Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Mechanisms of Polymyxin Resistance.

Polymyxin antibiotics are increasingly being used as last-line therapeutic options against a number of multidrug resistant bacteria. These antibiotics show strong bactericidal activity against a range of Gram-negative bacteria, but with the increased use of these antibiotics resistant strains are emerging at an alarming rate. Furthermore, some Gram-negative species, such as Neisseria meningitidis, Proteus mirabilis and Burkholderia spp., are intrinsically resistant to the action of polymyxins. Most identified polymyxin resistance mechanisms in Gram-negative bacteria involve changes to the lipopolysaccharide (LPS) structure, as polymyxins initially interact with the negatively charged lipid A component of LPS. The controlled addition of positively charged residues such as 4-amino-L-arabinose, phosphoethanolamine and/or galactosamine to LPS results in a reduced negative charge on the bacterial surface and therefore reduced interaction between the polymyxin and the LPS. Polymyxin resistant species produce LPS that intrinsically contains one or more of these additions. While the genes necessary for most of these additions are chromosomally encoded, plasmid-borne phosphoethanolamine transferases (mcr-1 to mcr-8) have recently been identified and these plasmids threaten to increase the rate of dissemination of clinically relevant colistin resistance. Uniquely, Acinetobacter baumannii can also become highly resistant to polymyxins via spontaneous mutations in the lipid A biosynthesis genes lpxA, lpxC or lpxD such that they produce no LPS or lipid A. A range of other non-LPS-dependent polymyxin resistance mechanisms has also been identified in bacteria, but these generally result in only low levels of resistance. These include increased anionic capsular polysaccharide production in Klebsiella pneumoniae, expression of efflux systems such as MtrCDE in N. meningitidis, and altered expression of outer membrane proteins in a small number of species.

Emergence of High-Level Colistin Resistance in an Acinetobacter baumannii Clinical Isolate Mediated by Inactivation of the Global Regulator H-NS.

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium Acinetobacter baumannii However, colistin-resistant A. baumannii isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 µg/ml and 128 µg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element ISAba125 within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an hns deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of hns We also provided 6009-2 with an intact copy of hns and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant hns mutant 6009-2. Importantly, the expression of eptA, encoding a second lipid A-specific pEtN transferase but not pmrC, was increased in the hns mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection.

Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiellaeffector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3×FLAG or MceA tagged with 2×hemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.

Does a mandatory rural exposure change medical students' rural practice intent?

While rural background has consistently been the strongest predictor of rural practice, the Australian medical schools have a vital role to play in changing rural intention. Research on rural exposure during medical training has to date resulted in inconsistent findings. The purpose of this paper is to examine what occurs during a short conscripted rural exposure. A post-test self-report questionnaire evaluating the University of Queensland Rural Medicine Rotation eight-week clinical rotation was completed by 1609 domestic medical students from 2007 to 2011. The most effective single learning outcome was students who strongly agree that the rural rotation experience had increased their appreciation of the greater depth of clinical responsibility inherent in rural practice. These students were more likely to indicate that their intention to practise rurally had been positively rather than negatively influenced and more like to indicate they were very encouraged by the experience rather than merely encouraged to practise rurally. The most powerful group of learning outcomes, in positively changing rural intention from a short mandated exposure were aligned with developing an understanding of the context of rural medical practise. These contribute to our understanding of the role rural exposure.

Coxiella burnetii: turning hostility into a home.

Coxiella burnetii, the causative agent of the human disease Q fever, is a unique intracellular bacterial pathogen. Coxiella replicates to high numbers within a pathogen-derived lysosome-like vacuole, thriving within a low pH, highly proteolytic and oxidative environment. In 2009, researchers developed means to axenically culture Coxiella paving the way for the development of tools to genetically manipulate the organism. These advances have revolutionized our capacity to examine the pathogenesis of Coxiella. In recent years, targeted and random mutant strains have been used to demonstrate that the Dot/Icm type IV secretion system is essential for intracellular replication of Coxiella. Current research is focused towards understanding the unique cohort of over 130 effector proteins that are translocated into the host cell. Mutagenesis screens have been employed to identify effectors that play important roles for the biogenesis of the Coxiella-containing vacuole and intracellular replication of Coxiella. A surprisingly high number of effector mutants demonstrate significant intracellular growth defects, and future studies on the molecular function of these effectors will provide great insight into the pathogenesis of Coxiella. Already, this expanse of new data implicates many eukaryotic processes that are targeted by the arsenal of Coxiella effectors including autophagy, apoptosis and vesicular trafficking.

