block and no further analgesics, and it was verified that the nociceptive threshold was lower on the incised side than nonincised side (p ≤ 0.05), and that von Frey filaments evoked a pain response. The 16 animals were divided into group A (ketamine, n = 9) and B (saline, n = 7). An epidural catheter was inserted 24 hours before the trials. The thigh was shaved bilaterally, and the right side was blocked (incised side) using lidocaine. Twenty‐five minutes later, ketamine (A) or saline (B) was administered epidurally. Five minutes later, a 10‐cm skin incision was made on the right side, and then sutured. Nociceptive threshold was determined with von Frey filaments at 1, 3, and 5 cm around the incision at 15‐minute intervals for 2 hours, then at 4, 6, and 8 hours. Behavioral alterations, heart and respiratory rates were recorded. Nociceptive thresholds from these points were averaged to obtain mean values at each time, converted to a logarithmic scale, and submitted to a nonparametric analysis (Mann–Whitney and one‐way repeated measures anova test, p ≤ 0.05). Results After 8 hours, the global range score revealed reduced hyperalgesia (p < 0.01) around the incision in 92% (4.65–4.27) of evaluated intervals in group A (ketamine). There were no significant changes in behavior, heart and respiratory rates. Conclusions It was concluded that pre‐emptive epidural ketamine reduced post‐incisional pain in the horse, and that von Frey filaments were able to quantify cutaneous sensitivity after tissue damage. Clinical relevance Epidural ketamine injection can reduce post‐incisional sensitivity in the horse.     

The Seroprevalence of Canine Influenza Virus H3N8 in Dogs Participating in a Flyball Tournament in Pennsylvania in 2010: A Follow‐Up Study

Background

Canine influenza virus (CIV) H3N8 infection is thought to be highly contagious and potentially deadly. Low seroprevalence in dogs participating in the sport of flyball in 2009 was reported. It remains unknown if prevalence is changing in dogs at risk.

Objective

To determine seroprevalence of antibodies to CIV H3N8 in a population of pet dogs that participated in an indoor flyball tournament in Pennsylvania in November 2010.

Animals

Dogs participating in a flyball tournament.

Methods

Medical, travel, and activity histories for previous 10.5 months were obtained from consenting owners and serum samples were collected from dogs. Antibodies to CIV H3N8 were measured using hemagglutination inhibition.

Results

Of 251 competing dogs, 103 dogs were tested. Overall, seroprevalence of CIV H3N8 in dogs participating in flyball was 1.9% (95% CI, 0.05–6.8%). Thirty‐five dogs were tested in both 2009 (all titers <8) and 2010 (all titers <8). Ten of 103 dogs had been vaccinated for CIV H3N8. Two dogs had positive titers (>8) and both had been vaccinated twice in the previous month. Titers in remaining 8 vaccinated dogs were undetectable. Seroprevalence in vaccinated dogs was 20% (95% CI, 5.7–50.1%), whereas in unvaccinated dogs it was 0% (95% CI, 0–4.0%). Although 7 dogs had history of respiratory signs, only 1, which was vaccinated, was serologically positive for CIV H3N8.

Conclusions

Consistent with low prevalence of CIV H3N8 exposure previously reported in flyball dogs, unvaccinated dogs had 0% prevalence, suggesting a stable exposure rate. However, low seroprevalence may result in unprotected flyball dogs at risk for developing CIV H3N8.     

Postpartum Variations of Plasma IGF and IGFBPs,Oocyte Production and Quality in Dairy Cows: Relationships With Parity and Subsequent Fertility

