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Studies of collective motion have heretofore been dominated by a thermodynamic perspective in which the emergent "flocked" phases are analyzed in terms of their time-averaged orientational and spatial properties. Studies that attempt to scrutinize the dynamical processes that spontaneously drive the formation of these flocks from initially random configurations are far more rare, perhaps owing to the fact that said processes occur far from the eventual long-time steady state of the system and thus lie outside the scope of traditional statistical mechanics. For systems whose dynamics are simulated numerically, the nonstationary distribution of system configurations can be sampled at different time points, and the time evolution of the average structural properties of the system can be quantified. In this paper, we employ this strategy to characterize the spatial dynamics of the standard Vicsek flocking model using two correlation functions common to condensed matter physics. We demonstrate, for modest system sizes with 800 to 2000 agents, that the self-assembly dynamics can be characterized by three distinct and disparate time scales that we associate with the corresponding physical processes of clustering (compaction), relaxing (expansion), and mixing (rearrangement). We further show that the behavior of these correlation functions can be used to reliably distinguish between phenomenologically similar models with different underlying interactions and, in some cases, even provide a direct measurement of key model parameters.

Postdoctoral researchers (postdocs) are an essential component of the scientific workforce in German universities and research institutions and play a vital role in advancing knowledge and innovation. However, the experiences of postdocs and other early career researchers (ECRs) indicate that working conditions pose a significant challenge to the pursuit of a long-term research career in Germany-particularly for international scientists and those from marginalized groups. We examine how unstable working conditions as well as insufficient structural support for equal opportunities and diversity are significant obstacles for the career development of ECRs in German academia. We discuss these issues with the aid of an extensive survey recently conducted and published by PostdocNet, a target-group network representing the interests of postdocs across Germany's Max Planck Society. The survey drew responses from 659 postdoctoral researchers working at the Max Planck Society and represents one of the few datasets of postdoctoral researchers' perspectives in Germany. Building on these findings, we suggest actions at governmental, institutional, and individual levels to improve the working conditions of postdoctoral researchers in Germany., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Davidson, de Oliveira Lopes, Safaei, Hillemann, Russell and Schaare.)

Background: There is limited data on antibiotic treatment in hospitalized neonates in low- and middle-income countries (LMICs). We aimed to describe patterns of antibiotic use, pathogens, and clinical outcomes, and to develop a severity score predicting mortality in neonatal sepsis to inform future clinical trial design., Methods and Findings: Hospitalized infants <60 days with clinical sepsis were enrolled during 2018 to 2020 by 19 sites in 11 countries (mainly Asia and Africa). Prospective daily observational data was collected on clinical signs, supportive care, antibiotic treatment, microbiology, and 28-day mortality. Two prediction models were developed for (1) 28-day mortality from baseline variables (baseline NeoSep Severity Score); and (2) daily risk of death on IV antibiotics from daily updated assessments (NeoSep Recovery Score). Multivariable Cox regression models included a randomly selected 85% of infants, with 15% for validation. A total of 3,204 infants were enrolled, with median birth weight of 2,500 g (IQR 1,400 to 3,000) and postnatal age of 5 days (IQR 1 to 15). 206 different empiric antibiotic combinations were started in 3,141 infants, which were structured into 5 groups based on the World Health Organization (WHO) AWaRe classification. Approximately 25.9% (n = 814) of infants started WHO first line regimens (Group 1-Access) and 13.8% (n = 432) started WHO second-line cephalosporins (cefotaxime/ceftriaxone) (Group 2-"Low" Watch). The largest group (34.0%, n = 1,068) started a regimen providing partial extended-spectrum beta-lactamase (ESBL)/pseudomonal coverage (piperacillin-tazobactam, ceftazidime, or fluoroquinolone-based) (Group 3-"Medium" Watch), 18.0% (n = 566) started a carbapenem (Group 4-"High" Watch), and 1.8% (n = 57) a Reserve antibiotic (Group 5, largely colistin-based), and 728/2,880 (25.3%) of initial regimens in Groups 1 to 4 were escalated, mainly to carbapenems, usually for clinical deterioration (n = 480; 65.9%). A total of 564/3,195 infants (17.7%) were blood culture pathogen positive, of whom 62.9% (n = 355) had a gram-negative organism, predominantly Klebsiella pneumoniae (n = 132) or Acinetobacter spp. (n = 72). Both were commonly resistant to WHO-recommended regimens and to carbapenems in 43 (32.6%) and 50 (71.4%) of cases, respectively. MRSA accounted for 33 (61.1%) of 54 Staphylococcus aureus isolates. Overall, 350/3,204 infants died (11.3%; 95% CI 10.2% to 12.5%), 17.7% if blood cultures were positive for pathogens (95% CI 14.7% to 21.1%, n = 99/564). A baseline NeoSep Severity Score had a C-index of 0.76 (0.69 to 0.82) in the validation sample, with mortality of 1.6% (3/189; 95% CI: 0.5% to 4.6%), 11.0% (27/245; 7.7% to 15.6%), and 27.3% (12/44; 16.3% to 41.8%) in low (score 0 to 4), medium (5 to 8), and high (9 to 16) risk groups, respectively, with similar performance across subgroups. A related NeoSep Recovery Score had an area under the receiver operating curve for predicting death the next day between 0.8 and 0.9 over the first week. There was significant variation in outcomes between sites and external validation would strengthen score applicability., Conclusion: Antibiotic regimens used in neonatal sepsis commonly diverge from WHO guidelines, and trials of novel empiric regimens are urgently needed in the context of increasing antimicrobial resistance (AMR). The baseline NeoSep Severity Score identifies high mortality risk criteria for trial entry, while the NeoSep Recovery Score can help guide decisions on regimen change. NeoOBS data informed the NeoSep1 antibiotic trial (ISRCTN48721236), which aims to identify novel first- and second-line empiric antibiotic regimens for neonatal sepsis., Trial Registration: ClinicalTrials.gov, (NCT03721302)., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Members of the funder (MB, NK, SE) participated as authors on the study, reviewed the manuscript, and approved the final manuscript as submitted. All other authors have declared that no competing interests exist., (Copyright: © 2023 Russell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rapidly identifying likely Ebola patients is difficult because of a broad case definition, overlap of symptoms with common illnesses, and lack of rapid diagnostics. However, rapid identification is critical for care and containment of contagion. We analyzed retrospective data from 252 Ebola-positive and 172 Ebola-negative patients at a Sierra Leone Ebola treatment center to develop easy-to-use risk scores, based on symptoms and laboratory tests (if available), to stratify triaged patients by their likelihood of having Ebola infection. Headache, diarrhea, difficulty breathing, nausea/vomiting, loss of appetite, and conjunctivitis comprised the symptom-based score. The laboratory-based score also included creatinine, creatine kinase, alanine aminotransferase, and total bilirubin. This risk score correctly identified 92% of Ebola-positive patients as high risk for infection; both scores correctly classified >70% of Ebola-negative patients as low or medium risk. Clinicians can use these risk scores to gauge the likelihood of triaged patients having Ebola while awaiting laboratory confirmation.