Using evaluation to improve medical student rural experience.

Objective The aim of this evaluation was to see whether interventions implemented to improve the Rural Medicine Rotation made this a more effective rural medical education experience. Multiple interventions targeting the student experience, lecturers and preceptors were implemented. Methods A quasi-experimental design using pre- and post-measures was used. The participants were all University of Queensland, School of Medicine, Rural Medicine Rotation students who completed the 2009 and 2010 rural medicine rotation evaluations. There were 769 students, with an 84% response rate in 2009 and an 80% response rate in 2010. In addition, all the 25 program preceptors who were visited in 2009 and the 34 who were visited in 2010 participated in the study. Results The implementation of interventions resulted in significant improvement in three outcome measures, namely teaching effectiveness, provision of an environment supportive of learning in a rural/remote setting and opportunities for professional growth. Two of the three other outcome measures - ensuring a safe clinical placement and opportunities for procedural skills experience and development - were very positively evaluated in both 2009 and 2010. Conclusions The interventions contributed to a more effective rural medical education experience, providing students with the opportunity to develop skills and knowledge relevant for rural medicine and to gain an understanding of the context in which rural medicine is practiced. What is known about the topic? Many Australian medical schools offer students rural-based educational opportunities based on the premise that placing medical students in a rural setting may ultimately lead to them choosing careers in rural medicine. However, there is a paucity of evidence on the factors that are considered necessary for medical students to gain a positive rural experience of short conscripted rural placements. What does the paper add? This paper identifies successful interventions to the rotation and placements that provide a positive experience of the rural clinical placement for students. These interventions occurred within an ongoing evaluation program embedded in the rotation. What are the implications for practitioners? Through ongoing evaluation, interventions can be selected and implemented that succeed in contributing to students having a positive rural clinical placement experience. This paper demonstrates how an embedded continuous improvement program serves to provide direction for ongoing modifications.

Lipopolysaccharide-deficient Acinetobacter baumannii shows altered signaling through host Toll-like receptors and increased susceptibility to the host antimicrobial peptide LL-37.

Infections caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious global health problem. We have shown previously that A. baumannii can become resistant to the last-line antibiotic colistin via the loss of lipopolysaccharide (LPS), including the lipid A anchor, from the outer membrane (J. H. Moffatt, M. Harper, P. Harrison, J. D. Hale, E. Vinogradov, T. Seemann, R. Henry, B. Crane, F. St. Michael, A. D. Cox, B. Adler, R. L. Nation, J. Li, and J. D. Boyce, Antimicrob. Agents Chemother. 54:4971-4977, 2010). Here, we show how these LPS-deficient bacteria interact with components of the host innate immune system. LPS-deficient A. baumannii stimulated 2- to 4-fold lower levels of NF-κB activation and tumor necrosis factor alpha (TNF-α) secretion from immortalized murine macrophages, but it still elicited low levels of TNF-α secretion via a Toll-like receptor 2-dependent mechanism. Furthermore, we show that while LPS-deficient A. baumannii was not altered in its resistance to human serum, it showed increased susceptibility to the human antimicrobial peptide LL-37. Thus, LPS-deficient, colistin-resistant A. baumannii shows significantly altered activation of the host innate immune inflammatory response.

Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-ß-1,6-N-acetylglucosamine.

We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-ß-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

Physician in practice clinic: educating GPs in endocrinology through specialist-outreach.

