Association of a P2RX7 gene missense variant with brachycephalic dog breeds.

Missense variants are associated with various phenotypic traits and disorders in dogs. The canine P2RX7 gene, coding the ATP-gated P2X7 receptor ion channel, contains four known missense variants. The current study aimed to examine the presence of these variants in a random sample of pedigree and mixed-pedigree dogs. Exons 3, 8, 11 and 13 of the P2RX7 gene, encoding these four respective variants, in 65 dogs were assessed by Sanger sequencing and combined with existing sequencing data from another 69 dogs. The distribution of these variants was then evaluated in all 134 dogs combined and separately within individual breeds including 35 different pure breeds. The rs23314713 (p.Phe103Leu) and rs23315462 (p.Pro452Ser) variants were present in 47 and 40% of all dogs studied respectively, with the rs23314713 variant associated with brachycephalic breeds. Among pedigree dogs, the rs23314713 and rs23315462 variants were associated with brachycephalic and non-brachycephalic breeds respectively. The rs851148233 (p.Arg270Cys) and rs850760787 (p.Arg365Gln) variants were present only in dogs of Cocker Spaniel and Labrador Retriever pedigrees respectively. No other missense variants were found in exons 3, 8, 11 and 13 of the P2RX7 gene within the dogs. In conclusion, the rs23314713 and rs23315462 missense variants of the P2RX7 gene are present in a large proportion of dogs, with the rs23314713 variant associated with a number of brachycephalic breeds. However, the association of this variant with dogs of bulldog ancestry, not brachycephaly per se, cannot be excluded.

Prevalence of healthcare-associated infections and antimicrobial use among inpatients in a tertiary hospital in Fiji: a point prevalence survey.

BACKGROUND: Healthcare-associated infections (HAIs) and antimicrobial use (AMU) are important drivers of antimicrobial resistance, yet there is minimal data from the Pacific region. We sought to determine the point prevalence of HAIs and AMU at Fiji's largest hospital, the Colonial War Memorial Hospital (CWMH) in Suva. A secondary aim was to evaluate the performance of European Centre for Diseases Prevention and Control (ECDC) HAI criteria in a resource-limited setting. METHODS: We conducted a point prevalence survey of HAIs and AMU at CWMH in October 2019. Survey methodology was adapted from the ECDC protocol. To evaluate the suitability of ECDC HAI criteria in our setting, we augmented the survey to identify patients with a clinician diagnosis of a HAI where diagnostic testing criteria were not met. We also assessed infection prevention and control (IPC) infrastructure on each ward. RESULTS: We surveyed 343 patients, with median (interquartile range) age 30 years (16-53), predominantly admitted under obstetrics/gynaecology (94, 27.4%) or paediatrics (83, 24.2%). Thirty patients had one or more HAIs, a point prevalence of 8.7% (95% CI 6.0% to 12.3%). The most common HAIs were surgical site infections (n = 13), skin and soft tissue infections (7) and neonatal clinical sepsis (6). Two additional patients were identified with physician-diagnosed HAIs that failed to meet ECDC criteria due to insufficient investigations. 206 (60.1%) patients were receiving at least one antimicrobial. Of the 325 antimicrobial prescriptions, the most common agents were ampicillin (58/325, 17.8%), cloxacillin (55/325, 16.9%) and metronidazole (53/325, 16.3%). Use of broad-spectrum agents such as piperacillin/tazobactam (n = 6) and meropenem (1) was low. The majority of prescriptions for surgical prophylaxis were for more than 1 day (45/76, 59.2%). Although the number of handwashing basins throughout the hospital exceeded World Health Organization recommendations, availability of alcohol-based handrub was limited and most concentrated within high-risk wards. CONCLUSIONS: The prevalence of HAIs in Fiji was similar to neighbouring high-income countries, but may have been reduced by the high proportion of paediatric and obstetrics patients, or by lower rates of inpatient investigations. AMU was very high, with duration of surgical prophylaxis an important target for future antimicrobial stewardship initiatives.

Case of the month: Cutaneous myiasis in a returning traveller from the Algarve: first report of tumbu maggots, Cordylobia anthropophaga, acquired in Portugal.

A 61 year old woman returning to the UK from the Algarve and complaining of boil-like lesions was found to have cutaneous myiasis caused by tumbu maggots, Cordylobia anthropophaga. This is apparently the first report of acquisition of this form of myiasis in Portugal.

Collisional studies of some C3H 7O (+) ions.

The C3H7O(+) ions of nominal structures 1, 2, 3, and 4, produced by protonation of acetone, propanal, propylene oxide, and oxetan, respectively, have been studied by a variety of collisional techniques. The nominal isomers 2 and 3 give, within experimental error, identical high-energy collision-induced dissociation (CID) mass spectra. In addition, the breakdown graphs for the two isomers obtained from low, variable energy CID are identical, as are the neutrahzation-reionization mass spectra. The results are consistent with a facile isomerization of 3 to 2. By contrast, the ions of nominal structures 1 and 4 are shown by each technique to be distinct stable species in the gas phase. While the differences in the highenergy CID mass spectrum of 4 compared to 2 and 3 are relatively small, these differences are more pronounced in the low-energy CID mass spectra and become very large in the neutralization-reicnization mass spectra.

Stem cell biology in the lung and lung cancers: using pulmonary context and classic approaches.

Classic stem cell biology approaches tailored specifically with lung biology in mind are needed to bring the field of lung stem cell biology up to speed with that in other tissues. The infrequent cellular turnover, the diversity of cell types, and the necessity of daily cell function in this organ must be considered in stem cell studies. Previous work has created a base from which to explore transplantation, label retention, and more sophisticated lineage-tracing schemes to identify and characterize stem cell populations in the normal lung. These approaches are also imperative for building on precedents set in other tissues in the exploration of the cancer stem cell hypothesis in lung cancers. Additionally, recent studies provide key leads to further explore the molecular mechanisms that regulate lung homeostasis. Here, we discuss strategies to advance the field of lung stem cell biology with an emphasis on developing new, lung-specific tools.

