Mechanical ventilation in snake envenomation of dogs and cats.

Envenomation by snakes in Elapidae and Viperidae families have been associated with respiratory failure in dogs and cats. Mechanical ventilation may be required for hypoventilation due to neuromuscular paralysis or hypoxemia due to pulmonary hemorrhage or aspiration pneumonia. Median incidence of dogs and cats with snake envenomation that require mechanical ventilation is 13% (0.06-40%). Standard treatment of snake envenomation in dogs and cats includes prompt administration of appropriate antivenom and management of envenomation complications such as coagulopathy, rhabdomyolysis and acute kidney injury. When mechanical ventilation is required, overall prognosis is good with appropriate treatment. Standard anesthetic protocols and mechanical ventilator settings are generally appropriate, with lung protective ventilation strategies typically reserved for patients with pulmonary disease. Median survival to discharge for cats and dogs with elapid envenomation is 72% (76-84%) with 33 h (19.5-58 h) median duration of mechanical ventilation and 140 h (84-196 h) median hospitalization. This article reviews indications for mechanical ventilation in cats and dogs with snake envenomation, and discusses ventilator settings, anesthetic and nursing considerations, complications and outcomes specific to this disease.

Microdeletions/duplications involving TBX1 gene in fetuses with conotruncal heart defects which are negative for 22q11.2 deletion on fluorescence in-situ hybridization.

OBJECTIVES: Conotruncal heart defects (CTD) are associated with del22q11.2 syndrome, which is often diagnosed by fluorescence in-situ hybridization (FISH). However, in those negative for del22q11.2 on FISH, the etiology is usually obscure. We aimed to use high-resolution array comparative genomic hybridization (array CGH) to clarify the underlying genetic causes in these cases. METHODS: In this retrospective study, fetal samples of amniocytes or fibroblasts, taken either for prenatal diagnosis by amniocentesis or for postnatal survey after termination of pregnancy, were obtained from 45 fetuses with CTD and were investigated by cytogenetic analysis including karyotyping and FISH for del22q11.2 syndrome. Eight fetuses with no findings on karyotyping and FISH were investigated further by array CGH, real-time quantitative polymerase chain reaction (qPCR) and Sanger sequencing of TBX1. RESULTS: Array CGH revealed that three of the eight fetuses carried submicroscopic genomic imbalances. Of these, two cases showed similar small microdeletions/duplications in 22q11.2 (one 0.85 kb microdeletion and one 8.51 kb microduplication). The minimal shared region spanned exon 2 of TBX1, a candidate gene responsible for cardiovascular defects in del22q11.2 syndrome. In all eight cases, the array CGH results were confirmed by qPCR, and Sanger sequencing did not detect other molecular pathologies. CONCLUSION: Our findings indicate an association between TBX1 variations and fetal CTD. The results also demonstrate the power of array CGH to further scrutinize the critical gene(s) of del22q11.2 syndrome responsible for heart defects. Array CGH apparently has diagnostic sensitivity superior to that of FISH in fetuses with CTD associated with del22q11.2 (and dup22q11.2) syndrome.

Short-term outcomes following intrauterine transfusion in Scotland.

AIM: To describe neonatal outcomes following intrauterine transfusion (IUT) for severe Rhesus isoimmunisation from 1993 to 2004. RESULTS: 116 neonates who had undergone 457 IUTs (median 4, range 1-9) were identified. Three neonates died, all before 1995 (two because of hypoxic ischaemic multiorgan failure and one because of overwhelming Escherichia coli sepsis). 13 neonates (11%) were delivered by emergency Caesarean section following either IUT complication or spontaneous onset of preterm labour. They were more likely to require intubation (p<0.0001), on-going respiratory support (p=0.0007) and an exchange transfusion (p=0.007). 23 (20%) required an exchange transfusion and 63 (54%) at least one top-up transfusion. CONCLUSIONS: Management of severe Rhesus disease is associated with encouraging neonatal outcomes and most infants can be managed with phototherapy and a few top-up transfusions. IUT complications are rare but significantly increase neonatal mortality and morbidity. Antenatal counselling should address the likely postnatal course for these infants.