UNLABELLED: The escalation in the incidence of diabetes mellitus type 2 requires innovative approaches to manage the increasing burden of service provision, a particularly challenging issue for rural communities. Whereas shared care through specialist outreach clinics is a traditional approach to the management of chronic disease, the results on effectiveness are mixed. Where there is a joint consultation with both the General Practitioner (GP) and specialist present during the patient consultation, benefits are reported; however, this model of specialist outreach is uncommon when compared with the more typical model where a specialist sees the patient alone but at the GP's rooms, and later communicates with the GP. The explicit long-term goal of the Physician in Practice Clinic, which emulates the joint consultation model, is improved patient outcomes through better educated and more confident GPs, easier service access for patients and reduced waiting lists. The education of GPs in endocrinology, an early goal, is the focus of the article. METHODS: Fifteen GPs were sampled purposively on sex, rural/regional location, place of training, practice size and length of time practising locally. Semi-structured interviews were conducted, transcribed then thematically analysed. RESULTS: General practitioners reported substantial educational benefits. One aspect is the face-to-face contract with the endocrinologist which promotes an interactive learning process. All GPs reported that they acquired new knowledge. An important aspect of this new knowledge is that it could be used quickly, often immediately, and also used in the longer term when generalised for use with other patients. A follow-on effect from the new knowledge and its short- and long-term application was an increase in professional confidence. A benefit with the potential for a long-term effect was improved relationships between the GPs and specialist, and the GPs reporting that they were making fewer referrals. The greatest benefits reported by those in small practices were the interactive learning and being able to generalise the new knowledge to other patients. For rural doctors most benefit was from the interactive learning, whereas for regional doctors it was increased confidence. Australian trained doctors reported mostly the benefits of being able to use the knowledge quickly and the interactive learning. By contrast, doctors not trained in Australia favoured the increased confidence and the generalisability of the new knowledge. Those who had practised locally for up to 10 years benefited most from the new knowledge and the increased confidence, and females benefited most from increased confidence and receiving new knowledge. CONCLUSIONS: From the GPs' perspective, the goal of creating better educated and more confident GPs in endocrinology in this rural/regional setting was achieved. Therefore this easily replicated but novel approach to specialist outreach has the potential to improve health outcomes in chronic disease in rural communities. In addition, a more tailored approach to shaping the Clinics based on the socio-demographic categories reported here could have additional short- and long-term benefits.

Epidermal Microbiomes of Leopard Sharks (Triakis semifasciata) Are Consistent across Captive and Wild Environments.

Characterizations of shark-microbe systems in wild environments have outlined patterns of species-specific microbiomes; however, whether captivity affects these trends has yet to be determined. We used high-throughput shotgun sequencing to assess the epidermal microbiome belonging to leopard sharks (Triakis semifasciata) in captive (Birch Aquarium, La Jolla California born and held permanently in captivity), semi-captive (held in captivity for <1 year in duration and scheduled for release; Scripps Institute of Oceanography, San Diego, CA, USA) and wild environments (Moss Landing and La Jolla, CA, USA). Here, we report captive environments do not drive epidermal microbiome compositions of T. semifasciata to significantly diverge from wild counterparts as life-long captive sharks maintain a species-specific epidermal microbiome resembling those associated with semi-captive and wild populations. Major taxonomic composition shifts observed were inverse changes of top taxonomic contributors across captive duration, specifically an increase of Pseudoalteromonadaceae and consequent decrease of Pseudomonadaceae relative abundance as T. semifasciata increased duration in captive conditions. Moreover, we show captivity did not lead to significant losses in microbial α-diversity of shark epidermal communities. Finally, we present a novel association between T. semifasciata and the Muricauda genus as Metagenomes associated genomes revealed a consistent relationship across captive, semi-captive, and wild populations. Since changes in microbial communities is often associated with poor health outcomes, our report illustrates that epidermally associated microbes belonging to T. semifasciata are not suffering detrimental impacts from long or short-term captivity. Therefore, conservation programs which house sharks in aquariums are providing a healthy environment for the organisms on display. Our findings also expand on current understanding of shark epidermal microbiomes, explore the effects of ecologically different scenarios on benthic shark microbe associations, and highlight novel associations that are consistent across captive gradients.

Insertion sequence ISAba11 is involved in colistin resistance and loss of lipopolysaccharide in Acinetobacter baumannii.

Infections caused by Acinetobacter baumannii are of increasing concern, largely due to the multidrug resistance of many strains. Here we show that insertion sequence ISAba11 movement can result in inactivation of the A. baumannii lipid A biosynthesis genes lpxA and lpxC, resulting in the complete loss of lipopolysaccharide production and high-level colistin resistance.

Different surface charge of colistin-susceptible and -resistant Acinetobacter baumannii cells measured with zeta potential as a function of growth phase and colistin treatment.

