Urine and fecal excretion patterns of dairy cows divergent for milk urea nitrogen breeding values consuming either a plantain or ryegrass diet.

Environmental degradation has been attributed to inefficient nitrogen utilization from pastoral dairy production systems. This degradation has especially been associated with the urine patch, which has been identified as a key component of nitrate leaching to waterways. However, a lack of information exists regarding the pattern of urination events and individual urination characteristics across the day, which would help inform strategic management decisions. The aim of this study was therefore to evaluate and report the patterns and characteristics of fecal and urination events throughout the day for cows divergent for milk urea nitrogen breeding values (MUNBV) on either a plantain [Plantago lanceolata L. (PL)] or ryegrass [Lolium perenne L. (RG)] diet as ways to reduce environmental impact. Sixteen multiparous lactating Holstein Friesian × Jersey cows divergent for MUNBV were housed in metabolism crates for 72 h, with all excretion events captured and analyzed. Cows selected as low for MUNBV consistently had a 65.2-kg lower urinary urea nitrogen (UUN) load (kg/ha) than high MUNBV cows for all hours of the day when consuming RG. The association between lower urinary urea loading rates and less N leaching implies a reduced environmental impact from low MUNBV cows consuming RG. When cows consumed PL, regardless of MUNBV, they had on average a 137.5-kg (UUN/ha) lower loading rate compared with high MUNBV cows on RG and a 72.2-kg (UUN/ha) lower loading rate compared with low MUNBV cows consuming RG across the day. Cows on PL also exhibited a different diel pattern of UUN load compared with cows consuming RG. Differences in the diel pattern of N excreted in feces were also detected based on MUNBV and by diet, with low MUNBV cows excreting on average 3.06 g more N in feces per event for the majority of the day compared with high MUNBV cows when consuming RG. Lower UUN loading rates and more N excreted in feces indicate a potentially lower environmental impact from low MUNBV cows when consuming RG compared with high MUNBV cows. The use of the PL diet also resulted in lower UUN loading rates and greater levels of N excreted in feces compared with RG, therefore also indicating its ability to reduce environmental impact compared with RG.

Evaluation of PEETER V1.0 urine sensors for measuring individual urination behavior of dairy cows.

Due to environmental concerns around N leaching and NO2 emissions from intensive pastoral dairying systems, there has been an increase in research focused on mitigation strategies and on-animal technologies to evaluate urination behavior of grazing dairy cows. Nitrogen leaching and NO2 emissions are associated with urine nitrogen loading onto pasture, which is a function of urine nitrogen concentration and urine volume per urination event. The PEETER V1.0 urine sensor (Lincoln University, Christchurch, New Zealand) is a promising on-animal measurement technology; however, it has yet to be validated in vivo. The objective of this work was to validate the PEETER V1.0 urine sensor's estimations of individual urination events (i.e., urine volume). We fitted 15 Holstein-Friesian × Jersey lactating dairy cows (506 ± 35 kg of live weight, body condition score of 3.75 ± 0.25, and 150.4 ± 20.7 d in milk) with individual PEETER V1.0 sensors and placed them in metabolism crates for 72 h. Every urination event (n = 480) was collected manually and compared with the urine volume estimated by the PEETER V1.0 sensor to determine precision and accuracy using Lin's concordance correlation coefficient (CCC). The CCC is calculated as a function of the Pearson's correlation (precision) and bias correction factor (Cb; Cb = 1 is perfect), and it demonstrates how far the values of the 2 methods are from perfect agreement (accuracy; i.e., a 45° line). The mean urination event volume (mean ± standard deviation) was 2.7 ± 0.94 and 2.6 ± 0.92 L for the actual and PEETER V1.0 sensor, respectively. The PEETER V1.0 sensor showed excellent precision (r = 0.90) with near-perfect accuracy (Cb = 1.00), and the CCC value was high (CCC = 0.90), indicating excellent agreement. Based on these results, the PEETER V1.0 urine sensor provides estimates that are precise and accurate. We conclude that the PEETER V1.0 sensor can be used to evaluate urination behavior of grazing dairy cows.

Grazing dairy cows with low milk urea nitrogen breeding values excrete less urinary urea nitrogen.