Aims: The UK Medicines and Healthcare products Regulatory Agency (MHRA) runs a national spontaneous reporting system (Yellow Card [YC] Scheme) to collect 'suspected' adverse drug reaction (ADR) data. We aim to describe the content and utility of YC reports received for patients aged <2 years., Methods: Data on all ADRs reported using YC in infants aged <2 years from the years 2001-10 were supplied by the MHRA., Results: For infants age <2 years, 3496 suspected ADRs were reported using YC (paternal medication pre-conception n = 3, transplacental n = 246, transmammary n = 30, neonates n = 97, infant n = 477, and vaccinations n = 2673), averaging 0.96 YC per day. There was a male preponderance (male 49.1%, female 44.4%, unknown 6.5%), and only 34 (1.0%) of YC reports stated a gestational age. The medications most frequently reported were: transplacental and transmammary (fluoxetine, n = 21 and n = 4 respectively), neonate (swine flu vaccine, n = 8) infant (oseltamivir, n = 37) and vaccines (meningococcal vaccine, n = 693). Paternal, transmammary, neonatal and infant YC did not reflect clinical concerns raised by the UK regulator. Transplacental and vaccination reports did correlate with some of the changes in practice and clinical alerts received., Conclusions: The frequency of YC reports for those <2 years is low, neonates are poorly represented, and recording of gestational age is poor. With the exception of vaccinations, spontaneous reports alone are not currently generating the data required, and important safety messages from the regulator do not match reporting patterns. Additional reporting strategies are required to improve the quantity and quality of suspected ADR data in young children., (© 2016 The British Pharmacological Society.)

A 4-year-old female spayed domestic longhair cat was referred for dyspnea. Further diagnostics revealed severe pleural effusion and a peritoneopericardial diaphragmatic hernia (PPDH). Following surgical correction of the PPDH the pleural effusion persisted. Re-check echocardiogram 4 weeks after initial evaluation revealed leftward deviation of the interventricular septum and interatrial septum occurring with inspiration. There were also exaggerated phasic changes in trans-tricuspid flow velocities suggestive of constrictive pericardial disease. Cardiac catheterization was performed and revealed elevated pressures in the right atrium and right ventricle. Constrictive pericarditis (CP) and epicarditis was confirmed at surgery, where subtotal pericardiectomy was performed with epicardial decortication. The cat continued to develop recurrent pleural effusion after surgery, although the volume and frequency of recurrence slowed over time. This is the first reported case of CP following PPDH repair in a cat., (© ISFM and AAFP 2014.)

Antarctic microbial biodiversity is the result of a balance between evolution, extinction and colonization, and so it is not possible to gain a full understanding of the microbial biodiversity of a location, its biogeography, stability or evolutionary relationships without some understanding of the input of new biodiversity from the aerial environment. In addition, it is important to know whether the microorganisms already present are transient or resident - this is particularly true for the Antarctic environment, as selective pressures for survival in the air are similar to those that make microorganisms suitable for Antarctic colonization. The source of potential airborne colonists is widespread, as they may originate from plant surfaces, animals, water surfaces or soils and even from bacteria replicating within the clouds. On a global scale, transport of air masses from the well-mixed boundary layer to high-altitude sites has frequently been observed, particularly in the warm season, and these air masses contain microorganisms. Indeed, it has become evident that much of the microbial life within remote environments is transported by air currents. In this review, we examine the behaviour of microorganisms in the Antarctic aerial environment and the extent to which these microorganisms might influence Antarctic microbial biodiversity.

Lymphangiosarcoma is a malignant neoplasm of the lymphatic endothelium that is rare in cats. This report describes two cases of feline lymphangiosarcoma that originated in the distal limb, causing intractable lymphoedema and serosanguineous discharge with ecchymoses in local and distant sites. In association with the neoplasia, one cat had cortical bone lysis of multiple metacarpal bones of the affected limb and the other had severe immune-mediated haemolytic anaemia (IMHA). The disease in both cases affected young cats and progressed rapidly. Persistent distal limb lymphoedema with serosanguineous discharge is suggestive of lymphangiosarcoma especially when local or distal ecchymoses are evident.

Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included alpha-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein.

The phenotypic adaptation of membrane lipids in seven strains of the food-poisoning bacterium Bacillus cereus, isolated from Bangladeshi rice, is reported in relation to their ability to grow under conditions of low water activity (a(w)), reduced temperature and the presence of soluble rice starch. The strains have different membrane phospholipid head-group and fatty acyl compositions, and they display individual differences in their responses to both low a(w) and reduced temperature. The extent of the increase in anionic membrane lipids in response to low a(w) varies from strain to strain, is solute specific and in one strain does not occur. Growth is stimulated by the presence of soluble rice starch and results in a large rise in the proportion of diphosphatidylglycerol (DPG) at the expense of phosphatidylglycerol (PG), without any change in the proportion of total anionic phospholipids. Growth at 15 degrees C compared with 37 degrees C increases the proportions of DPG and phosphatidylethanolamine at the expense of PG. At the lower temperature there are changes in phospholipid fatty acyl composition characteristic of those expected to maintain membrane fluidity, including increases in the amount of total branched fatty acids and the anteiso-/iso-branched ratio, and a decrease in the equivalent chain-length, but there are strain differences in how those changes were achieved. In contrast to some other bacilli, there are persistent large increases in the proportions of unsaturated fatty acyl chains in phospholipids during growth at 15 degrees C.

The aim of this study was to identify Antarctic microorganisms with the ability to produce cold-active antimicrobial compounds with potential for use in chilled food preservation. Colonies (4496) were isolated from 12 Antarctic soil samples and tested against Listeria innocua, Pseudomonas fragi and Brochothrix thermosphacta. Thirteen bacteria were confirmed as being growth-inhibitor producers (detection rate 0.29%). When tested against a wider spectrum of eight target organisms, some of the isolates also inhibited the growth of L. monocytogenes and Staphylococcus aureus. Six inhibitor producers were psychrotrophic (growth optima between 18 and 24 degrees C), halotolerant (up to 10% NaCl) and catalase-positive; all but one were Gram-positive and oxidase-positive. The inhibitors produced by four bacteria were sensitive to proteases, suggesting a proteinaceous nature. Four of the inhibitor-producers were shown to be species of Arthrobacter, Planococcus and Pseudomonas on the basis of their 16S rRNA gene sequences and fatty acid compositions. It was concluded that Antarctic soils represent an untapped reservoir of novel, cold-active antimicrobial-producers.

Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylcitrate synthase and also possess 2-methylcitrate synthase activity. The data are discussed with respect to the structure, function and evolution of citrate synthase and 2-methylcitrate synthase.

The hypothesis that biodegradable surfactants stimulate the attachment of biodegradation-competent bacteria to surfaces has been re-evaluated using a variant of the surfactant-degrading bacterium Pseudomonas sp. DES1 designated Pseudomonas sp. DES2. This variant was identical to the parental strain in terms of its carbon-utilization patterns and alcohol dehydrogenase and alkylsulfatase complements (enzymes involved in surfactant biodegradation), but differed markedly in its growth characteristics when using sodium dodecyl triethoxysulfate or triethylene glycol dodecyl ether as secondary carbon sources. Pseudomonas sp. DES1 exhibited diauxie in these surfactant-based culture media in contrast to Pseudomonas sp. DES2, which exhibited single-phase growth. Pseudomonas sp. DES2 did not attach to river sediment in a microcosm system when challenged with a dose of either surfactant, although it did biodegrade the substrate. In contrast, Pseudomonas sp. DES1 attached to the river sediment whilst biodegrading the test substrate. It is concluded that the ether-scission system, which is responsible for primary biodegradation of both substrates, is deregulated in Pseudomonas sp. DES2 in contrast to that in Pseudomonas sp. DES1, and that, contrary, to a previous hypothesis, biodegradable surfactants do not necessarily stimulate the attachment of biodegradation-competent bacteria during their biodegradation.

A nisin-resistant (NISr) variant of Listeria monocytogenes Scott A was isolated by stepwise exposure to increasing concentrations of nisin in brain heart infusion (BHI) broth. The NISr strain was about 12 times more resistant to nisin than was the wild-type (WT) strain. Accordingly, higher nisin concentrations were required to dissipate both components of the proton motive force in the NISr strain than in the WT strain. Comparison of the membrane fatty acyl composition of the sensitive strain with that of its NISr derivative revealed no significant differences. From phospholipid head group composition analysis and phospholipid biosynthesis measurements during growth in the absence and presence of nisin, it could be inferred that the NISr strain produces relatively more phosphatidylglycerol (PG) and less diphosphatidylglycerol (DPG) than the parent strain does. Monolayer studies with pure lipid extracts from both strains showed that nisin interacted more efficiently with lipids derived from the WT strain than with those derived from the NISr strain, reflecting qualitative differences in nisin sensitivity. Involvement of the cell wall in acquisition of nisin resistance was excluded, since the WT and NISr strains showed a comparable sensitivity to lysozyme. Recently, it has been demonstrated that nisin penetrates more deeply into lipid monolayers of DPG than those of other lipids including PG, phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol (R.A. Demel, T. Peelen, R.J. Siezen, B. de Kruijff, and O.P. Kuipers, Eur. J.Biochem. 235:267-274, 1996). Collectively, the mechanism of nisin resistance in this L. monocytogenes NISr strain is attributed to a reduction in the DPG content of the cytoplasmic membrane.