Internal cycling of nitrogen, potassium and magnesium in young Sitka spruce.

Potassium (K) and magnesium (Mg) are essential macro-nutrients, but little is known about how they are cycled within plants. Stable isotope studies have shown that the internal cycling of nitrogen (N) is independent of current nutrient supply in temperate tree species. This is ecologically significant because it allows trees to produce rapid shoot growth in spring independent of current soil N uptake. We used stable isotopes to quantify N, K and Mg in new shoots of Sitka spruce (Picea sitchensis (Bong.) Carr.) seedlings and to compare the relative contributions from current uptake and internal cycling. Two-year-old Sitka spruce seedlings were labeled with (15)N, (41)K and (26)Mg in an abundant or a limited supply for one growing season. The trees were repotted in the subsequent dormant season to prevent further root uptake of enriched isotopes and provided with an abundant or a limited supply of unlabeled nutrients until they were harvested in early summer of the following year. The supply was switched for half the trees in the second year to create four nutrient regimes. Enrichment of (15)N, (41)K and (26)Mg in current-year growth was attributed to internally cycled N, K and Mg uptake from the previous year. The internal cycling of N, K and Mg in new growth was significantly affected by the first-year nutrient treatments. The second-year nutrient supply affected the growth rates of the trees, but had no effect on the amounts of N, K or Mg contributed from internal cycling. Thus, internal cycling of K and Mg in Sitka spruce are, like that of N, independent of current nutrient supply.

First-trimester combined ultrasound and biochemical screening for Down syndrome in routine clinical practice.

OBJECTIVES: To assess the effectiveness of combined ultrasound and biochemical (CUB) screening for chromosome abnormalities in singleton pregnancies in a routine antenatal clinic and laboratory setting. METHODS: Women whose pregnancies fell within the gestational age range of 11 to 14 weeks by ultrasound assessment were offered CUB screening on the basis of measurement of nuchal translucency (NT), maternal serum free beta-human chorionic gonadotrophin (FbetahCG) and pregnancy-associated plasma protein A (PAPP-A). NT measurements were obtained using a standardised method defined by the Fetal Medicine Foundation and FbetahCG, and PAPP-A were measured using the DELFIA immunoassay system. Each screening marker measurement was converted to a multiple of the appropriate gestational median and a risk was derived using previously published parameters for each marker in chromosomally abnormal and unaffected pregnancies. A combined risk of Down syndrome and of trisomy 18/13, incorporating the maternal age risk, was calculated for all women. Invasive diagnostic testing was offered to women whose combined risk exceeded the cut-off risk of 1 in 250 (term). RESULTS: Five thousand and eighty-four women accepted a first-trimester screening test for Down syndrome, representing 75% of the eligible booking population. Out of the population eligible for CUB screening at the time of booking, NT measurements were obtained from 93% at the first clinic visit and 7% had to return for a second attempt. After excluding women who defaulted on a return visit, satisfactory NT measurements were obtained in 99.5% of pregnancies. Fifteen cases of Down syndrome and eleven pregnancies with other chromosome abnormalities were ascertained. The detection rate for Down syndrome was 93% (14/15) at a false-positive rate of 5.9% and for all chromosome abnormalities it was 96% (25/26) at an overall false-positive rate of 6.3%. CONCLUSIONS: CUB screening offers a significant improvement in sensitivity over second-trimester biochemical screening and is deliverable within a routine prenatal clinical setting.

Neonatal respiratory morbidity at term and the risk of childhood asthma.