There is an increasing pressure on temperate pastoral dairy production systems to reduce environmental impacts, coming from the inefficient use of N by cows in the form of excessive urinary N excretion and subsequent N leaching to the waterways and NO2 emissions to the atmosphere, these impacts have spurred research into various mitigation strategies, which have so far overlooked animal-based solutions. The objectives of this study were first, to investigate the relationship between MUN breeding values (MUNBV) and urinary urea N (UUN) concentrations and total excretion in grazing dairy cows; and secondly, to evaluate such a potential relationship in the context of different sward compositions and stage of lactation. Forty-eight multiparous, lactating Holstein-Friesian dairy cows genetically divergent for MUNBV were strip-grazed on either a ryegrass-white clover (24 cows) or ryegrass, white clover and plantain sward (24 cows), during both early and late lactation. Cows were fitted with Lincoln University PEETER sensors to evaluate urination behaviour by measuring frequency and volume of urination, as well as daily urine excretion. Urine and faeces were sampled for urea N content. Milk yield and composition were measured for individual cows in both periods. There was a positive relationship between MUNBV and MUN (R2 = 0.67, P ≤ 0.05), with MUN decreasing 1.61 ± 0.19 mg/dL per unit decrease in MUNBV across both sward types and stages of lactation. Urinary urea N concentration decreased 0.67 ± 0.27 g/L (R2 = 0.46, P ≤ 0.05) per unit decrease of MUNBV, with no effect on urine volume or frequency (number of urination events per day), which resulted in a 165.3 g/d difference in UUN excretion between the animal with the highest and the lowest MUNBV. At the same milk yield, percentage of protein in milk increased by 0.09 ± 0.03 (R2 = 0.61, P ≤ 0.05,) per unit decrease in MUNBV. Our results suggest that breeding and selecting for dairy cows with low MUNBV can reduce urinary urea N deposition onto pasture and consequently the negative environmental impact of pastoral dairy production systems in temperate grasslands. Moreover, reducing MUNBV of dairy cows can potentially increase farm profitability due to greater partitioning of N to milk in the form of protein.

Effect of cane use on bone marrow lesion volume in people with medial tibiofemoral knee osteoarthritis: randomized clinical trial.

OBJECTIVE: To evaluate effects of daily cane use for 3 months on medial tibiofemoral bone marrow lesion (BML) volumes in people with medial tibiofemoral osteoarthritis (OA). DESIGN: In this randomized controlled trial (RCT), 79 participants with medial tibiofemoral OA were randomized to either a cane group (using a cane whenever walking) or control group (not using any gait aid) for 3 months. The cane group received a single training session by a physiotherapist, using a biofeedback cane to teach optimal technique and body weight support and motor learning principles to facilitate retention of learning. The primary outcome was change in total medial tibiofemoral BML volume (per unit bone volume) measured from magnetic resonance imaging (MRI) at 3 months. Secondary outcomes were BML volumes (per unit bone volume) of the medial tibia and femur, and patient-reported outcomes of overall knee pain, knee pain on walking, physical function, perceived global symptom changes and health-related quality of life. MRI analyses were performed by a blinded assessor. RESULTS: Seventy-eight participants (99%) completed the primary outcome. Mean (standard deviation) daily cane use was 2.3 (1.7) hours over 3 months. No evidence of between-group differences was found for change in total medial tibiofemoral BML volume (mean difference: -0.0010 (95% confidence intervals: -0.0022, 0.0003)). Most secondary outcomes showed minimal differences between groups. CONCLUSION: Daily use of a cane during walking for 3 months aiming to reduce knee joint loading did not change medial tibiofemoral BML volumes compared to no use of gait aids. CLINICAL TRIAL REGISTRATION: Australian New Zealand Clinical Trial Registry (ACTRN12614000909628).

Reliability and measurement error of the Osteoarthritis Research Society International (OARSI) recommended performance-based tests of physical function in people with hip and knee osteoarthritis.

OBJECTIVE: To estimate the reliability and measurement error of performance-based tests of physical function recommended by the Osteoarthritis Research Society International (OARSI) in people with hip and/or knee osteoarthritis (OA). DESIGN: Prospective repeated measures between independent raters within a session and within-rater over a week interval. Relative reliability was estimated for 51 people with hip and/or knee OA (mean age 64.5 years, standard deviation (SD) 6.21 years; 47% females; 36 (70%) primary knee OA) on the 30s Chair Stand Test (30sCST), 40m Fast-Paced Walk Test (40mFPWT), 11-Stair Climb Test (11-step SCT), Timed Up and Go (TUG), Six-Minute Walk Test (6MWT), 10m Fast-Paced Walk Test (10mFPWT) and 20s Stair Climb Test (20sSCT) using intra-class correlation coefficients (ICC). Absolute reliability was calculated using standard error of measurement (SEM) and minimal detectable change (MDC). RESULTS: Measurement error was acceptable (SEM < 10%) for all tests. Between-rater reliability was: optimal (ICC > 0.9, lower 1-sided 95% CI > 0.7) for the 40mFPWT, 6MWT and 10mFPWT; sufficient (ICC >0.8, lower 1-sided 95% CI > 0.7) for 30sCST, 20sSCT; unacceptable (lower 1-side 95% CI < 0.7) for 11-step SCT and TUG. Within-rater reliability was optimal for 40mFPWT, and 6MWT; sufficient for 30sCST and 10mFPWT and unacceptable for 11-step SCT, TUG and 20sSCT. CONCLUSIONS: The 30sCST, 40mFPWT, 6MWT and 10mFPWT, demonstrated, at minimum, acceptable levels of both between and within-rater reliability and measurement error. All tests demonstrated sufficiently small measurement error indicating they are adequate for measuring change over time in individuals with knee/hip OA.

