Assessment of the effects of prepartum anti-inflammatory therapies on type 1/type 2 immunity ratio using a rapid blood test.

The objective of this study was to assess (1) the effects of prepartum administration of anti-inflammatory therapies on type 1/type 2 immunity ratio using a rapid blood test (D2Dx immunity test; Nano Discovery Inc.), and (2) correlations between rapid blood test scores and daily milk yield in Holstein dairy cows. At 14 d before the expected calving date, cows (n = 64) and heifers (n = 23) were blocked by body condition score (optimal = 3.25-3.5; high ≥3.75) and parity (nulliparous, parous), and randomly allocated to one of 3 treatment groups (1) ASA (n = 29) = receive one oral treatment with administration of acetylsalicylic acid (4 boluses; 480 grain/bolus); (2) MEL (n = 31) = receive one oral administration with meloxicam (1 mg/kg of body weight), or (3) PLC (n = 27) = receive one oral treatment with 4 gelatin capsules filled with water. Blood samples were collected weekly starting 1 wk before treatment until 3 wk after calving for assessment of type 1/type 2 immunity ratio using a rapid blood test (i.e., D2Dx immunity test). A higher D2Dx score corresponds to a higher type 1/type 2 ratio. Furthermore, blood samples were collected within 72 h before and after calving by farm personnel. Daily milk yield for the first 60 d in milk (DIM) was collected from on-farm computer records. The data were analyzed using MIXED procedure of SAS (SAS Institute Inc.) as a randomized complete block design. On average enrolled cows received treatment administration 10 d before the actual calving date (standard deviation = 5.10 d). There was a tendency for a treatment by day interaction. Cows treated with ASA had higher type 1/type 2 ratio within 3 d after calving compared with MEL and PLC cows (ASA = 0.065 ± 0.002; MEL = 0.059 ± 0.002; PLC = 0.053 ± 0.002). Similarly, ASA and MEL cows had a higher type 1/type 2 ratio at 7 ± 3 DIM compared with PLC cows (ASA = 0.062 ± 0.002; MEL = 0.064 ± 0.002; PLC = 0.056 ± 0.002). Regardless of treatment, there was an interaction between parity and day. Parous cows had higher type 1/type 2 ratios compared with nulliparous cows at 14 ± 3 d before calving and at 7 ± 3, 14 ± 3, and 21 ± 3 d after calving. Furthermore, there was a positive correlation between D2Dx scores at 14 ± 3 DIM and average daily milk yield in the first 60 DIM. These results suggest that prepartum anti-inflammatory therapies may cause an increased shift in type 1 immunity around calving. Similarly, parous cows may have an increased shift in type 1 immunity after calving. Interestingly, higher type 1/type 2 ratios may be associated with higher milk yields in the first 60 DIM. Larger studies are needed to identify associations between the D2Dx immunity test and cow health and performance, as well as to assess the applicability of these types of tests in a conventional farm setting.

Local spectroscopic evidence for a nodeless magnetic kagome superconductor CeRu2.

We report muon spin rotation (µSR) experiments on the microscopic properties of superconductivity and magnetism in the kagome superconductor CeRu2withTc≃5 K. From the measurements of the temperature-dependent magnetic penetration depthλ, the superconducting order parameter exhibits nodeless pairing, which fits best to an anisotropics-wave gap symmetry. We further show that theTc/λ-2ratio is comparable to that of unconventional superconductors. Furthermore, the powerful combination of zero-field (ZF)-µSR and high-fieldµSR has been used to uncover magnetic responses across three characteristic temperatures, identified asT1∗≃110 K,T2∗≃65 K, andT3∗≃40 K. Our experiments classify CeRu2as an exceedingly rare nodeless magnetic kagome superconductor.

Adaptive slit beam shaping for direct laser written waveguides.