The gene encoding citrate synthase from a novel bacterial isolate (DS2-3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2-3R, are cold-active, with a temperature optimum of 31 degrees C. In addition the enzymes are rapidly inactivated at 45 degrees C, and show significant activity at 10 degrees C and below. Comparison of amino acid sequences indicates that DS2-3R citrate synthase is most closely related to the enzyme from gram-positive bacteria. The amino acid sequence of the DS2-3R enzyme shows several features previously recognised in other cold-active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.

Pseudomonas sp. strain SC25A, previously isolated for its ability to grow on alcohol ethoxylates (PEG dodecyl ethers) as sole source of carbon and energy, was shown to be capable of growth on the dodecyl ethers of mono-, di, tri- and octaethylene glycols. Comparative growth yields for this series of alcohol ethoxylate nonionic surfactants indicated that, whereas all of the carbon of monoethylene glycol dodecyl ether (MEGDE) was assimilable, only the alkyl chains were assimilated from the higher ethoxamers. These results are interpreted in terms of a primary biodegradation mechanism in which the scission of the dodecyl-ether bond is the first step. In the case of MEGDE this step separates the dodecyl chain from a C2 fragment, both of which are readily assimilable; for the higher ethoxamers, the assimilable dodecyl chain is accompanied by an ether-containing PEG derivative which would require further rounds of either scission before assimilation. Whole cells and cell extracts converted [1-14C]MEGDE initially and very rapidly to radiolabelled dodecanol. Disappearance of [14C]dodecaol was accompanied by production of [14C]dodecanal. [14C]Dodecanoic acid was present at relatively low concentrations throughout the incubation periods. [14C]Dodecan-1, 12-dioic acid was produced in significant quantities (up to 25% radiolabel), and the onset of its production coincided with the peak concentration of dodecanal, the disappearance of which mirrored the appearance of the dioic acid. Under anaerobic conditions in the presence of cell extracts, dodecanol (55% of radiolabel) and dodecanal (22%) accumulated rapidly from MEGDE, but there was little subsequent conversion to mono- or dicarboxylic acids. These results are interpreted in terms of a pathway initiated by dodecyl-ether cleavage to produce dodecanol, which is subsequently oxidized to dodecanal and dodecanoic acid. The formation of dodecan-1, 12-dioic acid, probably from dodecanal, may represent a means of harbouring carbon under non-growing conditions.

The fatty acid composition and temperature/growth characteristics of a psychrophilic bacterium, strain ACAM 456, isolated from Antarctic sea-ice is reported. The bacterium produced acyl components that may be grouped in three different carbon chain types: even-chain, odd-chain and iso -branched odd-chain. The proportions of these chain types varied according to growth temperature, and were manipulated by growth on L-serine, t-leucine or propionic acid as sole carbon sources. De novo fatty acid synthesis was investigated using sodium [1- 14 C]acetate, L-[U- 14 C]leucine and L-[U- 14 C]serine as radioactive precursors. Compared with a control culture, resuspension of midexponential phase cells in artificial seawater led to a change in the selection and/or intracellular availability of acyl chain primer molecules. The proportion of radiolabel incorporated into even-chain length components from cells declined, whereas the percentage of radiolabel present in odd-chain length components increased. An increase in incubation temperature augmented this effect, and also elicited a rise in the proportion of label present in branchedchain products. ACAM 456 manipulated the utilization of acyl chain primer molecules as an adaptive response to changes in environmental conditions. In particular, the regulation of odd-chain length fatty acids is described as a novel adaptational response.

The Biolog microtitre plate assay, which is based on tetrazolium dye reduction as an indicator of sole-carbon-source utilization, has been evaluated as a rapid method to investigate the biodegradation of five classes of anionic surfactant by pure and mixed cultures of bacteria. The assay gave reproducible results over a fourfold range of inoculum optical density, and the surfactant concentration was selected to provide a compromise between the length of the lag period prior to colour production and the maximum colour produced. A kinetic model was developed and used to analyse the appearance of colour in the assay and was found to give rise to three biologically significant parameters describing the processes underlying the assay. No false-positives were obtained with environmental isolates. The small number of false-negatives obtained (< 8% of the total) could be explained by the methodology used to prepare the bacterial inoculum. All isolates which were positive in the Biology assay were shown to be both primary and ultimate degraders of the test surfactant. These results show that the method provides a useful means of studying the biodegradation of anionic surfactants by both pure and mixed cultures of bacteria and will find use in the rapid analysis of biodegradation kinetics and specificities of larger numbers of individual isolates than hitherto possible. In addition, an important benefit of the methodology is that it can be used for direct analysis of the biodegradation potential of whole bacterial communities without having to make an artificial selection during laboratory growth.

The effects of 6-amino-2-n-pentylthiobenzothiazole (APB), a new antifungal agent, on ergosterol biosynthesis in Candida albicans and Saccharomyces cerevisiae were studied, using [14C]acetate incorporation. In C. albicans, the inhibition of growth was accompanied by a marked inhibition of acetate incorporation in 4-desmethylsterols, with a significant portion of the radiolabel being incorporated in 4,4-dimethylsterols, lanosterol, and 4,4-dimethylzymosterol and minor amounts being incorporated in 4-methylsterols and squalene. The data are interpreted as evidence of a block of the ergosterol biosynthesis pathway at the level of 4-demethylation of 4,4-dimethylzymosterol, with partial inhibition of lanosterol 14-dimethylation and squalene epoxidation also being possible. In 6-amino-2-n-pentylthiobenzothiazole-treated S. cerevisiae, a significant amount of the radiolabel was incorporated also in 4-methylsterols, 4-methylzymosterol, and 4-methylfecosterol, indicating that in this microorganism there are different sensitivities of the two 4-demethylations and that the pathway is blocked at the level of 4-demethylation of 4-methylsterols.