A 9 kDa antifreeze protein from the Antarctic springtail, Gomphiocephalus hodgsoni.

A 9 kDA antifreeze protein (AFP) was isolated and purified from the Antarctic springtail, Gomphiocephalus hodgsoni. By combining selective sampling procedures and a modified ice affinity purification protocol it was possible to directly isolate a single AFP protein without recourse to chromatographic separation techniques. Mass spectrometry identified a single 9 kDa component in the purified ice fraction. Intramolecular disulphide bonding was suggested by the presence of 12 cysteine residues. The specific amino acid composition is unique, particularly with regard to the presence of histidine (11.5%). But it also shows noticeable commonalities with insect AFPs in the abundance of cysteine (13.8%), while simultaneously hinting, through the presence of glycine (11.5%), that the metabolic building blocks of AFPs in Collembola may have a phylogenetically-determined component.

Antifreeze proteins in the Antarctic springtail, Gressittacantha terranova.

Antarctic springtails are exemplars of extreme low temperature adaptation in terrestrial arthropods. This paper represents the first examination of such adaptation in the springtail, Gressittacantha terranova. Acclimatization state was measured in field-fresh samples over a 22-day period at the beginning of the austral summer. No evidence of temperature tracking was observed. Mean temperature of crystallization (T(c)) for all samples was -20.67 ± 0.32°C and the lowest T(c) recorded was -32.62°C. Ice affinity purification was used to collect antifreeze proteins (AFPs) from springtail homogenate. The purified ice fraction demonstrated both thermal hysteresis activity and recrystallisation inhibition. Growth-melt observations revealed that ice crystals grow normal to the c-axis (basal plane). Reverse-phased HPLC produce one clearly resolved peak (P1) and one compound peak (P2). Mass spectrometry identified the molecular mass of P1 as 8,599 Da. The P1 protein was also the most prominent in P2, although additional peptides of 6-7 KDa were also prominent. The main AFP of the Antarctic springtail, G. terranova has been isolated, although like other AFP-expressing arthropods, it shows evidence of expressing a family of AFPs.

Ice-active proteins from New Zealand snow tussocks, Chionochloa macra AND C. rigida.

The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process.

Integrin-linked kinase has a critical role in ErbB2 mammary tumor progression: implications for human breast cancer.

Elevated expression of the integrin-linked kinase (ILK) has been observed in a variety of cancers and has been further correlated with poor clinical outcome. Here, we show that mammary epithelial disruption of ILK results in a profound block in mammary tumor induction. Consistent with these observations, inhibition of ILK function in ErbB2-expressing cells with small molecule inhibitor or RNA interference resulted in profound block in their in vitro invasive properties due to the induction of apoptotic cell death. The rare ILK-deficient tumors that eventually arose overcame this block in tumor induction by an upregulation of ErB3 phosphorylation. These observations provide direct evidence that ILK has a critical role in the initiation phase of ErbB2 tumor induction.

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata.

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.

Cold tolerance of an Antarctic nematode that survives intracellular freezing: comparisons with other nematode species.

Panagrolaimus davidi is an Antarctic nematode with very high levels of cold tolerance. Its survival was compared with that of some other nematodes (P. rigidus, Rhabditophanes sp., Steinernema carpocapsae, Panagrellus redivivus and Ditylenchus dipsaci) in both unacclimated samples and those acclimated at 5 degrees C. Levels of recrystallization inhibition in homogenates were also compared, using the splat-cooling assay. The survival of P. davidi after the freezing of samples was notably higher than that of the other species tested, suggesting that its survival ability is atypical compared to other nematodes. In general, acclimation improved survival. Levels of recrystallization inhibition were not associated with survival but such a relationship may exist for those species that are freezing tolerant.

One hundred million years of chemical warfare by insects.