We demonstrate an improved method for fabricating optical waveguides in bulk materials by means of femtosecond laser writing. We use an LC spatial light modulator (SLM) to shape the beam focus by generating adaptive slit illumination in the pupil of the objective lens. A diffraction grating is applied in a strip across the SLM to simulate a slit, with the first diffracted order mapped onto the pupil plane of the objective lens while the zeroth order is blocked. This technique enables real-time control of the beam-shaping parameters during writing, facilitating the fabrication of more complicated structures than is possible using nonadaptive methods. Waveguides are demonstrated in fused silica with a coupling loss to single-mode fibers in the range of 0.2 to 0.5 dB and propagation loss <0.4 dB/cm.

Diurnal cycles of embolism formation and repair in petioles of grapevine (Vitis vinifera cv. Chasselas).

The impact of water deficit on stomatal conductance (g(s)), petiole hydraulic conductance (K(petiole)), and vulnerability to cavitation (PLC, percentage loss of hydraulic conductivity) in leaf petioles has been observed on field-grown vines (Vitis vinifera L. cv. Chasselas). Petioles were highly vulnerable to cavitation, with a 50% loss of hydraulic conductivity at a stem xylem water potential (Ψ(x)) of -0.95 MPa, and up to 90% loss of conductivity at a Ψ(x) of -1.5 MPa. K(petiole) described a daily cycle, decreasing during the day as water stress and evapotranspiration increased, then rising again in the early evening up to the previous morning's K(petiole) levels. In water-stressed vines, PLC increased sharply during the daytime and reached maximum values (70-90%) in the middle of the afternoon. Embolism repair occurred in petioles from the end of the day through the night. Indeed, PLC decreased in darkness in water-stressed vines. PLC variation in irrigated plants showed the same tendency, but with a smaller amplitude. The Chasselas cultivar appears to develop hydraulic segmentation, in which petiole cavitation plays an important role as a 'hydraulic fuse', thereby limiting leaf transpiration and the propagation of embolism and preserving the integrity of other organs (shoots and roots) during water stress. In the present study, progressive stomatal closure responded to a decrease in K(petiole) and an increase in cavitation events. Almost total closure of stomata (90%) was measured when PLC in petioles reached >90%.

A free-standing low-risk maternity unit in the United Kingdom: does it have a role?

This study aimed to evaluate the safety of this birth setting for low-risk deliveries based on our hospital protocol. The study was carried out at Heatherwood Hospital, Ascot (a low-risk unit) and Wexham Park Hospital, Slough, Berkshire (a consultant-led unit). This was a retrospective analysis of the computerised records and statistics of low-risk women delivered at Heatherwood Hospital, Ascot, UK following the unit protocol between July 1995 and December 2001. Women were assessed to be at low risk in accord with the unit protocol. Those who had antenatal and intrapartum care at Heatherwood Hospital and those who were transferred to the consultant unit for delivery were included in this study. We analysed the appropriateness of the structure of the unit with its medical staff input, reviewed the inclusion and exclusion criteria, analysed the perinatal and maternal mortality rates and evaluated the safety of this birth setting. We have had a total of 5468 women delivered at this low-risk maternity unit since the unit was opened. Approximately 1950 women were transferred to consultant care during this period. The intrapartum transfer in the first 18 months was 7.9%. However, since 1997 it has been static at 2.7% as confidence has grown in this model of care. The antenatal transfer rate has been static around 23%. Our emergency caesarean section rate was around 6% and the normal delivery rate was around 85%. For the first time we noted a rise in the emergency caesarean rate in 2001 at 9.5%. There were no maternal deaths. We had no serious postpartum complications accounting for long-term maternal morbidity. The antepartum stillbirths accounted for the majority of the perinatal mortality for 19/23 babies. Intrauterine growth retardation accounted for 4/23 babies in this group. The perinatal mortality rate in this low-risk population was 4.2 per 1000 total births and the stillbirth rate was 3.6 per 1000 total births. We conclude that this birth setting is safe to deliver low-risk women with less intrapartum intervention and a low transfer rate and should be setting an example for any future similar birth centre in this country.

Membrane dynamics in the malpighian tubules of the house cricket, acheta domesticus.