The bacterial biodegradation of a secondary sulphonate, sulphosuccinate, has been shown to occur by direct desulphonation. A bacterium, designated Pseudomonas sp. BS1, was isolated from activated sewage sludge, for its capacity to grow on sulphosuccinate as the sole source of carbon and energy. Cultures grown on sulphosuccinate were able to convert this substrate to sulphite which was subsequently oxidized rapidly to sulphate. The sequence of desulphonation and carbon-chain catabolism of sulphosuccinate was determined from measurements of the kinetics of sulphite and 14CO2 release from specifically radiolabelled sulpho[1,4-14C]succinate and sulpho[2,3-14C]succinate, which were synthesized from the corresponding maleic anhydrides. When each radiolabelled compound was incubated separately with washed-cell suspensions of Pseudomonas BS1, sulphite was released before 14CO2, as shown by chemical assay and radiorespirometry, respectively. Differences in the kinetics and extent of 14CO2 release from the 1,4- and 2,3-labelled substrates were consistent with entry of the intact C4 chain into the citric acid cycle. When carrier oxaloacetate was added to incubation mixtures containing resting-cell suspensions and radiolabelled sulphosuccinate, a radiolabelled metabolite with the same HPLC retention time as oxaloacetate accumulated. No radioactive metabolites accumulated when carrier oxaloacetate was replaced with succinate, fumarate or malate. Collectively, the data indicated co-production of sulphite and oxaloacetate from sulphosuccinate, which is interpreted in terms of an oxidative desulphonation mechanism.

A laboratory-scale river microcosm was used to investigate the effect of the anionic surfactant sodium dodecyl sulphate (SDS) on the attachment of five Pseudomonas strains to natural river-sediment surfaces. Three of the Pseudomonas strains were chosen for their known ability to express alkylsulphatase enzymes capable of hydrolysing SDS, and the other two for their lack of such enzymes. One strain from each category was isolated from the indigenous bacterial population present in the river sediment used; other isolates were from soil or sewage. The alkylsulphatase phenotypes were confirmed by gel zymography of cell extracts. Addition of SDS to mixed suspensions of river sediment with any one of the biodegradation-competent strains stimulated the attachment of bacteria to the sediment particles. In contrast, the attachment of biodegradation-incompetent strains was weak and, moreover, was unaffected by SDS. The SDS-stimulated attachment for competent organisms coincided with rapid biodegradation of the surfactant. The primary intermediate of SDS biodegradation, dodecan-1-ol, accumulated transiently, and the numbers of attached bacteria correlated closely with the amount of dodecan-1-ol present. Direct addition of dodecan-1-ol also stimulated attachment but the effect was more immediate compared with SDS, when there was a lag period of approximately 2 h. To account for these observations, a model is proposed in which SDS stimulates the attachment of biodegradation-competent bacteria through its conversion to dodecan-1-ol, and it is hypothesized that the observed reversibility of the attachment is due to the subsequent removal of dodecan-1-ol by further bacterial metabolism.

The Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell & P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.

GDP-mannose dehydrogenase (GMD) is a key regulatory enzyme and the committal step in alginate biosynthesis. In this study, a metabolic approach has been used to investigate GMD activity in non-mucoid and isogenically related mucoid strains of Pseudomonas aeruginosa. Intracellular concentrations of GDP-mannose and GDP-mannuronate have been quantified using HPLC separation methods, and their concentrations have been related to GMD activity and total alginate production. In all strains of P. aeruginosa tested, GDP-mannose accumulated particularly during the exponential phase of growth in batch culture; the GDP-mannose concentrations in mucoid strains were significantly lower compared with isogenic non-mucoid strains. The product of GMD activity, GDP-mannuronate, was detectable only in mucoid strains, albeit at low but relatively constant levels irrespective of growth phase. The GDP-mannose concentrations in mucoid strains were always significantly greater than those of GDP-mannuronate, indicating that GMD is a rate-limiting enzyme in the biosynthesis of alginate. Significant GMD activity and extracellular alginate production were detected only in mucoid strains. The metabolic data reported here, together with previous genetic studies, provide strong evidence that GMD is the key regulatory enzyme controlling alginate biosynthesis in mucoid strains of P. aeruginosa.

The isolation and characterization of a mucoid, alginate-producing strain of Pseudomonas aeruginosa from a nonhuman host, namely, in chondroids from an equine guttural pouch, is reported for the first time. Pure cultures of P. aeruginosa 12534 were isolated from a 17-month-old pony mare with a history of chronic bilateral mucopurulent nasal discharge from the right guttural pouch. Transmission electron microscopy of chondroids showed mucoid P. aeruginosa growing as microcolonies within a matrix of extracellular material. On the basis of expression of the mucoid phenotype under different growth conditions, P. aeruginosa 12534 belongs to group 1 and resembles other isolates carrying the muc-23 mutation. The bulk of the extracellular material was characterized as being alginate by chemical and 1H nuclear magnetic resonance analyses, which showed that it had a composition similar to that produced by isolates of P. aeruginosa from human patients with cystic fibrosis.