The aim of this study was to determine whether postpartum variations of plasma IGF‐1 and IGFBP concentrations, oocyte production and quality were related to parity and subsequent conception rate in Holstein dairy cows. Holstein dairy cows [10 primiparous (PP) and 22 multiparous (MP)] were allotted in six batches and sampled once weekly between calving and oestrous synchronization treatment started at 71.2 ± 2.0 days postpartum. During the 3 weeks before treatment, ovum pick‐up (OPU) was performed twice weekly. Oocytes were scored on a 4‐point scale, and oocytes from OPU1, 3 and 5 were fertilized in vitro. Seventeen cows became pregnant after first and second AI and were considered as fertile (F), while the others were considered to be subfertile (SF). Logistic regression was carried out to investigate the relationships between repeated measurements and fertility including parity and batch effects in the models. Likelihood of fertility significantly increased when plasma urea and IGFBP‐3 concentrations decreased and was higher in PP compared with MP cows. There was a trend for fertility to increase when plasma IGF‐1 concentrations increased (p = 0.07). In vitro cleavage and development rates were similar between SF and F cows (46.4% and 28.3% in SF vs 55.0% and 22.1% in F). Parity had an effect on plasma IGF‐1 concentrations (PP: 61.65 ± 2.67 vs MP: 41.63 ± 5.81 ng/ml, p < 0.001), mean number of follicles aspirated per session (PP: 5.7 ± 1.3 vs MP: 9.5 ± 0.8, p < 0.05) and fertility (PP: 8/10 = 80% vs MP: 9/22 = 41%, p < 0.05) but not on the number of oocytes recovered per session nor their quality. In conclusion, postpartum plasma urea and IGFBP‐3 concentrations, but not oocyte production and quality before breeding, were related to subsequent conception rate in our experimental design. Parity had a significant effect on energy status, follicular growth and fertility and needs to be considered when investigating relationships between nutrition and reproduction.     

Environmental mastitis in intensive high‐producing dairy herds in New South Wales

Objective To determine the prevalence of mastitis pathogens in high‐producing intensive dairy herds in New South Wales. Design Field survey. Procedure Milk samples from the mastitis‐affected quarter were collected from cows on five high‐producing dairy farms in NSW. The 820 samples were cultured using standard microbiological culture techniques. Results Bacteria or fungi were isolated from 83.3% of samples (683/820). More than two colony types were isolated from 16.7% of samples (137/820), two types from 6.6% (54/820), and one type from 52.3% (429/820). No bacteria were isolated from 24.4% (200/820) of the primary cultures, but enrichment cultures of these samples yielded single colony type bacterial isolates from 36.5% (73/200) of samples. Environmental pathogens, including coliforms, environmental Streptococcus and Staphylococcus spp., made up 91% (555/610) of isolates and accounted for 33.6% (205/610), 41.6% (254/610) and 15.7% (96/610), respectively, of isolates. Escherichia coli accounted for 76.1% (156/205) of the coliform isolates, Streptococcus uberis and Streptococcus dysgalactiae accounted for 32.3% (82/254) and 28.0% (71/254), respectively, of the environmental streptococcal isolates. Contagious pathogens were uncommon, comprising only 2.5% (15/610) of the total isolates. Conclusion The incidence and causes of mastitis are largely influenced by farm management. The relatively high prevalence of coliform mastitis in the intensive high‐producing herds in this survey contrasts with the low incidence reported in surveys of pasture‐based herds in Victoria. If the Australian dairy industry continues its current trend of intensification, coliform intra‐mammary infections may emerge as an increasingly important cause of mastitis.     

Efficacy of doxorubicin‐based chemotherapy for non‐resectable canine subcutaneous haemangiosarcoma

Eighteen dogs with measurable subcutaneous haemangiosarcoma (SQHSA) were treated with doxorubicin‐based chemotherapy. Response assessment was evaluated and compared using World Health Organization (WHO), Response Evaluation Criteria in Solid Tumours (RECIST) and tumour volume criteria. The overall response rate for all dogs was 38.8% using WHO criteria, 38.8% using RECIST criteria and 44% using tumour volume criteria. One dog had a complete response. The median response duration for all dogs was 53 days (range 13–190 days). Four dogs had complete surgical excision after neoadjuvant chemotherapy. The median progression‐free interval for dogs with complete surgical excision after neoadjuvant chemotherapy was significantly longer than those not having surgical excision (207 days versus 83 days, respectively) (P = 0.003). No significant difference in metastasis‐free interval or survival time was found between the groups. Doxorubicin‐based chemotherapy appears to be effective for non‐resectable canine SQHSA, although the response duration is relatively short.     