OBJECTIVE: To determine whether neonatal respiratory morbidity at term is associated with an increased risk of later asthma and whether this may explain previously described associations between caesarean delivery and asthma. DESIGN: Retrospective cohort study using Scottish Morbidity Record (SMR) data of maternity (SMR02), neonatal (SMR11), and acute hospital (SMR01) discharges. SETTING: Scotland. PARTICIPANTS: All singleton births at term between 1992-1995 in 23 Scottish maternity hospitals. MAIN OUTCOME MEASURES: Hospital admission with a diagnosis of asthma in the principal position between 1992 and 2000. RESULTS: Children who had a diagnosis of transient tachypnoea of the newborn or respiratory distress syndrome were at increased risk of being admitted to hospital with a diagnosis of asthma (hazard ratio (HR) 1.7, 95% confidence interval (95% CI) 1.4 to 2.2, p<0.001). This association was observed both among children delivered vaginally (HR 1.5, 95% CI 1.1 to 2.0, p = 0.007) and among those delivered by caesarean section (HR 2.2, 95% CI 1.6 to 3.0, p<0.001). In the absence of neonatal respiratory morbidity, delivery by caesarean section was weakly associated with the risk of asthma in childhood (HR 1.1, 95% CI 1.0 to 1.2, p = 0.004). The strengths of the associations were similar whether the caesarean delivery was planned or emergency and were not significantly altered by adjustment for maternal, obstetric, and other neonatal characteristics. CONCLUSIONS: Neonatal respiratory morbidity at term is associated with an increased risk of asthma in childhood which may explain previously described associations between caesarean delivery and later asthma.

Effect of direct fetal opioid analgesia on fetal hormonal and hemodynamic stress response to intrauterine needling.

BACKGROUND: Whether the fetus can experience pain remains controversial. During the last half of pregnancy, the neuroanatomic connections for nociception are in place, and the human fetus mounts sizable stress responses to physical insults. Analgesia has been recommended for intrauterine procedures or late termination, but without evidence that it works. The authors investigated whether fentanyl ablates the fetal stress response to needling using the model of delayed interval sampling during intrahepatic vein blood sampling and transfusion in alloimmunized fetuses undergoing intravascular transfusion between 20 and 35 weeks. METHODS: Intravenous fentanyl (10 microg/kg estimated fetal weight x 1.25 placental correction) was given once at intrahepatic vein transfusion in 16 fetuses, and changes (posttransfusion - pretransfusion) in beta endorphin, cortisol, and middle cerebral artery pulsatility index were compared with intrahepatic vein transfusions without fentanyl and with control transfusions at the placental cord insertion. RESULTS: Fentanyl reduced the beta endorphin (mean difference in changes, -70.3 pg/ml; 95% confidence interval, -121 to -19.2; P = 0.02) and middle cerebral artery pulsatility index response (mean difference, 0.65; 95% confidence interval, 0.26-1.04; P = 0.03), but not the cortisol response (mean difference, -10.9 ng/ml, 95% confidence interval, -24.7 to 2.9; P = 0.11) in fetuses who had paired intrahepatic vein transfusions with and without fentanyl. Comparison with control fetuses transfused without fentanyl indicated that the beta endorphin and cerebral Doppler response to intrahepatic vein transfusion with fentanyl approached that of nonstressful placental cord transfusions. CONCLUSIONS: The authors conclude that intravenous fentanyl attenuates the fetal stress response to intrahepatic vein needling.

The effect of type 1 diabetes mellitus on vascular responses to endothelin-1 in pregnant women.