Biofilm formation on presterile slate-discs placed at a pristine source site and at three polluted sites located upstream from, at, and down-stream from a sewage-works outfall in a South Wales river, was monitored over 14 days. Viable bacterial cell densities in biofilms increased with time at all sites but more rapidly at the polluted sites, and stabilized after 1-4 days at levels equal to those of stones indigenous to the corresponding sites, and similar to those seen in other comparable pristine and polluted rivers. Biofilm cell densities were elevated about 1,000-fold at the sewage works outfall compared with the site immediately upstream. Downstream from the outfall, the cell densities were intermediate between the upstream and outfall values. Epilithon resuspended from the slates during colonization was tested for its capacity to biodegrade the surfactant sodium dodecyl sulphate (SDS) in die-away tests. No biodegradation of this common pollutant was observed for the samples from the pristine source. In contrast, even after only 1 day, all polluted sites produced samples capable of biodegrading SDS. Longer exposure in the river led to more rapid onset of biodegradation in the die-away tests, indicating an adaptation mechanism(s). Die-away kinetics were fitted by computerized non-linear regression analysis to one of several models. The model of best fit involved biodegradation of SDS by a bacterial population growing at the expense of endogenous carbon. The regression parameter reflecting SDS-degrading activity of the epilithic samples increased markedly during Days 0-4 for all three polluted sites. The stabilized values (Days 4-14) increased from the upstream site to the outfall, then decreased to intermediate values downstream. Although this pattern corresponded to the changes in viable cell numbers, the effect of the sewage input was less marked for the SDS-degrading activities than for bacterial cell densities. In addition, there was little variation in growth characteristics throughout colonization at all three polluted sites. Collectively the results indicate that the observed adaptation during exposure in the river is attributable to colonization of the epilithon by an existing SDS-degrading population, rather than the acquisition or adaptation of this biodegradative capability.

We present new data obtained by 23Na nuclear magnetic resonance spectroscopy, which can distinguish free intracellular sodium from cell-bound sodium, showing that the intracellular concentration of Na+ the halophilic eubacterium Vibrio costicola is only 5 to 20% of that in the extracellular medium. Previous methods could not distinguish free intracellular Na+ from that bound to cell structures, and it was believed that in halophilic eubacteria the total monovalent cation concentration inside matched that of the NaCl outside. Information obtained by the newer technology raises fundamental questions about the ways in which these organisms and others which live in hypersaline environments function and cope with osmotic stress.

A microcosm containing resuspended river sediment was used to investigate the effect of anionic surfactants on the distribution of bacteria between planktonic and attached populations. Freshwater river sediment containing viable bacteria was preequilibrated in the microcosm, which was subsequently supplemented with biodegradable or recalcitrant surfactants and a non-surface-active carbon and energy source. Population dynamics of both free-living and attached bacteria were measured by epifluorescence microscopy with simultaneous analysis of the residual solution concentration of the xenobiotic carbon source. The addition of the readily biodegradable anionic surfactants sodium decyl sulfate and sodium dodecyl sulfate in separate experiments caused an increase in the number of attached bacteria and a concomitant decrease in the number of free-living bacteria. As biodegradation of the surfactants progressed, these trends reversed and the bacterial populations had returned to their preaddition values by the time when biodegradation was completed. In contrast, sodium tetradecyl sulfate or sodium dodecane sulfonate did not stimulate bacterial association with sediment, nor were they biodegraded in the microcosm. Sodium pyruvate, a non-surface-active carbon and energy source, was readily utilized but caused no bacterial attachment to the sediment. These results indicate that for an anionic surfactant to induce bacterial attachment to river sediment, it must be biodegradable. The bacterial attachment to the sediment appears to be reversible and may be dependent on the accumulation of the surfactant at the surface or as a result of alteration of the surface free energies.

The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.

The capacities of epilithic and planktonic river bacterial populations to degrade sodium dodecyl sulfate (SDS) in samples taken at two times during 1987 from one clean and four polluted sites in a South Wales river were estimated in die-away tests under simulated environmental conditions. There was a relatively slow disappearance of SDS in die-away tests for both planktonic and epilithic populations taken from the clean source site, as compared with those taken from the downstream polluted sites, for which the rate of biodegradation was accelerated, sometimes after an apparent initial lag period. The kinetic components contributing to the die-away curves were quantified by nonlinear regression analysis in which the experimental data were fitted to a variety of possible kinetic models. All samples except for one from the polluted sites best fitted a model which describes the biodegradation of SDS at concentrations well below its K(m) by bacteria whose growth is exponential and unaffected by the addition of a test substrate. Each sample from the clean source site fitted a different model, but there was generally little or no growth on endogenous carbon. A consideration of the numerical values of constants derived from the modeling of epilithic and planktonic populations from polluted sites indicated clearly that the biodegradative capacity of epilithic bacterial populations towards SDS is more stable than that of planktonic bacterial populations.

The delta 9-desaturase of the psychrophilic bacterium Micrococcus cryophilus is shown to be a membrane-bound enzyme that is probably linked to a cyanide- (and azide-) sensitive respiratory chain with oxygen as the final acceptor. It has a pH optimum of 8.7 and contains an essential thiol group, but has no special ion requirements. The desaturase activity of washed membranes could not be increased by adding supernatant or NADH and NADPH, possibly owing to the endogenous generation of reduced cofactors by the membranes. The substrate for the desaturase is not acyl-CoA and is probably not acyl-acyl-carrier protein. Evidence is presented that the substrate in vivo is saturated phospholipid and a scheme for the possible routes of incorporation of exogenous stearic acid into oleoyl-phospholipid is presented.