Invasive group A streptococcal infection in injecting drug users and non-drug users in a single UK city.

OBJECTIVES: Injecting drug users (IDU) represent an increasing proportion of patients with invasive group A streptococcal (GAS) disease. Our aims were to characterise the clinical presentation and strains causing GAS bacteremia in IDU from a single UK city (Brighton and Hove), and to compare this patient group with non-drug users (non-DU) with GAS bacteremia. METHODS: Consecutive GAS blood culture isolates from twenty-two IDU and twenty-two non-DU presenting to the city hospital were studied. Clinical features, strain emm typing and superantigen toxin genotyping were investigated. RESULTS: GAS invasive disease presented differently in IDU compared to non-DU with a predominance of injection site abscesses and lower mortality in IDU. GAS strains from IDU were predominantly emm82 and emm83 types, which are uncommon in the UK and emm82 strains appeared clonal. The non-DU GAS strains demonstrated a broader range of emm types including most frequently emm1 and emm89. There was no major difference in superantigen gene profile between the isolate groups. CONCLUSION: The distinct presentation of invasive GAS disease in IDU compared with non-DU was associated with distinct emm types, a predominance of abscesses, and low mortality, although the small numbers preclude definitive conclusions. Further study is required to establish if these findings reflect strain differences or epidemiological differences in colonisation patterns and injecting practice.

Mechanism of energy coupling for transport of D-ribose in Escherichia coli.

In Escherichia coli ML 308-225, d-ribose is transported into the cell by a constitutive active transport system of high activity. The activity of this transport system is severely reduced in cells subjected to osmotic shock, and the system is not present in membrane vesicles. The mechanism by which metabolic energy is coupled to transport of ribose was investigated. Substrates which generate adenosine 5'-triphosphate primarily through oxidative phosphorylation are poor energy sources for ribose uptake in DL-54, a mutant of ML 308-225 which lacks activity for the membrane-bound Ca(2+), Mg(2+)-dependent adenosine triphosphatase required for oxidative phosphorylation. Arsenate severely inhibits ribose uptake, whereas, under the same conditions, uptake of l-proline is relatively insensitive to arsenate. Anaerobiosis does not significantly inhibit ribose uptake in ML 308-225 or DL-54 when glucose is the energy source. A significant amount of ribose uptake is resistant to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol. These results indicate that the phosphate bond energy of adenosine 5'-triphosphate, rather than an energized membrane state, couples energy to ribose transport in ML 308-225.

Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.

Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.

Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase.

Genetic studies show that Escherichia coli has three enzymes capable of phosphorylating glucose: soluble adenosine 5'-triphosphate-dependent glucokinase, which plays only a minor role in glucose metabolism; an enzyme II, called glucosephosphotransferase, with high specificity for the D-glucose configuration; and another enzyme II, called mannosephosphotransferase, with broader specificity. The former enzyme II is active on glucose and methyl-alpha-glucopyranoside, whereas the latter is active on D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, and D-mannosamine. Mutations leading to loss of glucosephosphotransferase activity and designated by the symbol gpt are between the purB and pyrC markers in a locus previously called cat. The locus of mutations to loss of mannosephosphotransferase, mpt, is between the eda and fadD genes. Mutations to loss of glucokinase, glk, are between the ptsI and dsd genes.

Purification of penicillin-binding protein 2 of Escherichia coli.

Penicillin-binding protein 2 (PBP-2) of Escherichia coli K-12 was purified by covalent affinity chromatography using 6-aminopenicillanic acid covalently coupled to carboxymethyl-Sepharose (6-APA-CM-Sepharose). Purification of PBP-2 was accomplished by prebinding the methoxy cephalosporin, cefoxitin, to the Triton X-100-solubilized PBPs of E. coli and then incubating the PBPs with 6-APA-CM-Sepharose. Cefoxitin readily binds to all the E. coli PBPs except PBP-2 and, thus, in the presence of cefoxitin, only PBP-2 could bind to the 6-APA-CM-Sepharose. The purification of a mixture of all of the PBPs of E. coli by affinity chromatography is also described.

Mutational evidence for identity of penicillin-binding protein 5 in Escherichia coli with the major D-alanine carboxypeptidase IA activity.

The defect in D-alanine carboxypeptidase IA activity in the dacA11191 mutant of Escherichia coli was correlated with a defect in the release of penicillin G from penicillin-binding protein 5. The results suggest that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity of the wild type and that the mutation results in a defect in the deacylation step catalyzed by this enzyme.

Involvement of the glucose enzymes II of the sugar phosphotransferase system in the regulation of adenylate cyclase by glucose in Escherichia coli.

The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues. Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g. alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Analogues with changes in other parts of the molecule (e.g. 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system. This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems. Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively. The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase. In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated. In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated. These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system.

Haemoprotein b-590 (Escherichia coli); redesignation of a bacterial 'cytochrome a1'.

A 'soluble' fraction from anaerobically grown Escherichia coli contains a haemoprotein with spectral properties, notably an alpha-band in the reduced form at 585 to 595 nm, similar to cytochrome a1. Haem extraction of either the soluble preparation or whole cells yields haem b, but not haem a. In view of this, and the spectral similarities of the a1-like component to well-known high-spin haem b proteins, we propose that the name 'haemoprotein b-590' be used to describe cytochrome a1-like pigments in bacteria.