Diabetes is associated with vascular dysfunction, which may be due in part to altered vascular responses to endogenous peptides such as endothelin-1. These altered responses may also contribute to the decreased maternal peripheral resistance in pregnancy. The aim of this study was to examine the effect of diabetes on the vasoconstrictor response to endothelin-1 in pregnant women. Small arteries were isolated from nine healthy pregnant, seven type 1 diabetic pregnant women, and five healthy nonpregnant women. Contraction curves were performed on a wire myograph for noradrenaline (1 nM to 30 microM) and endothelin-1 (1 pM to 0.3 microM). Maximum responses and sensitivity were compared by t test. No differences in maximum response to noradrenaline or potassium were seen among the three groups. The maximum response to endothelin-1 was significantly increased in pregnancy (P < 0.05), whereas endothelin-1 sensitivity was reduced in the diabetic compared with the nondiabetic pregnant women (P < 0.05). Pregnant women have an increased maximum vasoconstriction response to endothelin-1 compared with nonpregnant women, whereas diabetic pregnant women demonstrate reduced sensitivity to endothelin-1. These observations suggest that endothelin-1 may play a role in maintaining peripheral vascular tone in normal pregnancy, and the decreased sensitivity seen in pregnant women with diabetes may reflect abnormal vascular reactivity.

The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 A resolution.

We present the three-dimensional structure of Trichoderma reesei endoglucanase 3 (Cel12A), a small, 218 amino acid residue (24.5 kDa), neutral pI, glycoside hydrolase family 12 cellulase that lacks a cellulose-binding module. The structure has been determined using X-ray crystallography and refined to 1.9 A resolution. The asymmetric unit consists of six non-crystallographic symmetry-related molecules that were exploited to improve initial multiple isomorphous replacement phasing, and subsequent structure refinement. The enzyme contains one disulfide bridge and is glycosylated at Asp164 by a single N-acetyl glucosamine residue. The protein has the expected fold for a glycoside hydrolase clan-C family 12 enzyme. It contains two beta-sheets, of six and nine strands, packed on top of one another, and one alpha-helix. The concave surface of the nine-stranded beta-sheet forms a large substrate-binding groove in which the active-site residues are located. In the active site, we find a carboxylic acid trio, similar to that of glycoside hydrolase families 7 and 16. The strictly conserved Asp99 hydrogen bonds to the nucleophile, the invariant Glu116. The binding crevice is lined with both aromatic and polar amino acid side-chains which may play a role in substrate binding. The structure of the fungal family 12 enzyme presented here allows a complete structural characterization of the glycoside hydrolase-C clan.

Congenital parvovirus B19 infection: experience of a recent epidemic.

OBJECTIVE: We report the management and outcome of 6 cases of non-immune fetal hydrops secondary to parvovirus B19 infection presenting over a 5-month period. METHODS: The Queen Mothers Hospital is a tertiary referral centre for fetal medicine. All cases were suspected on the basis of ultrasound evidence of hydrops. Two cases were managed conservatively owing to the presence of an active fetus with evidence of resolving hydrops. Fetal blood sampling intra-uterine transfusion and drainage of ascitic fluid were performed in 3 cases. The 6th case unfortunately resulted in an intra-uterine death prior to fetal blood sampling. RESULTS: Maternal parvovirus specific B19 was identified in all cases. Fetal parvovirus B19 IgM was identified in the 3 cases in whom fetal blood sampling was performed. A single intra-uterine transfusion was performed in these 3 cases; fetal hydrops resolved in 2 of these pregnancies progressing to the birth of a healthy baby at term, whereas 1 case was complicated by intra-uterine death. Fetal hydrops resolved in both cases managed conservatively, leading to the birth of a healthy baby at term. CONCLUSIONS: Parvovirus B19 infection should always be suspected in cases of non-immune hydrops. Conservative management will be appropriate in some cases and should involve weekly ultrasonography. The outlook for pregnancies presenting with gross hydrops remains guarded, even if intra-uterine transfusion is performed successfully.

Variations in the investigation and management of group B streptococcus in Scotland.

This article describes the practice of Scottish obstetricians in terms of their investigation and treatment of group B streptococcus (GBS). This was a postal questionnaire survey of all 125 consultant obstetricians in Scotland. We recorded indications for testing for GBS, categories of women to whom treatment is given empirically and following confirmed infection, type of antibiotic used, and timing and route of administration. No respondents screened all pregnant women but 97% screened some or all of those at highest risk. Three-quarters administered antibiotics empirically to women with intrapartum pyrexia. However, other high-risk groups were unlikely to receive treatment without confirmation of colonisation. Only one-third of respondents gave antibiotics to all women with confirmed GBS, and up to one-half withheld them from some colonised women in high-risk groups. Contrary to US guidelines, only 29% tested for GBS using low vaginal swabs and only 13% administered intrapartum antibiotics intravenously. There are wide variations in investigating and treating GBS throughout Scotland. It is likely that similar variations exist throughout the United Kingdom. UK guidelines are required to reduce variations and ensure appropriate and effective management.

The active-site residue tyr-175 in human glyoxalase II contributes to binding of glutathione derivatives.

Tyrosine-175 located in the active site of human glyoxalase II was replaced by phenylalanine in order to study the contribution of this residue to catalysis. The mutation had a marginal effect on the k(cat) value determined using S-D-lactoylglutathione as substrate. However, the Y175F mutant had an 8-fold higher K(m) value than the wild-type enzyme. The competitive inhibitor S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione had a 30-fold higher K(i) value towards the mutant, than that of the wild-type. Pre-equilibrium fluorescence studies with the inhibitor showed that this was due to a significantly increased off-rate for the mutant enzyme. The phenolic hydroxyl group of tyrosine-175 is within hydrogen bonding distance of the amide nitrogen of the glycine in the glutathione moiety and the present study shows that this interaction makes a significant contribution to the binding of the active-site ligand.

Human parvovirus B19 in pregnancy.

Parvovirus B19 infection can result in an adverse outcome when acquired during pregnancy. However, in the majority of cases a successful outcome can be anticipated. Public awareness of this condition is essential and obstetricians should be familiar with the options available to them if they are presented with this clinical problem.

Prenatal diagnosis and management of nonimmune hydrops fetalis.

We examined the incidence, aetiological factors and outcome in 40 cases of nonimmune hydrops fetalis (NIH) and suggest a rational approach to management. The incidence of NIH was 1 in 830 deliveries during the last 10-year period. In spite of extensive antenatal and postnatal investigation no cause could be established in 14 (35%) cases. A probable aetiological factor was found in 65% of cases. These included viral infection (7), cardiovascular (6), twin-to-twin transfusion (3), chromosomal abnormality (3), other malformation syndromes (4), renal dysplasia (1), laryngeal atresia (1) and severe fetomaternal haemorrhage (1). Five of the 40 fetuses survived, 2 treated antenatally for tachyarrhythmia, 2 had spontaneous resolution and the fifth fetus had repeated intrauterine transfusions because of human parvovirus B19-induced anaemia. After diagnosis of nonimmune hydrops fetalis, early referral to a tertiary centre is to be encouraged for investigation and provision of intensive perinatal care. Investigation allows parents to be counselled appropriately that the mortality is no longer 100% and a steadily growing number may be amenable to some form of fetal therapy.

Reaction mechanism of glyoxalase I explored by an X-ray crystallographic analysis of the human enzyme in complex with a transition state analogue.

The structures of human glyoxalase I in complexes with S-(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione (HIPC-GSH) and S-p-nitrobenzyloxycarbonylglutathione (NBC-GSH) have been determined at 2.0 and 1.72 A resolution, respectively. HIPC-GSH is a transition state analogue mimicking the enediolate intermediate that forms along the reaction pathway of glyoxalase I. In the structure, the hydroxycarbamoyl function is directly coordinated to the active site zinc ion. In contrast, the equivalent group in the NBC-GSH complex is approximately 6 A from the metal in a conformation that may resemble the product complex with S-D-lactoylglutathione. In this complex, two water molecules occupy the liganding positions at the zinc ion occupied by the hydroxycarbamoyl function in the enediolate analogue complex. Coordination of the transition state analogue to the metal enables a loop to close down over the active site, relative to its position in the product-like structure, allowing the glycine residue of the glutathione moiety to hydrogen bond with the protein. The structure of the complex with the enediolate analogue supports an "inner sphere mechanism" in which the GSH-methylglyoxal thiohemiacetal substrate is converted to product via a cis-enediolate intermediate. The zinc ion is envisioned to play an electrophilic role in catalysis by directly coordinating this intermediate. In addition, the carboxyl of Glu 172 is proposed to be displaced from the inner coordination sphere of the metal ion during substrate binding, thus allowing this group to facilitate proton transfer between the adjacent carbon atoms of the substrate. This proposal is supported by the observation that in the complex with the enediolate analogue the carboxyl group of Glu 172 is 3.3 A from the metal and is in an ideal position for reprotonation of the transition state intermediate. In contrast, Glu 172 is directly coordinated to the zinc ion in the complexes with S-benzylglutathione and with NBC-GSH.

Crystal structure of human glyoxalase II and its complex with a glutathione thiolester substrate analogue.

BACKGROUND: Glyoxalase II, the second of two enzymes in the glyoxalase system, is a thiolesterase that catalyses the hydrolysis of S-D-lactoylglutathione to form glutathione and D-lactic acid. RESULTS: The structure of human glyoxalase II was solved initially by single isomorphous replacement with anomalous scattering and refined at a resolution of 1.9 A. The enzyme consists of two domains. The first domain folds into a four-layered beta sandwich, similar to that seen in the metallo-beta-lactamases. The second domain is predominantly alpha-helical. The active site contains a binuclear zinc-binding site and a substrate-binding site extending over the domain interface. The model contains acetate and cacodylate in the active site. A second complex was derived from crystals soaked in a solution containing the slow substrate, S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione. This complex was refined at a resolution of 1.45 A. It contains the added ligand in one molecule of the asymmetric unit and glutathione in the other. CONCLUSIONS: The arrangement of ligands around the zinc ions includes a water molecule, presumably in the form of a hydroxide ion, coordinated to both metal ions. This hydroxide ion is situated 2.9 A from the carbonyl carbon of the substrate in such a position that it could act as the nucleophile during catalysis. The reaction mechanism may also have implications for the action of metallo-beta-lactamases.

Induced fit on sugar binding activates ribokinase.

The enzyme ribokinase phosphorylates ribose at O5* as the first step in its metabolism. The original X-ray structure of Escherichia coli ribokinase represented the ternary complex including ribose and ADP. Structures are presented here for the apo enzyme, as well as the ribose-bound state and four new ternary complex forms. Combined, the structures suggest that large and small conformational changes play critical roles in the function of this kinase. An initially open apo form can allow entry of the ribose substrate. After ribose binding, the active site lid is observed in a closed conformation, with the sugar trapped underneath. This closure and associated changes in the protein appear to assist ribokinase in recognition of the co-substrate ATP as the next step. Binding of the nucleotide brings about further, less dramatic adjustments in the enzyme structure. Additional small movements are almost certainly required during the phosphoryltransfer reaction. Evidence is presented that some types of movements of the lid are allowed in the ternary complex, which may be critical to the creation and breakdown of the transition state. Similar events are likely to take place during catalysis by other related carbohydrate kinases, including adenosine kinase.

Errors and reproducibility in electron-density map interpretation.

Three investigators, with varying levels of experience, independently built and refined the structure of Escherichia coli ribokinase at 2.6 A resolution. At the end of the refinement/rebuilding processes the models had essentially converged, although each had its own particular pattern of remaining errors. The subsequent refinement of the same structure at 1.8 A resolution allowed an overall quality check of each of the lower resolution models, and an analysis of which graphics-based tools were generally most efficient in locating these errors. Criteria which are useful in the application of Ramachandran, main-chain and side-chain database and real-space fit analyses are presented.