In Acheta domesticus, the Malpighian tubules (Mt) are composed of three morphologically distinct regions (proximal, mid and distal), each consisting of a single cell type. The bulk of the Mt is composed of the midtubule, which shows the greatest response to corticotropin releasing factor-related diuretic peptides (CRF-DP). We know from previous laboratory studies that the second messenger cAMP and its analog dibutyryl cAMP (db-cAMP) cause an approximate doubling in the secretion rate and that this is accompanied by notable ultrastructural changes in the midtubule, especially membrane reorganization in the basal area and extensive vesiculation of the cytoplasm. In this study, we examined the morphological changes in membranes both at the cell surface and internally. By enzymatically removing the basal lamina, we examined the increase in spacing between infolded membranes initiated by db-cAMP stimulation. To examine the intracellular membranes, we used a technique developed for use in invertebrate tissues. This allowed the removal of the cytoplasm for high resolution scanning electron microscopy (HR-SEM) while maintaining the integrity of the lipid constituents of the cell. By using HR-SEM and confocal laser scanning microscopy (CLSM), we gained a unique three-dimensional perspective of the complexity of the internal membrane system of the A. domesticus Mt in both the unstimulated and db-cAMP-stimulated states.

Conservation of Hox/ParaHox-related genes in the early development of a cnidarian.

To clarify the relationship between axial patterning in cnidarians and bilaterians, we have investigated the embryonic development of the hydrozoan Podocoryne carnea. The expression of Hox-like homeobox genes was analyzed by RT-PCR and in situ hybridization. Cnox1-Pc, an anterior Hox gene, is a maternal message. It is present throughout larval development, first weakly in all blastomeres and later restricted mostly to the anterior pole of the planula. Gsx, an anterior ParaHox gene, is first seen in the anterior endoderm but also extends into posterior regions. Cnox4-Pc, an orphan Hox-like gene, is expressed in the egg as a ring-shaped cloud around the germinal vesicle. After fertilization, the message remains in most animal blastomeres. When the embryo elongates in late blastula, staining is restricted to a few cells at the posterior pole where gastrulation will start. However, once gastrulation starts, the Cnox4-Pc signal disappears and is absent in later stages of larval development. Phylogenetic analysis shows that not all cnidarian Hox-like genes have recognizable orthologues in bilaterian groups. However, the expression analysis of Cnox1-Pc and Gsx correlates to some extent with the expression pattern of cognate genes of bilaterians, confirming the conservation of genes involved in organizing animal body plans and their putative common ancestral origin.

Ultrastructural changes in the Malpighian tubules of the house cricket, Acheta domesticus, at the onset of diuresis: A time study.

The Malpighian tubules (Mt) of insects are responsible for maintaining osmotic homeostasis and eliminating waste from the hemolymph. When stimulated by diuretic factors the tubule cells are able to transport extraordinary volumes of fluid over short periods of time. We have been studying the changes that occur within the cells that accompany and facilitate this phenomenon. We present the ultrastructural changes that occur in the mid-tubule of the house cricket, Acheta domesticus, following exposure to the second messenger analog, dibutyryl cAMP, over the period from 15-420 sec. Vacuolation of the cytoplasm begins as early as 30 sec poststimulation with a significant increase in vacuolation occurring after 120 sec. As expected, there is an increase in the surface area of the basolateral membrane to facilitate the rapid movement of fluid into the cells. Other ultrastructural changes noted to accompany the onset of diuresis include the movement of mitochondria into areas adjacent to transport membranes, the vesiculation of Golgi, mobilization of CaPO(4) spherites, and a direct interaction of these spherites with active mitochondria. We discuss several possible roles for these changes in terms of rapid fluid transport.

The mesoderm specification factor twist in the life cycle of jellyfish.

The basic helix-loop-helix (bHLH) transcription factor Twist is highly conserved from Drosophila to vertebrates and plays a major role in mesoderm specification of triploblasts. The presence of a Twist homologue in diploblasts such as the cnidarian Podocoryne carnea raises questions on the evolution of mesoderm, the third cell layer characteristic for triploblasts. Podocoryne Twist is expressed in the early embryo until the myoepithelial cells of the larva differentiate and then again during medusa development. There, the gene is detected first when the myoepithelial cells of the polyp dedifferentiate to form the medusa bud and later Twist is found transiently in the entocodon, a mesoderm-like cell layer which differentiates into the smooth muscle and striated muscle of the bell. On the other hand, in later bud stages and the medusa, expression is seen where non-muscle tissues differentiate. Experimental analysis of in vitro transdifferentiation and regeneration demonstrates that Twist activity is not needed when isolated striated muscle regenerate medusa organs. Developmental roles of Twist are discussed with respect to early animal evolution from a common ancestor of cnidarians and bilaterians.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Evidence in favour of ancient octaploidy in the vertebrate genome.

Vertebrate genomes are larger than invertebrates and show evidence of extensive gene duplication, including many collinear chromosomal segments. On the basis of this intra-genomic synteny, it has been proposed that two rounds of whole genome duplication (octaploidy) occurred early in the vertebrate lineage. Recently, this early vertebrate octaploidy has been challenged on the basis of gene trees. We report new linkage groups encompassing the matrilin (MATN), syndecan (SDC), Eyes Absent (EYA), HCK kinase and SRC kinase paralogous gene quartets. In contrast to other studies, the sequence trees are weakly supportive of ancient octaploidy. It is concluded that there is no strong evidence against the octaploidy, provided that consecutive genome duplication was rapid.

Modes of control of insect Malpighian tubules: synergism, antagonism, cooperation and autonomous regulation.

Rates of fluid and ion secretion by insect Malpighian tubules are controlled by peptides, including CRF-related peptides and kinins, and in some species by serotonin. It now appears to be a general rule that tubule secretion rate is controlled through the interaction of two or more haemolymph-borne factors. In this review we suggest that these interactions may be classified as synergistic, cooperative, or antagonistic. When presented together, two diuretic factors may act in synergism, so that fluid secretion is stimulated to a greater extent than the sum of their individual effects. Synergism may involve one or more second messenger systems. Alternatively, diuretic factors may act in cooperation, so that although their overall effects are additive, cation and anion transport pathways are controlled separately by distinct second messenger systems. There is also one example of antagonism between factors controlling tubule secretion and between their respective second messengers; one factor is stimulatory, the other is inhibitory. In addition to the complex control of fluid and ion transport by haemolymph-borne factors, sophisticated autonomous regulatory mechanisms have been identified in Malpighian tubules. When triggered by appropriate stimuli, these mechanisms play homeostatic roles, preserving haemolymph osmolality or ionic composition.

Tests of potential adipokinetic hormone precursor related peptide (APRP) functions: lack of responses.

The adipokinetic hormone (AKH) precursor related peptides (APRPs) are end products of the synthesis of the well-conserved AKHs. The large amount of metabolic energy devoted to APRP synthesis suggests they have an important function(s) in the insects. Several functions have been proposed, but currently none are known. We tested whether the APRPs stimulate hyperlipemia, hypertrehalosemia, fat body glycogen phosphorylase activation, Malpighian tubule secretion, and hindgut myotropia. Surprisingly, none of these responses were stimulated by APRPs isolated from the lubber grasshopper, Romalea microptera (= guttata). In addition, the APRPs delivered in concert with AKHs did not significantly increase hyperlipemia, hypertrehalosemia, or phosphorylase activation over the AKHs alone. Our data discount several proposed functions for the APRPs. Arch.

The homeobox gene Otx of the jellyfish Podocoryne carnea: role of a head gene in striated muscle and evolution.

In many bilaterian animals members of the Otx gene family are expressed in head or brain structures. Cnidarians, however, have no clearly homologous head and no distinct brain; but an Otx homolog from the jellyfish Podocoryne carnea is highly conserved in sequence and domain structure. Sequence similarities extend well beyond the homeodomain and Podocoryne Otx can be aligned over its entire length to human OTX1, OTX2, and CRX. The overall structure of Otx is better conserved from Podocoryne to deuterostomes while protostomes appear to be more derived. In contrast, functions seem to be conserved from protostomes to vertebrates but not in Podocoryne or echinoderms. Podocoryne Otx is expressed only during medusa bud formation and becomes restricted to the striated muscle of medusae. Cnidaria are the most basal animals with striated muscle. Podocoryne polyps have no striated muscle and no Otx expression; both appear only during the asexual medusa budding process. The common ancestor of all animals that gave rise to cnidarians, protostomes, and deuterostomes already had an Otx gene more similar to today's Podocoryne and human homologs than to Drosophila otd, while the head-specific function appears to have evolved only later.

A toxin homology domain in an astacin-like metalloproteinase of the jellyfish Podocoryne carnea with a dual role in digestion and development.

Metalloproteinases of the astacin family such as tolloid play major roles in animal morphogenesis. Cnidarians are thought to be evolutionary simple organisms and, therefore, a metalloproteinase from the marine hydrozoan Podocoryne carnea was analysed to evaluate the role of this conserved gene familiy at the base of animal evolution. Surprisingly, the proteinase domain of Podocornyne PMP1 is more similar to human meprin than to HMP1 from another hydrozoan, the freshwater polyp Hydra vulgaris. However, PMP1 and HMP1 both contain a small C-terminal domain with six cysteines that distinguishes them from other astacin-like molecules. Similar domains have been described only recently from sea anemone toxins specific for potassium channels. This toxin homology (Tox1) domain is clearly distinct from epidermal growth factor (EGF)-like domains or other cysteine-rich modules and terminates with the characteristic pattern CXXXCXXC with three out of six cysteines in the last eight residues of the protein. PMP1 is transiently expressed at various sites of morphogenetic activity during medusa bud development. In the adult medusa, however, expression is concentrated to the manubrium, the feeding organ, where the PMP1 gene is highly induced upon feeding. These disparate expression patterns suggest a dual role of PMP1 comparable to tolloid in development and, like astacin in the crayfish, also for food digestion. The Tox1 domain of PMP1 could serve as a toxin to keep the pray paralysed after ingestion, but as a sequence module such Tox1 domains with six cysteines are neither restricted to cnidarians nor to toxins.

Hormonal regulation in insects: facts, gaps, and future directions.

There are two main classes of hormones in insects: 1) the true hormones produced by epithelial glands and belonging to the ecdysteroids or juvenile hormones and 2) the neuropeptide hormones produced by neurosecretory cells. Members of these classes regulate physiological, developmental, and behavioral events in insects. Detailed accounts are given on isolation, identification, structure-activity relationships, mode of action, biological function, biosynthesis, inactivation, metabolism, and feedback for hormones involved in 1) metabolic regulation such as the adipokinetic/hypertrehalosemic peptides and the diuretic and antidiuretic peptides; 2) stimulation or inhibition of muscle activity such as the myotropic peptides; 3) control of reproduction, growth, and development such as allatotropins, allatostatins, juvenile hormones, ecdysteroids, folliculostimulins and folliculostatins, ecdysis-triggering and eclosion hormones, pheromone biosynthesis activating neuropeptides, and diapause hormones; and 4) regulation of tanning and of color change. Because of the improvements in techniques for isolation and structure elucidation, there has been rapid progress in our knowledge of the chemistry of certain neuropeptide families. With the employment of molecular biological techniques, the genes of some neuropeptides have been successfully characterized. There are, however, areas that are still quite underdeveloped. These are, for example, 1) receptor studies, which are still in their infancy; 2) the hormonal status of certain sequenced peptides is not clarified; and 3) functional studies are lacking even for established hormones. The authors plead for a concerted effort to continue research in this field, which will also advance our knowledge into the use of insect hormones as safer and species-specific molecules for insect pest management.