Effect of Pre‐Treatment of Ovine Sperm on Male Pronuclear Formation and Subsequent Embryo Development Following Intracytoplasmic Sperm Injection

The objective of this study was to determine the effects of various methods of sperm pre‐treatment on male pronuclear (MPN) formation and subsequent development of ovine embryos derived from in vitro‐matured oocytes and intracytoplasmic sperm injection (ICSI). The effect of treatment of injected oocytes with dithiothreitol (DTT) on embryo development was also assessed. In Exp. 1, the injected oocytes with non‐treated sperm were activated with three different procedures. The cleavage and blastocyst rates in those activated with DTT was lower (p < 0.05) than those activated with either ionomycin (Io) + 6‐dimethylaminopurine (6‐DMAP) or DTT + I + 6‐DMAP. In Exp. 2, the effects of sperm pre‐incubated with DTT, sodium dodecyl sulphate (SDS) or DTT + SDS as well as two‐time frozen/thawed sperm (without cryoprotectant) on MPN formation and oocyte activation were examined. The non‐treated sperm served as controls. The MPN formation in DTT + SDS group was higher (p < 0.05) than other groups except for freeze–thaw group. No difference in the rate of activated ICSI oocytes was observed among groups. In Exp. 3, the effect of pre‐treatment of sperm on subsequent development of ICSI embryos and blastocyst cell numbers were examined. The rates of cleavage and blastocyst formation as well as the blastocyst cell numbers were similar among the pre‐treated and control groups. In conclusion, pre‐treatment of sperm with DTT + SDS positively affected MPN formation, although the subsequent development capacity of the resulting embryos remained limited. Moreover, DTT was not effective on oocyte activation compared with Io + 6‐DMAP after ICSI.     

Insect-mediated cross-pollination in soybean [Glycine max (L.) Merrill] : I. Agronomic performance

In soybean, manual cross-pollination to produce large quantities of F₁ hybrid seed for yield trials is difficult and time-consuming. Conversely, insect-mediated cross-pollination has been shown to produce large quantities of hybrid seed in soybean and could facilitate the identification of heterotic patterns. The objective of our study was: (1) evaluate F₁ hybrid soybean plants from single crosses for yield and agronomic traits over several environments and (2) compare hybrid performance of the single crosses to lines developed from three-way crosses and backcrosses. In 2003, F₁ seed of single-crosses and their parent lines were evaluated in replicated experiments at three locations. Also in 2003, three-way crosses, and BC₁F₁ seed were produced. In 2004, three-way crosses, BC₁F₁ crosses, and their parent lines were evaluated at one location. High-parent heterosis (HPH) in single-crosses for grain yield ranged from −41.11% to +11.19%; for protein content from −4.34% to +3.53%, and for oil content from −13.22% to −0.84%. In three-way crosses, HPH for yield ranged from −25.21% to −4.50%, for protein from −2.72% to +1.92%, and for oil from −5.87% to −1.20%. For BC₁F₁ crosses, HPH for yield ranged from −15.65% to +41.97%, for protein from −2.57% to +1.69%, and for oil from −2.47% to +2.22%. Although positive heterosis levels were observed across all populations tested to determine the economic feasibility it is imperative that more tests of more cross-combinations be evaluated in replicated environments. Extensive research in different environments must be conducted to determine what parental combinations will produce the highest heterosis levels, and to develop criteria for selecting the parents with the best combining ability. This will be important to maximize agronomic performance that can economically justify the use of hybrids in soybean production.     

Black soil blindness: A new mycotoxicosis of cattle grazing Corallocytostroma-infected Mitchell grass (Astrebla spp)

A new, fatal mycotoxicosis of cattle has been recognised in north-western Australia. A feeding trial confirmed the toxicity of a previously unknown species of Corallocytostroma that grows on Mitchell grass (Astrebla spp). The disease has been colloquially named ‘black soil blindness’ because its most prominent features are its confinement to pastures on black soil, and blindness and death of affected animals. Over 500 cattle have died and considerable subclinical disease is present. Above average wet season rainfall and extended growing seasons may explain the emergence of the fungus. The disease is important because cattle production in large areas of Australia utilise Mitchell grass pastures.