OBJECTIVES: electrostatic forces mediate the initial interaction between cationic colistin and Gram-negative bacterial cells. Lipopolysaccharide (LPS) loss mediates colistin resistance in some A. baumannii strains. Our aim was to determine the surface charge of colistin-susceptible and -resistant A. baumannii as a function of growth phase and in response to polymyxin treatment. METHODS: the zeta potential of A. baumannii ATCC 19606 and 10 clinical multidrug-resistant strains (MICs 0.5-2 mg/L) was assessed. Colistin-resistant derivatives (MIC >128 mg/L) of wild-type strains were selected in the presence of 10 mg/L colistin, including the LPS-deficient lpxA mutant, ATCC 19606R. To determine the contribution of LPS to surface charge, two complemented ATCC 19606R derivatives were examined, namely ATCC 19606R + lpxA (containing an intact lpxA gene) and ATCC 19606R + V (containing empty vector). Investigations were conducted as a function of growth phase and polymyxin treatment (1, 4 and 8 mg/L). RESULTS: wild-type cells exhibited a greater negative charge (-60.5  ±â€Š 2.36 to -26.2  ±â€Š 2.56 mV) thancolistin-resistant cells (-49.2  ±â€Š 3.09 to -19.1  ±  2.80 mV) at mid-log phase (ANOVA, P  <  0.05). Opposing growth-phase trends were observed for both phenotypes: wild-type cells displayed reduced negative charge and colistin-resistant cells displayed increased negative charge at stationary compared with mid-logarithmic phase. Polymyxin exposure resulted in a concentration-dependent increase in zeta potential. Examination of ATCC 19606R and complemented strains supported the importance of LPS in determining surface charge, suggesting a potential mechanism of colistin resistance. CONCLUSIONS: zeta potential differences between A. baumannii phenotypes probably reflect compositional outer-membrane variations that impact the electrostatic component of colistin activity.

Barriers to the up-take of telemedicine in Australia--a view from providers.

INTRODUCTION: The continued poorer health status of rural and remote Australians when compared with their urban counterparts is cause for concern. The use of advanced technology to improve access to health care has the potential to assist in addressing this problem. Telemedicine is one example of such technology which has advanced rapidly in its capacity to increase access to healthcare services or provide previously unavailable services. The important anticipated benefits of greater access to healthcare services are improved health outcomes and more cost-effective delivery. METHODS: A national study was conducted to investigate the current perceived use and usefulness of telemedicine from the perspective of users and providers, and their views on how telemedicine could be expanded in Australia. In one component of this national study, the expert opinion of experienced providers of telemedicine services was elicited using a Grounded Theory approach and using semi-structured interviews which were analysed thematically. This article reports on the barriers to the up-take of telemedicine identified by this sub-sample. RESULTS: The primary barriers identified were: funding; time; infrastructure; equipment; skills; and preference for the traditional approach. While funding is a well-known barrier to the up-take of telemedicine, the extra time required for a telemedicine consultation has particular implications for the workload of rural doctors. The comparatively poor internet access available in rural Australia combines with difficulties accessing some items such as a computer, to make equipment an issue. Even though lack of equipment skills was identified as a barrier, the providers in this study reported that rural doctors are adept at using the telephone/teleconferencing and facsimile. A preference for a traditional approach can reflect a lack of interest in learning computer skills or difficulty acquiring this skill set. CONCLUSIONS: These results raise issues in the domains of policy, funding priorities, and education and training. This indicates an inter-related set of challenges that would require a targeted multifaceted approach to address. The results suggest that not using telemedicine is, in the current climate, a rational response--it is quicker, easier and more cost-effective not to use telemedicine.

Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production.

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.

The reported benefits of telehealth for rural Australians.

OBJECTIVE: A literature review was conducted to identify the reported benefits attributed to telehealth for people living and professionals working in rural and remote areas of Australia. DATA SOURCES: Scopus and relevant journals and websites were searched using the terms: telemedicine, telehealth, telepsychiatry, teledermatology, teleradiology, Australia, and each state and territory. Publications since 1998 were included. STUDY SELECTION: The initial search resulted in 176 articles, which was reduced to 143 when research reporting on Australian rural, regional or remote populations was selected. DATA SYNTHESIS: A narrative review was conducted using an existing 'benefits' framework. Patients are reported to have benefited from: lower costs and reduced inconvenience while accessing specialist health services; improved access to services and improved quality of clinical services. Health professionals are reported to have benefits from access to continuing education and professional development; provision of enhanced local services; experiential learning, networking and collaboration. DISCUSSION: Rural Australians have reportedly benefited from telehealth. The reported improved access and quality of clinical care available to rural Australians through telemedicine and telehealth may contribute to decreasing the urban-rural health disparities. The reported professional development opportunities and support from specialist through the use of telehealth may contribute to improved rural medical workforces recruitment and retention.

Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA.

Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA(-) mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome.