Multilocus sequence typing of human and canine C. upsaliensis isolates.

Risk of Campylobacter infection in humans has been associated with many sources, including dogs. C. upsaliensis is the most common species found in canines, and has been occasionally isolated from symptomatic humans. This study aimed to investigate the genetic diversity of 41 C. upsaliensis isolates carried by dogs and from nine isolates carried by humans using Multilocus sequence typing (MLST). We identified considerable genetic diversity amongst the C. upsaliensis isolates from both dogs and humans, identifying 45 different sequence types (STs). All STs were new, apart from that of the reference strain. Only three STs were found in more than one isolate: ST-72 (2 isolates), ST-98 (2 isolates) and ST-104 (3 isolates). ST-104 was the only ST to be encountered in both dogs and humans. Thirty-one of the 45 STs were assigned to one of 13 clonal complexes (CCs). Four of these CCs contained STs originating from both humans and dogs. None of the CCs contained exclusively human isolates, and two isolates from dogs within the same kennel belonged to the same CC. The large amount of diversity found in both dog and human isolates of C. upsaliensis, combined with the relatively small database, made it difficult to assign strains to sources of infection. This emphasizes the need to increase the size of the database. Dog and human isolates occasionally grouped together, however there were insufficient human-derived isolates to determine whether or not dogs are a common source of infection. Although C. upsaliensis infection is rare in humans, dogs still remain a potential source, and are therefore a possible zoonotic risk. Further work is needed to investigate the epidemiology of C. upsaliensis infection in humans.

Prevalence of Campylobacter spp. in a cross-sectional study of dogs attending veterinary practices in the UK and risk indicators associated with shedding.

Campylobacteriosis is a major cause of gastroenteritis in humans and some studies have suggested that dog ownership is a risk factor for the condition. To determine the prevalence, species distribution, and risk indicators for Campylobacter spp. infecting dogs attending veterinary practices in UK, faecal samples were collected in a cross-sectional study from 249 dogs with and without clinical signs. The prevalence of Campylobacter spp. was 38% (95% CI 32, 44), with Campylobacter upsaliensis accounting for 94 (98%) of the isolates and Campylobacter jejuni for the remainder. Multivariable analysis indicated that younger dogs were more likely to carry C. upsaliensis and the high prevalence of this pathogen supports the hypothesis that dogs, particularly younger animals, may be an important source of C. upsaliensis infection for humans. However the prevalence of C. jejuni, the most common Campylobacter spp. associated with disease in humans, was low (1.2%, 95% CI 0.3, 3).

Risk factors for the carriage of Campylobacter upsaliensis by dogs in a community in Cheshire.

Samples of faeces were taken from 183 healthy pet dogs in a census-based, cross-sectional study in Cheshire; culture methods were used to detect any Campylobacter species and a direct PCR was used to detect Campylobacter upsaliensis. Forty-six of the dogs were positive for C upsaliensis by either culture or direct PCR, giving a prevalence of 25.1 per cent (95 per cent confidence interval [CI] 19.0 to 32.1 per cent). One sample was positive by culture for Campylobacter jejuni (95 per cent CI 0.0 to 3.0 per cent) and one for Campylobacter lari. Multivariable logistic regression identified risk factors for the carriage of C upsaliensis by a dog as: living with another dog that also carried C upsaliensis; being small rather than medium-sized; being less than three years old; living in a household that kept fish; being fed commercial dog treats; and being fed human food titbits, particularly in the dog's bowl.

Typing of Campylobacter jejuni isolates from dogs by use of multilocus sequence typing and pulsed-field gel electrophoresis.

Campylobacter is a major cause of human gastroenteritis worldwide. Risk of Campylobacter infection in humans has been associated with many sources, including dogs. This study aimed to investigate whether C. jejuni carried by dogs could potentially be a zoonotic risk for humans and if there were common sources of C. jejuni infection for both humans and dogs. Multilocus sequence typing (MLST) together with macrorestriction analysis of genomic DNA using SmaI and pulsed-field gel electrophoresis (PFGE) were both used to analyze 33 C. jejuni isolates obtained from various dog populations, including those visiting veterinary practices and from different types of kennels. MLST data suggested that there was a large amount of genetic diversity between dog isolates and that the majority of sequence types found in isolates from these dogs were the same as those found in isolates from humans. The main exception was ST-2772, which was isolated from four samples and could not be assigned to a clonal complex. The most commonly identified clonal complex was ST-45 (11 isolates), followed by ST-21 (4 isolates), ST-508 (4 isolates), and ST-403 (3 isolates). The profiles obtained by macrorestriction PFGE were largely in concordance with the MLST results, with a similar amount of genetic diversity found. The diversity of sequence types found within dogs suggests they are exposed to various sources of C. jejuni infection. The similarity of these sequence types to C. jejuni isolates from humans suggests there may be common sources of infection for both dogs and humans. Although only a small number of household dogs may carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel dog populations, where the prevalence may be higher.

Partly-PEGylated Poly-L-lysine dendrimers have reduced plasma stability and circulation times compared with fully PEGylated dendrimers.

The current study was performed to examine the effect of 50% surface PEGylation on the plasma circulation, biodistribution and metabolism of intravenously administered Generation 4 poly-L-lysine dendrimers in rats. Partial surface PEGylation blocked the rapid and extensive vascular binding previously described for the non-PEGylated dendrimer and slowed metabolism of the core, however the effect was less than that observed for total surface coverage of PEG. Capping the residual surface amines with acetyl groups further improved plasma stability.

Vitamin D status in renal transplant recipients.

Vitamin D plays an important role in calcium homeostasis. Renal transplant recipients may be more susceptible to reduced levels because of decreased sun exposure and steroid therapy. This study aimed to determine vitamin D status after renal transplantation and its effect on parathyroid hormone (PTH) and bone mineral density (BMD). We measured serum 25-hydroxyvitamin D levels (25-OHD) in 244 renal transplant recipients, divided into two groups, 104 recently transplanted (less than 1 year) and 140 long-term. Vitamin D status was defined according to NKF/KDOQI guidelines. Mean 25-OHD levels were 33 +/- 19 nmol/L and 42 +/- 20 nmol/L, respectively, for the recent and long-term transplant recipients. Vitamin D insufficiency was present in 29% and 43%, deficiency in 56% and 46% and severe deficiency in 12% and 5%, respectively. An inverse correlation was found between logPTH and 25-OHD (r=-0.2, p= 0.019) in long-term but not in recently transplanted patients. No correlation was found between 25-OHD levels and BMD. Hypercalcaemia was present in 40% of the recently transplanted recipients and 25% of the long-term. In conclusion 25-OHD was low in virtually all of our renal transplant recipients and may aggravate secondary hyperparathyroidism, but its correction may be difficult in patients with hypercalcaemia.

An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA.

In Bacillus subtilis, the termination of DNA replication via polar fork arrest is effected by a specific protein:DNA complex formed between the replication terminator protein (RTP) and DNA terminator sites. We report the crystal structure of a replication terminator protein homologue (RTP.C110S) of B. subtilis in complex with the high affinity component of one of its cognate DNA termination sites, known as the TerI B-site, refined at 2.5 A resolution. The 21 bp RTP:DNA complex displays marked structural asymmetry in both the homodimeric protein and the DNA. This is in contrast to the previously reported complex formed with a symmetrical TerI B-site homologue. The induced asymmetry is consistent with the complex's solution properties as determined using NMR spectroscopy. Concomitant with this asymmetry is variation in the protein:DNA binding pattern for each of the subunits of the RTP homodimer. It is proposed that the asymmetric "wing" positions, as well as other asymmetrical features of the RTP:DNA complex, are critical for the cooperative binding that underlies the mechanism of polar fork arrest at the complete terminator site.

NMR analysis of G7-18NATE, a nonphosphorylated cyclic peptide inhibitor of the Grb7 adapter protein.

G7-18NATE is a nonphosphorylated, cyclic peptide that specifically inhibits the Grb7 adapter protein implicated in several pathways critical to cell proliferation and migration. It has been shown that G7-18NATE is able to compete with natural ligands for the Grb7 SH2 phosphotyrosine binding site, and to attenuate cell migration in a pancreatic cancer cell line. It is thus an important lead in the development of a selective inhibitor of Grb7 and potential novel anticancer therapeutics. The current study reports the solution properties of G7- 18NATE determined using NMR spectroscopy, in both water (pH 2-3) and phosphate buffer (pH 6.0), with 100 mM NaCl. The spectra reveal that G7-18NATE exists in two distinguishable conformational states on the NMR timescale, most likely due to cis-trans proline isomerization. In addition, the chemical shift data are consistent with a tendency of G7-18NATE to form a turn about the YDN motif, known to be important for binding, and suggest that this turn is stabilized in low salt and low pH conditions. Low NH temperature coefficients of Tyr-5 and Asn-7 amide protons may reflect their involvement in the formation of hydrogen bonds that stabilize such a turn. Overall, however, the peptide does not form a rigid structure, but exists in a highly flexible state in solution. Averaged 3JNH-H coupling constants and a lack of interresidue NOEs are characteristic of such peptide solution behavior. This suggests that there is scope for increasing the rigidity of the peptide that may enhance its binding affinity and specificity for Grb7.

Lethal outbreak of disease associated with feline calicivirus infection in cats.

Recently, in the USA, virulent mutants of feline calicivirus (FCV) have been identified as the cause of a severe and acute virulent systemic disease, characterised by jaundice, oedema and high mortality in groups of cats. This severe manifestation of FCV disease has so far only been reported in the USA. However, in 2003, an outbreak of disease affected a household of four adult cats and an adult cat from a neighbouring household in the UK. Three of the adult cats in the household and the neighbouring cat developed clinical signs including pyrexia (39.5 to 40.5 degrees C), lameness, voice loss, inappetence and jaundice. One cat was euthanased in extremis, two died and one recovered. A postmortem examination of one of the cats revealed focal cellulitis around the right hock and right elbow joints. The principal finding of histopathological examinations of selected organs from two of the cats was disseminated hepatocellular necrosis with mild inflammatory infiltration. Immunohistology identified FCV antigen in parenchymal and Kupffer cells in the liver of both animals and in alveolar macrophages of one of them. In addition, calicivirus-like particles were observed by electron microscopy within the hepatocytes of one cat. FCV was isolated from two of the dead cats and from the two surviving cats. Sequence analysis showed that they were all infected with the same strain of virus, but that it was different from strains of FCV associated with the virulent systemic disease in cats in the USA. The outbreak was successfully controlled by quarantine in the owner's house.

Recombination of Feline calicivirus within an endemically infected cat colony.

To understand the evolution of the family Caliciviridae, the persistence of Feline calicivirus (FCV) was studied within an endemically infected cat colony. Polymerase and capsid sequences were analysed for 34 FCV isolates obtained over a 4 year period. Initially, the colony was infected with one strain of virus, but a second distinct strain was later identified. Subsequently, the emergence of a recombinant virus was observed, containing elements of both of the strains circulating within the colony. The recombination event mapped close to the ORF1/ORF2 junction. This is consistent with recombination in other caliciviruses, suggesting a common mechanism within this family. This is the first report of recombination within the genus Vesivirus in the family Caliciviridae and the first time that a recombination event has been observed where the parental strains have also been identified.

Acquired QT prolongation associated with esophagitis and acute weight loss: how to evaluate a prolonged QT interval.

When the physician is confronted with a patient having significant QT prolongation, it is critical to determine whether the patient harbors a genetic defect and a transmissible form of long QT syndrome (LQTS) or whether the QT prolongation has an acquired cause. The distinction has profound ramifications for the type of care provided to the patient and family. We report the case of a previously healthy 14-year-old boy who presented with a 10-day history of painful swallowing, a 10-lb weight loss, and chest pain. A 12-lead electrocardiogram (ECG) showed marked QT prolongation. Endoscopy and culture identified a Herpes simplex esophageal ulcer. After treatment with acyclovir, the patient recovered completely. Three weeks after the resolution of his symptoms and recovery from his acute weight loss, a follow-up ECG showed complete normalization of the QT interval. This case illustrates yet another potential mechanism for acquired QT prolongation. We also provide a diagnostic algorithm for the careful evaluation of a prolonged QT interval.

Formation of an alphaCP1-KH3 complex with UC-rich RNA.

The alphaCP family of proteins [also known as poly(C)-binding or heterogeneous nuclear ribonucleoprotein E proteins] are involved in the regulation of messenger RNA (mRNA) stability and translational efficiency. They bind via their triple heterologous nuclear ribonucleoprotein K homology (KH) domain structures to C-rich mRNA, and are thought to interact with other mRNA-binding proteins as well as provide direct nuclease protection. In particular, alphaCP1 and alphaCP2 have been shown to bind to a specific region of androgen receptor (AR) mRNA, resulting in its increased stability. The roles of each of the KH motifs in the binding affinity and the specificity is not yet understood. We report the beginning of a systematic study of each of the alphaCP KH domains, with the cloning and expression of alphaCP1-KH2 and alphaCP1-KH3. We report the ability of alphaCP1-KH3, but not alphaCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alphaCP1-KH3/AR mRNA complex for structural studies. (1)H-(15)N heteronuclear single quantum correlation NMR spectra of (15)N-labelled alphaCP1-KH3 verified the integrity and good solution behaviour of the purified domain. The titration of the 11-nucleotide RNA target sequence from AR mRNA resulted in a rearrangement of the (1)H-(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.

Structure and RNA binding of the third KH domain of poly(C)-binding protein 1.

Poly(C)-binding proteins (CPs) are important regulators of mRNA stability and translational regulation. They recognize C-rich RNA through their triple KH (hn RNP K homology) domain structures and are thought to carry out their function though direct protection of mRNA sites as well as through interactions with other RNA-binding proteins. We report the crystallographically derived structure of the third domain of alphaCP1 to 2.1 A resolution. alphaCP1-KH3 assumes a classical type I KH domain fold with a triple-stranded beta-sheet held against a three-helix cluster in a betaalphaalphabetabetaalpha configuration. Its binding affinity to an RNA sequence from the 3'-untranslated region (3'-UTR) of androgen receptor mRNA was determined using surface plasmon resonance, giving a K(d) of 4.37 microM, which is indicative of intermediate binding. A model of alphaCP1-KH3 with poly(C)-RNA was generated by homology to a recently reported RNA-bound KH domain structure and suggests the molecular basis for oligonucleotide binding and poly(C)-RNA specificity.

Burns from illegal drug manufacture: case series and management.

This case series presents our experience with burns sustained while manufacturing illegal drugs. All adult burn admissions in an 18-month period were retrospectively reviewed. All patients suspected of sustaining burns from illegal drug manufacture were contacted. Information regarding the burn mechanism was sought. Nine of the 64 adult burn admissions were caused by explosions during the manufacture of cannabis oil. Young males with hand and face burns were heavily represented. First-aid treatment was often ignored in favor of hiding incriminating evidence. Only two patients gave honest admission histories. Illegal drug manufacture is becoming more common as synthetic drugs become more consumer desirable. Burns sustained may be thermal and/or chemical. Dishonest patient histories negatively influence burn management. A high level of suspicion is required for diagnosing and treating burns from illegal drug manufacture. Public education is unlikely to be effective as the financial rewards outweigh the perceived risks.

Role of amiodarone in the treatment of fetal supraventricular tachyarrhythmias and hydrops fetalis.

We report three consecutive hydropic fetuses with fetal tachyarrhythmias treated with amiodarone-two in combination with digoxin and one with digoxin, procainamide, and propranolol. Sinus rhythm was achieved in one case and ventricular rate control was achieved in two cases. All fetuses treated with amiodarone gradually improved. Observed side effects of amiodarone were a maternal rash in one mother and transient neonatal hypothyroidism in one infant. We conclude that amiodarone might be effective and safe for fetal tachyarrhythmias and impending hydrops. The small number of patients suggests that a multicenter cooperative approach is required in order to determine if this is correct.

Effect of dietary fatty acids on the intestinal permeability of marker drug compounds in excised rat jejunum.

The aim of this study was to explore the effects of diets containing saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and omega-3 and omega-6 polyunsaturated fatty acids (omega-3 and omega-6 PUFA, respectively) on the passive and active transport properties of rat jejunum using marker compounds. Rats were fed diets supplemented with 18.4% (w/w) lipid (4 groups) or standard rat chow (1 group) for a period of 30 days. At the end of the dietary period, mucosal scrapings were taken for the determination of membrane phospholipids, and the apparent jejunal permeability of radiolabelled marker compounds was determined using modified Ussing chambers. Changes in the phospholipid content of the brush border membrane reflected the different lipid content of the diets. The passive paracellular permeability of mannitol was not significantly affected by the fatty acid composition of the diet, although there was a trend toward decreased mannitol permeability in the rats fed both the omega-3 and omega-6 PUFA diets. In comparison, the transcellular diffusion of diazepam was reduced by 20% (P < 0.05) in rats fed diets supplemented with omega-3 and omega-6 PUFA. In the lipid-fed rats, the serosal to mucosal flux of digoxin, an intestinal P-glycoprotein substrate, was reduced by 20% (P < 0.05) relative to the chow-fed group, however there were no significant differences between the different lipid groups. The active absorption of D-glucose via the Na+-dependent transport pathway was highest in the SFA, MUFA and PUFA omega-3 dietary groups, intermediate in the low-fat chow group and lowest in the PUFA omega-6 group, and was positively correlated with short-circuit current. These studies indicate that dietary fatty acid changes can result in moderate changes to the active and passive transport properties of excised rat jejunum.

DNA vaccination against feline calicivirus infection using a plasmid encoding the mature capsid protein.

Feline calicivirus (FCV), a member of the diverse family Caliciviridae, is a respiratory and oral pathogen of cats. Although conventional FCV vaccines are available, there are some safety and efficacy problems associated with their use. The potential of DNA vaccination against FCV infection was therefore explored. Four cats were inoculated intramuscularly with three 100 microg doses, 2 weeks apart, with a plasmid (pF9VAC) containing the mature capsid protein gene of FCV strain F9. Four control cats received the same plasmid lacking the FCV gene insert. All eight cats showed clinical signs following heterologous challenge with FCV strain LS027. However, rectal temperatures and general clinical sign scores were significantly lower in vaccinates compared to controls, and there was a marked difference in ulcer distribution between the two groups. Although no serological responses were detected in either group prior to challenge, post-challenge titres in the vaccinated group were generally higher. The results indicate that partial protection against a calicivirus is possible by DNA vaccination but that other approaches to enhance efficacy such as the use of cytokine genes or prime-boost protocols may also be required.

A conscious dog model for assessing the absorption, enterocyte-based metabolism, and intestinal lymphatic transport of halofantrine.

Postprandial administration of halofantrine (Hf), an important antimalarial, leads to 3- and 12-fold increases in oral bioavailability in humans and beagles, respectively, and corresponding 2.4-fold and 6.8-fold decreases in metabolic conversion to desbutylhalofantrine (Hfm). Factors contributing to the decreased postprandial metabolism of Hf could include inhibition of presystemic CYP3A metabolism by food components and/or recruitment of the intestinal lymphatics as an absorption pathway. Although previous rat studies confirmed Hf base is a substrate for lymphatic transport, it is difficult to extrapolate such data to higher species, as the largely constant bile flow in a rat precludes attainment of representative pre- and postprandial states, and formulations administered to rats are often not relevant to higher species. These limitations have now been addressed by development of a conscious dog model that allows simultaneous study of intestinal lymphatic and nonlymphatic drug absorption and aspects of enterocyte-based drug metabolism. After oral administration of 100 mg Hf base, the mean fasted and postprandial lymphatic transport was 1.3% and 54% of the administered dose, respectively. Comparison of portal and systemic plasma Hfm concentration profiles suggested enterocyte-based conversion of Hf to Hfm; however, the proportion of Hf metabolized to Hfm was similar after fasted or postprandial administration. Hence, it appears that the previously observed decrease in the postprandial metabolism of Hf is largely a consequence of significant postprandial intestinal lymphatic transport (which bypasses first pass hepatic metabolism). This new dog model will facilitate identification of the key factors that impact bioavailability, lymphatic transport, and metabolic profiles of highly lipophilic drugs.

Anatomically aberrant forearm arteries: an absent radial artery with co-dominant median and ulnar arteries.

A radial-artery free flap was dissected from a traumatically amputated forearm. During dissection the arterial tree was found to be abnormal, with no radial artery. The arterial supply was based on co-dominant median and ulnar arteries. This description raises issues regarding anatomical vascular aberrations when planning forearm flaps.