Lipid biosynthesis has been studied in photosynthetic cultures of Rhodopseudomonas sphaeroides that had been synchronized by stationary-phase cycling or by a centrifugation selection procedure. Synchrony index values in the range 0.70-0.80 were obtained for the first cell cycle with both synchronization methods. The major membrane lipids phosphatidylethanolamine and phosphatidylglycerol were accumulated discontinuously during the cell cycle, their mass doubling immediately before cell division. This accumulation of lipid corresponded to peaks in incorporation of radioactivity from either [1-14C]acetate or [2-3H]glycerol into individual acyl lipids as measured in individual portions of bacteria. For phosphatidylglycerol an additional peak of incorporation of radioactivity from [2-3H]glycerol was found midway through the cell cycle. In spite of their rather similar endogenous fatty acid compositions, the individual phosphoacylglycerols showed distinctive patterns of incorporation of radioactivity from [1-14C]acetate into their acyl moieties. The discontinuous synthesis of acyl lipids observed in cultures of Rhodopseudomonas sphaeroides synchronized by either stationary-phase cycling or centrifugation selection procedures contrasted with the accumulation of chlorophyll-protein complexes whose amounts were found to increase throughout the cell cycle. The implications of these findings for the control of lipid synthesis in bacterial photosynthetic membranes are discussed.

The sterol content of germinating conidia of the opportunistic pathogenic fungus Aspergillus fumigatus has been correlated with germination phase and sensitivity to polyene antibiotics. The sterol and sterol ester contents of walls did not change during germination. The sterol ester content of membranes and cell sap remained constant during germination, whereas the sterol content increased during the outgrowth of germ tubes. On the basis of differential extraction studies it was concluded that the loss of resistance to polyenes that occurred in the early stages of swelling of conidia during germination was not due to a movement of sterol or sterol ester out of the wall. Radioactive-labelling experiments demonstrated that, although the amounts of conidial wall sterol and sterol ester did not change during germination, they were metabolically active. Changes in the turnover rate of wall and membrane sterol and sterol ester during germination were investigated and their relationship to a possible mechanism for the change from resistance to sensitivity to polyene antibiotics is discussed.

A system for the rapid and relatively synchromous germination of conidia from a clinical isolate of Aspergillus fumigatus is described. The polyene-mediated release of K plus from germinating conidia has been determined. Ungerminated conidia were insensitive to amphotericin B methyl ester (AME) at concentrations greater than 50 mug/ml, but rapidly became sensitive to 1 to 2 mug AME/ml during the intitial stages of germination. These findings have been correlated with minimum inhibitory concentration values obtained in studies of conidial germination and hyphal outgrowth using a variety of growth tests.

The total lipid content of Candida albicans (serotype A: NCPF 3153) exponential-phase mycelial cultures grown in tissue-culture medium 199 (containing 10%, v/v, foetal calf serum) was 29.8 +/- 8 mg (g dry weight)-1 (mean +/- SD). The weight ratios of phospholipid to neutral lipid and phospholipid to non-esterified sterol were 2.6 +/- 0.4 and 24.9 +/- 0.5, respectively. The major phospholipid was phosphatidylcholine with smaller amounts of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and diphosphatidylglycerol; the most abundant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. The major neutral lipids comprised esterified sterol, triacylglycerol and non-esterified fatty acid with a smaller amount of non-esterified sterol. The fatty acid compositions of the three fatty-acid-containing neutral lipids were distinct from each other and the phospholipids. Comparison with previous data on yeast cultures of C. albicans A grown in glucose broth shows that mycelial cultures have a larger lipid content, lower phospholipid to neutral lipid ratio and higher phospholipid to non-esterified sterol ratio. We now show that mycelial cultures were more permeable to a [14C]triazole antifungal antibiotic compared with exponentially growing yeast cultures of several azole-sensitive strains. Taken together these data are consistent with there being a relationship between the phospholipid/non-esterified sterol ratio of a culture and its ability to accumulate a triazole.

Sodium dodecyltriethoxy sulphate (SDTES), either pure or as a component of commercial surfactant mixtures, underwent rapid primary biodegradation by mixed bacterial cultures in OECD screen and river-water die-away tests. Inoculation of [35S]SDTES-containing solutions with OECD screen test media acclimatized to surfactants or their degradation products led to production of various 35S-labelled glycol sulphates and their oxidation products, all known to occur during degradation of [35S]SDTES by pure bacterial isolates. Triethylene glycol monosulphate was the major catabolite together with smaller amounts of di- and monoethylene glycol monosulphates implying, by analogy with pure cultures, that ether-cleavage was the major primary biodegradation step. The oxidation product (carboxylate derivative) of each glycol sulphate was also detected together with metabolites tentatively identified as omega-/beta-oxidation products of the dodecyl chain. Relatively little SO2-4 was liberated directly from SDTES but mixed cultures derived from sewage could metabolize the sulphated glycols to SO2-4. The environmental relevance of these degradation routes was established by following metabolite production from [35S]SDTES in full-scale river-water die-away tests. Triethylene glycol sulphate was formed first, then rapidly oxidized to acetic acid 2-(diethoxy sulphate) which persisted as the major metabolite for 2-3 weeks. Small amounts of sulphated derivatives of di- and monoethylene glycols were also detected during the same period. Very little SO2-4 was formed directly from SDTES but large amounts accompanied the eventual disappearance of glycol sulphate derivatives. None of the 35S-labelled organic metabolites was persistent and, whenever [35S]SDTES was a component of a commercial mixture, all ester sulphate was completely mineralized to 35SO4(2-) within 28 d.