An important defensive strategy among animals is the use of chemical compounds with toxic or irritating properties. In this paper, we report the discovery of an Early Cretaceous soldier beetle (Coleoptera: Cantharidae) in Burmese amber that seemingly employed a chemical defense response against a potential predator. Six pairs of cuticular vesicles with associated gland reservoirs were extruded from the insect's abdomen, and a secretion released from one of these covers a portion of the antenna of a second insect species, considered to be the perpetrator of the response. This is the earliest fossil record of a putative chemical defense response and suggests that chemical defense mechanisms in beetles have been in existence for at least 100 Ma.

Ice-active proteins from the Antarctic nematode Panagrolaimus davidi.

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.

Freezing and cryoprotective dehydration in an Antarctic nematode (Panagrolaimus davidi) visualised using a freeze substitution technique.

The pattern of ice formation during the freezing of Panagrolaimus davidi, an Antarctic nematode that can survive intracellular ice formation, was visualised using a freeze substitution technique and transmission electron microscopy. Nematodes plunged directly into liquid nitrogen had small ice crystals throughout their tissues, including nuclei and organelles, but did not survive. Those frozen at high subzero temperatures showed three patterns of ice formation: no ice, extracellular ice, and intracellular ice. Nematodes subjected to a slow-freezing regime (at -1 degrees C) had mainly extracellular ice (70.4%), with the bulk of the ice in the pseudocoel. Some (24.8%) had no ice within their bodies, due to cryoprotective dehydration. Nematodes subjected to a fast-freezing regime (at -4 degrees C) had intracellular (54%) and extracellular (42%) ice. Intracellular ice was confined to the cytoplasm of cells, with organelles in the spaces in between ice crystals. The survival of nematodes subjected to the fast-freezing regime (53%) was less than those subjected to the slow-freezing regime (92%).

Integrin beta1 is required for the invasive behaviour but not proliferation of squamous cell carcinoma cells in vivo.

Integrin beta1 is both overexpressed and in an 'active' conformation in vulval squamous cell carcinomas (VSCCs) compared to matched normal skin. To investigate the significance of integrin beta1 deregulation we stably knocked-down integrin beta1 expression in the VSCC cell line A431. In vitro analysis revealed that integrin beta1 is required for cell adhesion, cell spreading and invasion. However, integrin beta1 is not required for cell growth or activation of FAK and ERK signalling in vitro or in vivo. Strikingly, while control tumours were able to invade the dermis, integrin beta1 knockdown tumours were significantly more encapsulated and less invasive.

Freezing rate affects the survival of a short-term freezing stress in Panagrolaimus davidi, an Antarctic nematode that survives intracellular freezing.

The ability of the Antarctic nematode Panagrolaimus davidi to survive a short-term freezing stress depended upon the rate of freezing of its surroundings, measured as the duration of the sample exotherm. The freezing rate increased as the sample volume and freezing temperature decreased and resulted in fewer nematodes surviving. This appears to be due to the greater risk of physical damage by ice crystal growth at high freezing rates. Once frozen the nematodes will then survive exposure to lower temperatures. The environment of the nematode is likely to produce the slow rate of freezing of its surroundings that is necessary for its survival.

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

Activation of the Ral and phosphatidylinositol 3' kinase signaling pathways by the ras-related protein TC21.

TC21 is a member of the Ras superfamily of small GTP-binding proteins that, like Ras, has been implicated in the regulation of growth-stimulating pathways. We have previously identified the Raf/mitogen-activated protein kinase pathway as a direct TC21 effector pathway required for TC21-induced transformation (M. Rosário, H. F. Paterson, and C. J. Marshall, EMBO J. 18:1270-1279, 1999). In this study we have identified two further effector pathways for TC21, which contribute to TC21-stimulated transformation: the phosphatidylinositol 3' kinase (PI-3K) and Ral signaling pathways. Expression of constitutively active TC21 leads to the activation of Ral A and the PI-3K-dependent activation of Akt/protein kinase B. Strong activation of the PI-3K/Akt pathway is seen even with very low levels of TC21 expression, suggesting that TC21 may be a key small GTPase-regulator of PI-3K. TC21-induced alterations in cellular morphology in NIH 3T3 and PC12 cells are also PI-3K dependent. On the other hand, activation of the Ral pathway by TC21 is required for TC21-stimulated DNA synthesis but not transformed morphology. We show that inhibition of Ral signaling blocks DNA synthesis in human tumor cell lines containing activating mutations in TC21, demonstrating for the first time that this pathway is required for the proliferation of human tumor cells. Finally, we provide mechanisms for the activation of these pathways, namely, the direct in vivo interaction of TC21 with guanine nucleotide exchange factors for Ral, resulting in their translocation to the plasma membrane, and the direct interaction of TC21 with PI-3K. In both cases, the effector domain region of TC21 is required since point mutations in this region can interfere with activation of downstream signaling.