1. The conduction velocities of axons supplying the intertransverse caudal muscles of 8-week-old rats were measured. The distribution of conduction velocities was found to be similar to the more commonly studied hind-limb innervation. 2. In animals in which the intertransverse caudal muscles had been tenotomized at birth, however, the conduction velocities attained by both the sensory and the motor nerves by 8 weeks of age were significantly reduced. 3. This effect is limited to growing animals since tenotomy of the intertransverse caudal muscles for the same period in adults had no effect on axonal conduction velocity. 4. Deafferentation of normal intertransverse caudal muscles during development also significantly reduced the conduction velocities attained by the motor innervation, to the same extent as tenotomy had done. 5. These results are discussed in relation to the role of impulse traffic in the development of neuronal dimensions.

Electrical stimulation of the cut distal end of the posterior articular nerve (p.a.n.) of the cat knee joint resulted in significant extravasation of plasma proteins and erythrocytes into the synovial cavity of the knee. This effect was mediated by group IV afferents (C fibres) since stimulation of p.a.n. suprathreshold for group II or III afferents but subthreshold for group IV afferents did not produce extravasation. Unmyelinated sympathetic efferent fibres in the joint nerve did not contribute to the extravasation and were responsible for a diminution of this response as shown by the enhanced extravasation occurring after adrenergic blockade. Plasma and erythrocyte extravasation was mediated by afferents containing substance P (SP), as demonstrated by the reversible abolition of extravasation when the substance P antagonist (D-Pro4,D-Trp7,9,10)-SP (4-11) was injected into the synovial cavity. In some animals it was observed that electrical stimulation of the cut distal end of p.a.n. in one limb resulted in extravasation in the contralateral knee joint. It is suggested that articular C fibre afferents could make a significant neurogenic contribution to the initiation or maintenance of inflammatory joint disease.

Conventional chest tomography and fibreoptic bronchoscopy were performed in 100 patients with a localised chest abnormality on their chest radiographs who had been referred with a possible diagnosis of bronchial carcinoma. Carcinoma was eventually confirmed in 74 cases and in 26 the lesion proved to be benign. The accuracy of tomography in the overall series was 83%, with a sensitivity of 92% and a specificity of 58%. These figures compare favourably with the results of other imaging techniques used in the diagnosis of bronchial carcinoma. In 50 patients bronchoscopy did not suggest carcinoma and in this group of patients 24 had a carcinoma and 26 a benign lesion. The accuracy of tomography in the bronchoscopy negative patients was 74%, the major source of error being the false positive category. The lesion eventually proved to be benign in 33% of the cases where tomography suggested malignancy, but when the tomograms suggested that the lesion was benign a tumour was found to be present in only 12% of cases.

The chest radiological findings and outcomes of 120 consecutive patients attending the Accident & Emergency Department with anterior chest pain were recorded prospectively to investigate the value of routine chest radiography in their management. Twenty-one patients (17.5%) were excluded because of incomplete information. Thirty-seven radiological abnormalities were identified in 33 (33%) of the remaining 99 chest X-rays. Seventeen of the abnormalities identified in 14 (14%) of the chest X-rays were clinically significant. The casualty officer's interpretation of 70 (70%) of the chest X-rays was correct, but 36 errors were made interpreting the other 29 chest X-rays. Of these errors, 19 were false negative errors, resulting in the mismanagement of two patients and 17 false positive errors, resulting in the mismanagement of four patients. It appears that a routine chest X-ray provides little information of practical value in the management of patients with anterior chest pain attending an Accident & Emergency Department, unless the training of medical students and junior doctors in the interpretation of chest X-rays is improved.

The phagocytosis of Staphylococcus epidermis by rabbit alveolar macrophages was measured in the absence or presence of a series of sensory irritants. The phagocytic index was determined in the presence of the sensory irritant dibenzoxazepine and 10 of its derivatives. All of the compounds tested inhibited phagocytosis to some extent. Dibenzoxazepine was the most potent inhibitor and the 1-chloro and 2-chloro derivatives were the least potent. With all the compounds tested, the inhibition of phagocytosis was dose dependent. The degree of inhibition of phagocytosis was not correlated with either the nature or the position of the substituent groups on the dibenzoxazepine molecule. There was no discernible relationship between the inhibition of phagocytosis and the irritancy of the compounds tested.

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.

When the incubation period of primary isolation plates was extended to 48 h, mucoid strains of Pseudomonas aeruginosa were found in specimens from various infected sites in patients who did not have cystic fibrosis. The 17 mucoid isolates were characterised in terms of mucoid type, pyocin type, and their sensitivity or resistance to seven beta-lactam and two aminoglycoside antibiotics. The carbohydrate, uronic acid (alginate) and protein content of the water-soluble extracellular material of 15 strains was determined. This material was fractionated by ion-exchange chromatography, and the presence of alginate confirmed by the chemical assay of uronic acids and their quantitation by gas-liquid chromatography. Uronic acids were absent from a non-mucoid revertant of one strain. The strains produced alginate with a high content of mannuronic acid and substituted with O-acetyl groups. By proton nuclear magnetic resonance (1H-nmr) analysis the alginate from three strains was shown to lack polyguluronate blocks in its structure. These properties are also found in the alginate of mucoid P. aeruginosa strains from patients with cystic fibrosis.