bluuebunny/crossref_metadata_2025 huggingface dataset:
Ecophysiology matters: linking inorganic carbon acquisition to ecological preference in four species of microalgae (Chlorophyceae)
Authors: Lachmann Sabrina C. , Maberly Stephen C. , Spijkerman Elly
Published: September 2016

The effect ofCO2supply is likely to play an important role in algal ecology. Since inorganic carbon (Ci) acquisition strategies are very diverse among microalgae and Ciavailability varies greatly within and among habitats, we hypothesized that Ciacquisition depends on the pH of their preferred natural environment (adaptation) and that the efficiency of Ciuptake is affected byCO2availability (acclimation). To test this, four species of green algae originating from different habitats were studied. The pH‐drift and Ciuptake kinetic experiments were used to characterize Ciacquisition strategies and their ability to acclimate tohighandlowCO2conditions andhighand lowpHwas evaluated. Results from pH drift experiments revealed that the acidophile and acidotolerantChlamydomonasspecies were mainly restricted toCO2, whereas the two neutrophiles were efficient bicarbonate users.CO2compensation points inlowCO2‐acclimated cultures ranged between 0.6 and 1.4 μMCO2and acclimation to different culture pH andCO2conditions suggested thatCO2concentrating mechanisms were present in most species. HighCO2acclimated cultures adapted rapidly to lowCO2condition during pH‐drifts. Ciuptake kinetics at different pH values showed that the affinity for Ciwas largely influenced by external pH, being highest under conditions whereCO2dominated the Cipool. In conclusion, Ciacquisition was highly variable among four species of green algae and linked to growth pH preference, suggesting that there is a connection between Ciacquisition and ecological distribution.
In vivo electrical application on titanium implants stimulating bone formation
Authors: Bins‐Ely L. M. , Cordero E. B. , Souza J. C. M. , Teughels W. , Benfatti C. A. M. , Magini R. S.
Published: September 2016

Background and Objective

The aim of the present in vivo study was to measure the bone implant contact area after electrical stimulation of dental implants.

Material and Methods

Ninety titanium dental implants (6 mm × 11.5 mm) with a smooth surface were placed in six male Beagle dogs and then the implant‐bone interfaces was assessed by histological analyses after 7 and 15 d. The 12‐mo‐old dogs, with a weight of 15 kg, were randomly divided into two groups based on the duration of bone healing: 7 and 15 d. Also, implants were divided into three groups based on electrical stimulation: group A, 10 μA; group B, 20 μA; and group C, control group. The electrical current was applied by an electrical device coupled to the implant connection.

Results

After 7 d of electrical stimulation, no statistical differences in bone–implant interface contact area were observed. However, a significantly higher bone–implant interface contact area was recorded for group B than for groups A and C (p < 0.01) after 15 d. No statistical difference was observed between groups A and C (p > 0.05).

Conclusion

The electrical stimulation of dental implants can generate a larger area of bone–implant interface contact as a result of bone formation. Factors such as different electrical current intensity and duration should be studied in further work to clarify the potential of this method.
Combined effects of starvation and butyrate on autophagy‐dependent gingival epithelial cell death
Authors: Evans M. , Murofushi T. , Tsuda H. , Mikami Y. , Zhao N. , Ochiai K. , Kurita‐Ochiai T. , Yamamoto M. , Otsuka K. , Suzuki N.
Published: September 2016

Background and Objective

Bacteria in the dental biofilm surrounding marginal gingival grooves cause periodontal diseases. Numerous bacteria within the biofilm consume nutrients from the gingival crevicular fluid. Furthermore, some gram‐negative bacteria in mature dental biofilms produce butyrate. Thus, gingival epithelial cells in close proximity to mature dental biofilms are at risk of both starvation and exposure to butyrate. In the present study, we determined the combined effects of starvation and butyrate exposure on gingival epithelial cell death and the underlying mechanisms.

Material and Methods

The Ca9‐22 cell line was used as an in vitro counterpart of gingival epithelial cells. Cell death was measured as the amount of total DNA in the dead cells using SYTOX Green dye, which penetrates through membranes of dead cells and emits fluorescence when it intercalates into double‐stranded DNA. AMP‐activated protein kinase (AMPK) activity, the amount of autophagy, and acetylation of histone H3 were determined using western blot. Gene expression levels of microtubule‐associated protein 1 light chain 3b (lc3b) were determined using quantitative reverse transcription–polymerase chain reaction.

Results

Butyrate‐induced cell death occurred in a dose‐dependent manner whether cells were starved or fed. However, the induction of cell death was two to four times higher when cells were placed under starvation conditions compared to when they were fed. Moreover, both starvation and butyrate exposure induced AMPK activity and autophagy. While AMPK inactivation resulted in decreased autophagy and butyrate‐induced cell death under conditions of starvation, AMPK activation resulted in butyrate‐induced cell death when cells were fed. Combined with the results of our previous report, which demonstrated butyrate‐induced autophagy‐dependent cell death, the results of this study suggest that the combination of starvation and butyrate exposure activates AMPK inducing autophagy and subsequent cell death. Notably, this combination markedly induced LC3B production and the induction was attenuated by AMPK inhibition. LC3B knockdown, in turn, significantly decreased butyrate‐induced cell death. Therefore, AMPK‐dependent LC3B induction apparently plays an important role in butyrate‐induced cell death. There was a lack of correspondence between the levels of AMPK activation and LC3B induction; this may reflect the histone deacetylase‐inhibitory capacity of butyrate on histone proteins.

Conclusion

Taken together, starvation and butyrate exposure promote autophagy via AMPK signaling, while the histone deacetylase‐inhibitory effects of butyrate alter chromatin to transcriptionally active state, resulting in strong LC3B induction and subsequent cell death. These findings may help improve the understanding of the cellular processes underlying periodontal disease initiation.
Soft tissue sarcoma in the dog – part 1: a current review
Authors: Bray J. P.
Published: September 2016

Soft tissue sarcomas are derived from tissues of mesenchymal origin. Although local recurrence following surgical resection is the characteristic challenge in their management, 40% dogs with high‐grade tumours may also develop metastatic disease, despite successful local control. Soft tissue sarcoma is a complex disease and there are many uncertainties regarding the biology and optimal clinical management. There are currently no diagnostic tests that can reliably predict the amount of surgical margin required for a particular tumour, so there can be a mismatch between treatment and disease. Historically, the tendency has been to always recommend wide excision margins but this is not fully supported by recent evidence. A selection bias for less aggressive soft tissue sarcomas in primary care practice can account for good outcomes that are achieved despite narrow surgical excision margins. On the other hand, inappropriately conservative treatment will adversely affect outcomes for patients with more aggressive disease. This review provides an update on the current understanding of management of canine soft tissue sarcomas.
Single‐molecule dynamics of the molecular chaperone trigger factor in living cells
Authors: Yang Feng , Chen Tai‐Yen , Krzemiński Łukasz , Santiago Ace George , Jung Won , Chen Peng
Published: September 2016

In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to assist the folding of nascent polypeptides. Studies in vitro have provided insights into the function and mechanism of TF. Much is to be elucidated, however, about how TF functions in vivo. Here, we use single‐molecule tracking, in combination with genetic manipulations, to study the dynamics and function of TF in living E. coli cells. We find that TF, besides interacting with the 70S ribosome, may also bind to ribosomal subunits and form TF‐polypeptide complexes that may include DnaK/DnaJ proteins. The TF‐70S ribosome interactions are highly dynamic inside cells, with an average residence time of ∼0.2 s. Our results confirm that the signal recognition particle weakens TF's interaction with the 70S ribosome, and further identify that this weakening mainly results from a change in TF's binding to the 70S ribosome, rather than its unbinding. Moreover, using photoconvertible bimolecular fluorescence complementation, we selectively probe TF2 dimers in the cell and show that TF2 does not bind to the 70S ribosome but is involved in the post‐translational interactions with polypeptides. These findings contribute to the fundamental understanding of molecular chaperones in assisting protein folding in living cells.
Antifungal susceptibility testing of Malassezia yeast: comparison of two different methodologies
Authors: Rojas Florencia D. , Córdoba Susana B. , de los Ángeles Sosa María , Zalazar Laura C. , Fernández Mariana S. , Cattana María E. , Alegre Liliana R. , Carrillo‐Muñoz Alfonso J. , Giusiano Gustavo E.
Published: September 2016

All Malassezia species are lipophilic; thus, modifications are required in susceptibility testing methods to ensure their growth. Antifungal susceptibility of Malassezia species using agar and broth dilution methods has been studied. Currently, few tests using disc diffusion methods are being performed. The aim was to evaluate the in vitro susceptibility of Malassezia yeast against antifungal agents using broth microdilution and disc diffusion methods, then to compare both methodologies. Fifty Malassezia isolates were studied. Microdilution method was performed as described in reference document and agar diffusion test was performed using antifungal tablets and discs. To support growth, culture media were supplemented. To correlate methods, linear regression analysis and categorical agreement was determined. The strongest linear association was observed for fluconazole and miconazole. The highest agreement between both methods was observed for itraconazole and voriconazole and the lowest for amphotericin B and fluconazole. Although modifications made to disc diffusion method allowed to obtain susceptibility data for Malassezia yeast, variables cannot be associated through a linear correlation model, indicating that inhibition zone values cannot predict MIC value. According to the results, disc diffusion assay may not represent an alternative to determine antifungal susceptibility of Malassezia yeast.
Beyond perceived ability: the contribution of psychosocial factors to academic performance
Authors: Dixson Dante D. , Worrell Frank C. , Olszewski‐Kubilius Paula , Subotnik Rena F.
Published: September 2016

In this study, we examined the contributions of grit, hope, and academic self‐efficacy to academic achievement in a sample of 609 academically talented students ranging in age from 10 to 18 years. Our hypothesis was that these variables would predict academic achievement above and beyond the contributions of demographic variables and perceived ability. After establishing the reliability and structural validity of scores for the three constructs in this sample, regression analyses indicated that hope and academic self‐efficacy were predictors of perceived ability and that academic self‐efficacy was the most robust predictor of academic achievement. Grit was not a meaningful predictor of either perceived ability or academic achievement. We concluded that psychosocial variables can contribute to performance in talented students, but research is needed to establish which psychosocial variables are the most useful.
Feeling lonely in an unfamiliar place: older people’s experiences of life close to death in a nursing home
Authors: Österlind Jane , Ternestedt Britt‐Marie , Hansebo Görel , Hellström Ingrid
Published: September 2016

Aim

The aim of the study was to deepen the understanding of how older persons living in a nursing home experience life close to death.

Background

A move to and a life in a nursing home while being close to death is a reality for many older people in Sweden. Being able to express thoughts and feelings about death has been described as both crucial for sustaining personhood as well as for establishing a meaningful existence at the end of life. Important are the experiences of older people living in nursing homes who are approaching death.

Method

Six older people were interviewed on one to four occasions. A total of 16 interviews were conducted with the participants. An interpretative approach was chosen.

Findings

The main interpretation, Feeling lonely in an unfamiliar place, is based on three themes (i) Waiting for death, with the subthemes death as a release and thinking of oneself as dead; (ii) Subordinate oneself to values and norms of the staff, with the subthemes feeling offended and feeling trapped; and (iii) Keep the courage up. The older people's lives were characterised by feelings of aloneness in an unfamiliar place which contributed to a sense of existential loneliness. They experienced few opportunities to discuss their thoughts of life and death, including preparations for passing away.

Conclusion and implication for practice

It is of importance for professionals to be able to meet older people as they are and respect them as human beings in their transitions, before, during and after the move to a nursing home. It is important to find ways to support older people's wellbeing and identity near death.
Atlantoaxial Joint Interlocking Following Type II Odontoid Fracture Associated with Posterolateral Atlantoaxial Dislocation: a Case Report and Review of Published Reports
Authors: He Deng‐wei , Huang Wen‐jun , Sheng Xiao‐yong , Wu Li‐jun , Fan Shun‐wu
Published: September 2016

A rare case of atlantoaxial lateral mass joint interlocking secondary to traumatic posterolateral C1,2 complete dislocation associated with type II odontoid fracture is herein reported and the impact of atlantoaxial joint interlocking on fracture reduction discussed. A 72‐year‐old man presented with traumatic atlantoaxial lateral mass joint interlocking without spinal cord signal change, the diagnosis being confirmed by radiography and 3‐D reconstruction digital anatomy. Posterior internal fixation was performed after failure to achieve closed reduction by skull traction. After many surgical attempts at setting had failed because of interlocking of the lateral mass joints, reduction was achieved by compressing the posterior parts of the atlantal and axial screws. Odontoid bone union and C1,2 posterior bone graft fusion were eventually obtained by the 12‐month follow‐up. The patient had a complete neurological recovery with no residual neck pain or radiculopathy.
Clinical Evaluation and Gait Characteristics before and after Total Knee Arthroplasty Based on a Portable Gait Analyzer
Authors: Zhang Hao‐hua , Yan Song‐hua , Fang Chen , Guo Xin‐yuan , Zhang Kuan
Published: September 2016

Objective

To evaluate the effects of surgery and rehabilitation on patients undergoing total knee arthroplasty (TKA).

Methods

Twelve patients and 12 healthy controls were enrolled and their clinical scores evaluated by a doctor. Gait data, including walking velocity, stride length, single support time, foot fall and swing power, were collected using a portable gait analyzer from 12 patients before and 6 weeks and 6 months after surgery and from 12 healthy controls. The gait data and clinical scores at selected time points were compared and correlations between gait characteristics and clinical scores assessed.

Results

Clinical knee and knee function scores increased significantly from before surgery to 6 weeks to 6 months after surgery (P < 0.001). The only significant differences identified were for single support time on the diseased side between before surgery and 6 months after surgery (P = 0.031) and for foot fall with the diseased side between 6 weeks and 6 months after surgery (P = 0.016). Foot fall and speed of the healthy or diseased sides were significantly different in patients at all time points from those of the healthy subjects (P < 0.05). Single support time on the diseased side was significantly different 6 months after surgery (P = 0.035) in patients than in healthy controls. Single support time on the healthy side before surgery was significantly different from that of healthy controls (P = 0.048) and 6 weeks after surgery (P = 0.042). Stride lengths differed significantly between patients and healthy subjects before surgery (healthy side: P = 0.007; diseased side: P = 0.008) and 6 weeks after surgery (healthy side: P = 0.001; diseased side: P = 0.001), but were not different at 6 months after surgery (healthy side: P = 0.088; diseased side: P = 0.077). The only significant correlations identified were between single support time with the diseased side of patients and their knee (r = 0.43, P = 0.032) and knee function scores (r = 0.493, P = 0.012).

Conclusions

A portable gait analyzer appears to be suitable for evaluating the effects of TKA. Single support time on the diseased side may be a sensitive quantitative index for determining the effect of TKA and rehabilitation.
Comorbid disorders associated with asthma in children in Germany – National analysis of pediatric primary care data
Authors: Jacob Louis , Keil Thomas , Kostev Karel
Published: September 2016

Background

Using routine healthcare data, the goal of this study was to examine the association between asthma and comorbid disorders in children in Germany.

Methods

Patients with a diagnosis of asthma documented by a German pediatrician in 2015 were identified. Demographic data included age and gender. Patients younger than 6 and older than 17 and individuals followed for less than a year before index date were excluded. Each patient was matched for age, sex, and physician with an asthma‐free control. A total of 34,305 cases and 34,305 controls were available for analysis. There were three different age groups: 6–9 years, 10–13 years, and 14–17 years. Several disorders known to be associated with asthma or found in more than 1% of children and adolescents were also determined on the basis of primary care diagnoses. A multivariate logistic regression model was used to estimate the association between asthma and comorbid disorders.

Results

The mean age was 10.9 years, and 61.6% of the subjects were boys. The most common diseases found in the three age groups were vasomotor and allergic rhinitis, chronic bronchitis, chronic rhinitis, pneumonia, and atopic dermatitis. These five comorbid disorders were associated with asthma to a significant extent.

Conclusions

Overall, this study, based on primary healthcare data, found a considerably high prevalence of several comorbid diseases in German children with asthma.
Production of avocado trees infected with Phytophthora cinnamomi under different management regimes
Authors: Ramírez‐Gil J. G. , Castañeda‐Sánchez D. A. , Morales‐Osorio J. G.
Published: September 2016

Avocado root rot is the most important disease of this fruit crop worldwide. This pathology may be caused by several biotic and abiotic agents, with the oomycete Phytophthora cinnamomi being the pathogen more frequently associated with poor phytosanitary conditions. There are disease control methods available that can reduce disease severity and allow plants to recover; however, they are not consistently and promptly applied. In addition, only chemical products are used by farmers as the preferred management method. This research aimed to evaluate different root rot management strategies in a commercial orchard. Data suggest that individual control methods are not as effective as when they are applied in combination, as in the T8 treatment (metalaxyl + mancozeb applied in drench; injection of potassium phosphite to each plant stem; potassium silicate applied in drench; addition of a layer of organic mulch and incorporation of 10 kg of composted substrate, both applied to the ground around the base of each tree). Using this strategy, the area under the disease progress curve for the avocado root rot was reduced by up to 68.6%, and the extra‐quality avocado fruit class increased by as much as 44% compared to the diseased control plants (T0) (P < 0.01). With the combined treatment T8, farm income showed a 9.5‐fold increase, probably due to an increase in the percentage of viable roots by up to 9.4‐fold, which would have improved nutrient and water uptake.
The Role of Rituals in Warfare during the Neo‐Assyrian Period
Authors: Melville Sarah C.
Published: September 2016

In imperial Assyria during the first millennium BCE, ritual pervaded every aspect of life. As the link between the divine realm and the earthly one, the king's primary duty was to discover the gods' will and enact it. To this end, an array of ritual experts in the art of reading and reacting to divine signs aided the king. Due to the risk involved, warfare required particular attention from the experts during every phase of operations. Based on evidence from cuneiform sources (ritual texts, royal inscriptions, and letters) and monumental art (narrative sculptured reliefs), this article focuses on how ritual activity at each campaign stage affected different audiences, including the enemy, the king's officials, and the Assyrian army.
Computer-Controlled Eucapnic Voluntary Hyperpnea Challenge Method
Authors: Al-Jumaily Ahmed M. , Wang Lulu
Published: September 2016

Eucapnic voluntary hyperpnea (EVH) challenge is a well-established and sensitive method of determining the degree of bronchoconstriction at hospitals and clinics. This paper presents the development of a computer-controlled system for assessing exercise-induced bronchoconstriction (EIB) in humans including a computer graphic user interface (GUI) to control a low-pressure demand valve for better efficiency. GUI is designed to monitor the severity of acute lung airway narrowing using a matlab software and to present the measurement data into a simple user-friendly program consisting of patient information, EVH test analysis, and detection of exercise-induced asthma (EIA) and EIB. The proposed system is assessed using human subjects. Typical outputs from this system showed that for a female participant, a 20.25% and a 15.61% decrease from baseline in her forced expiratory volume in 1 s (FEVl) after 10 and 15 min of the challenge commencement, respectively. Her actual expiratory flow rate (45.833 L/m) and actual total volume of gas respired (275 L) were smaller than the target values. This system widens the usage of EVH challenge in medical areas, and the GUI may serve as a new clinical computer-aided diagnostic tool to help healthcare professionals noninvasively monitor the severity of asthma, EIA, and EIB.
Imprecise Probabilities in Fatigue Reliability Assessment of Hydraulic Turbines
Authors: Berdai Mounia , Tahan Antoine , Gagnon Martin
Published: September 2016

Risk analyses are often performed for economic reasons and safety purposes. In some cases, these studies are biased by epistemic uncertainties due to the lack of information and knowledge, which justifies the need for expert opinion. In such cases, experts can follow different approaches for the elicitation of epistemic data, using probabilistic or imprecise theories. But how do these theories affect the reliability calculation? What are the influences of using a mixture of theories in a multivariable system with a nonexplicit limit model? To answer these questions, we propose an approach for the comparison of these theories, which was performed based on a reliability model using the first-order reliability method (FORM) approach and having the Kitagawa–Takahashi diagram as limit state. We also propose an approach, appropriate to this model, to extend the reliability calculation to variables derived from imprecise probabilities. For the chosen reliability model, obtained results show that there is a certain homogeneity among the considered theories. The study also concludes that priority should be given to expert opinions formulated according to unbounded distributions, in order to achieve better reliability calculation accuracy.
Effects of Averaging the Heat Transfer Coefficient on Predicted Material Temperature and Its Gradient
Authors: Lee Chien-Shing , Shih Tom I-P. , Bryden Kenneth Mark
Published: September 2016

The heat transfer coefficient (HTC) is often averaged spatially when designing heat exchangers. Since the HTC could vary appreciably about a heat transfer enhancement feature such as a pin fin or a rib, it is of interest to understand the effects of averaging the HTC on design. This computational study examines those effects via a unit problem—a flat plate of thickness H and length L, where L represents the distance between pin-fins or ribs. This flat plate is heated on one side, and cooled on the other. Variable HTC is imposed on the cooled side—a higher HTC (hH) over LH and a lower HTC (hL) over LL = L − LH. For this unit problem, the following parameters were studied: abrupt versus gradual transition between hH and hL, hH/hL, LH/L, and H/L. Results obtained show that if the averaged HTC is used, then the maximum temperature in the plate and the maximum temperature gradient in the plate can be severely underpredicted. The maximum temperature and the maximum temperature gradient can be underpredicted by as much as 36.3% and 542%, respectively, if the Biot number is less than 0.1 and as much as 13.0% and 570% if the Biot number is between 0.25 and 0.4. A reduced-order model was developed to estimate the underpredicted maximum temperature.
Structural basis for KCNE3 modulation of potassium recycling in epithelia
Authors: Kroncke Brett M. , Van Horn Wade D. , Smith Jarrod , Kang CongBao , Welch Richard C. , Song Yuanli , Nannemann David P. , Taylor Keenan C. , Sisco Nicholas J. , George Alfred L. , Meiler Jens , Vanoye Carlos G. , Sanders Charles R.
Published: September 2016

KCNE3 modulates the KCNQ1 K
+
channel in epithelia by directly stabilizing the voltage-sensor S4 segment in its activated state.
Efficacy of the Olyset Duo net against insecticide-resistant mosquito vectors of malaria
Authors: Ngufor Corine , N’Guessan Raphael , Fagbohoun Josias , Todjinou Damien , Odjo Abibath , Malone David , Ismail Hanafy , Akogbeto Martin , Rowland Mark
Published: September 2016

Olyset Duo, a bed net treated with a mixture of the insecticides pyriproxyfen and permethrin, provides improved control of mosquito vectors of malaria in regions of high insecticide resistance.
Multiplication of Legionella pneumophila Sequence Types 1, 47, and 62 in Buffered Yeast Extract Broth and Biofilms Exposed to Flowing Tap Water at Temperatures of 38°C to 42°C
Authors: van der Kooij Dick , Brouwer-Hanzens Anke J. , Veenendaal Harm R. , Wullings Bart A.
Published: September 2016

Legionella pneumophila
proliferates in freshwater environments at temperatures ranging from 25 to 45°C. To investigate the preference of different sequence types (ST) for a specific temperature range, growth of
L. pneumophila
serogroup 1 (SG1) ST1 (environmental strains), ST47, and ST62 (disease-associated strains) was measured in buffered yeast extract broth (BYEB) and biofilms grown on plasticized polyvinyl chloride in flowing heated drinking water originating from a groundwater supply. The optimum growth temperatures in BYEB were approximately 37°C (ST1), 39°C (ST47), and 41°C (ST62), with maximum growth temperatures of 42°C (ST1) and 43°C (ST47 and ST62). In the biofilm at 38°C, the ST47 and ST62 strains multiplied equally well compared to growth of the environmental ST1 strain and an indigenous
L. pneumophila
non-SG1 strain, all attaining a concentration of approximately 10
7
CFU/cm
−2
. Raising the temperature to 41°C did not impact these levels within 4 weeks, but the colony counts of all strains tested declined (at a specific decline rate of 0.14 to 0.41 day
−1
) when the temperature was raised to 42°C. At this temperature, the concentration of
Vermamoeba
vermiformis
in the biofilm, determined with quantitative PCR (qPCR), was about 2 log units lower than the concentration at 38°C. In columns operated at a constant temperature, ranging from 38 to 41°C, none of the tested strains multiplied in the biofilm at 41°C, in which also
V. vermiformis
was not detected. These observations suggest that strains of ST47 and ST62 did not multiply in the biofilm at a temperature of ≥41°C because of the absence of a thermotolerant host.

IMPORTANCE
Growth of
Legionella pneumophila
in tap water installations is a serious public health concern. The organism includes more than 2,100 varieties (sequence types). More than 50% of the reported cases of Legionnaires' disease are caused by a few sequence types which are very rarely detected in the environment. Strains of selected virulent sequence types proliferated in biofilms on surfaces exposed to warm (38°C) tap water to the same level as environmental varieties and multiplied well as pure culture in a nutrient-rich medium at temperatures of 42 and 43°C. However, these organisms did not grow in the biofilms at temperatures of ≥41°C. Typical host amoebae also did not multiply at these temperatures. Apparently, proliferation of thermotolerant host amoebae is needed to enable multiplication of the virulent
L. pneumophila
strains in the environment at elevated temperatures. The detection of these amoebae in water installations therefore is a scientific challenge with practical implications.
Genomic Analysis of Salmonella enterica Serovar Typhimurium from Wild Passerines in England and Wales
Authors: Mather Alison E. , Lawson Becki , de Pinna Elizabeth , Wigley Paul , Parkhill Julian , Thomson Nicholas R. , Page Andrew J. , Holmes Mark A. , Paterson Gavin K.
Published: September 2016

Passerine salmonellosis is a well-recognized disease of birds in the order Passeriformes, which includes common songbirds such as finches and sparrows, caused by infection with
Salmonella enterica
serovar Typhimurium. Previous research has suggested that some subtypes of
S
. Typhimurium—definitive phage types (DTs) 40, 56 variant, and 160—are host adapted to passerines and that these birds may represent a reservoir of infection for humans and other animals. Here, we have used the whole-genome sequences of 11 isolates from British passerines, five isolates of similar DTs from humans and a domestic cat, and previously published
S
. Typhimurium genomes that include similar DTs from other hosts to investigate the phylogenetic relatedness of passerine salmonellae to other
S
. Typhimurium isolates and investigate possible genetic features of the distinct disease pathogenesis of
S
. Typhimurium in passerines. Our results demonstrate that the 11 passerine isolates and 13 other isolates, including those from nonpasserine hosts, were genetically closely related, with a median pairwise single nucleotide polymorphism (SNP) difference of 130 SNPs. These 24 isolates did not carry antimicrobial resistance genetic determinants or the
S
. Typhimurium virulence plasmid. Although our study does not provide evidence of
Salmonella
transmission from passerines to other hosts, our results are consistent with the hypothesis that wild birds represent a potential reservoir of these
Salmonella
subtypes, and thus, sensible personal hygiene precautions should be taken when feeding or handling garden birds.

IMPORTANCE
Passerine salmonellosis, caused by certain definitive phage types (DTs) of
Salmonella
Typhimurium, has been documented as a cause of wild passerine mortality since the 1950s in many countries, often in the vicinity of garden bird feeding stations. To gain better insight into its epidemiology and host-pathogen interactions, we sequenced the genomes of a collection of 11 isolates from wild passerine salmonellosis in England and Wales. Phylogenetic analysis showed these passerine isolates to be closely related to each other and to form a clade that is distinct from other strains of
S
. Typhimurium, which included a multidrug-resistant isolate from invasive nontyphoidal
Salmonella
disease that shares the same phage type as several of the passerine isolates. Closely related to wild passerine isolates and within the same clade were four
S
. Typhimurium isolates from humans as well as isolates from horses, poultry, cattle, an unspecified wild bird, and a domestic cat and dog with similar DTs and/or multilocus sequence types. This suggests the potential for cross-species transmission, and the genome sequences provide a valuable resource to investigate passerine salmonellosis further.
Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail
Authors: Krishnamurthy Shruthi , Deng Bin , del Rio Roxana , Buchholz Kerry R. , Treeck Moritz , Urban Siniša , Boothroyd John , Lam Ying-Wai , Ward Gary E.
Published: September 2016

Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of
Toxoplasma gondii
that plays a critical role in host cell invasion. The ligand to which
T. gondii
AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion.

IMPORTANCE
Nearly one-third of the world’s population is infected with the protozoan parasite
Toxoplasma gondii
, which causes life-threatening disease in neonates and immunocompromised individuals.
T. gondii
is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.
Thrombin Cleavage of Plasmodium falciparum Erythrocyte Membrane Protein 1 Inhibits Cytoadherence
Authors: Gillrie Mark R. , Renaux Bernard , Russell-Goldman Eleanor , Avril Marion , Brazier Andrew J. , Mihara Koichiro , Di Cera Enrico , Milner Danny A. , Hollenberg Morley D. , Smith Joseph D. , Ho May
Published: September 2016

Plasmodium falciparum
malaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium by
Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy.

IMPORTANCE
Plasmodium falciparum
malaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion of
Plasmodium falciparum
-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria. While the recent implementation of artemisinin-based antimalarial therapy for severe malaria improves patient survival by targeting all parasite stages, antiparasite drugs alone may not immediately reverse pathophysiological processes in occluded vessels. Here we show that thrombin, an enzyme intimately involved in the clotting process, cleaves the main parasite adhesin expressed on the surface of IRBC, thereby preventing and reversing the binding of IRBC to endothelial cells. This beneficial effect of thrombin can be achieved by modified thrombins that cause significantly less clotting and vessel leakage while preserving the ability to cleave the parasite protein. Our results provide the basis for using modified thrombins as adjunctive therapy in severe malaria.
Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment
Authors: Fregoso Oliver I. , Emerman Michael
Published: September 2016

There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized.

IMPORTANCE
HIV-1 and HIV-2 belong to a family of viruses called lentiviruses that infect at least 40 primate species, including humans. Lentiviruses have been circulating in primates for at least 5 million years. In order to better fight HIV, we must understand the viral and host factors necessary for infection, adaptation, and transmission of these viruses. Using the natural variation of HIV-1, HIV-2, and related lentiviruses, we have investigated the role of the DNA damage response in the viral life cycle. We have found that the ability of lentiviruses to activate the DNA damage response is largely conserved. However, we also found that the SLX4 host factor is not required for this activation, as was previously proposed. This indicates that the DNA damage response is an important player in the viral life cycle, and yet the mechanism(s) by which HIV-1, HIV-2, and other primate lentiviruses engage the DNA damage response is still unknown.
Global Identification of Biofilm-Specific Proteolysis in Candida albicans
Authors: Winter Michael B. , Salcedo Eugenia C. , Lohse Matthew B. , Hartooni Nairi , Gulati Megha , Sanchez Hiram , Takagi Julie , Hube Bernhard , Andes David R. , Johnson Alexander D. , Craik Charles S. , Nobile Clarissa J.
Published: September 2016

Candida albicans
is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections.
C. albicans
cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new
C. albicans
infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by
C. albicans
biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by
C. albicans
. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in
C. albicans
biofilms; we also show that these activities are upregulated in other
Candida
clade pathogens. Deletion of the
SAP5
and
SAP6
genes in
C. albicans
compromised biofilm development
in vitro
in standard biofilm assays and
in vivo
in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for
Candida
biofilm formation.

IMPORTANCE
Biofilm formation by the opportunistic fungal pathogen
C. albicans
is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by
C. albicans
biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during
C. albicans
biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from
C. albicans
and also from additional pathogenic
Candida
species. Furthermore,
SAP5
and
SAP6
deletions confirm that both proteases are required for proper biofilm development
in vitro
and
in vivo
. We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of
Candida
biofilm-associated infections.
Does integrated training in evidence-based medicine (EBM) in the general practice (GP) specialty training improve EBM behaviour in daily clinical practice? A cluster randomised controlled trial
Authors: Kortekaas M F , Bartelink M E L , Zuithoff N P A , van der Heijden G J M G , de Wit N J , Hoes A W
Published: September 2016

Objectives

Evidence-based medicine (EBM) is an important element in the general practice (GP) specialty training. Studies show that integrating EBM training into clinical practice brings larger benefits than stand-alone modules. However, these studies have neither been performed in GP nor assessed EBM behaviour of former trainees in daily clinical practice.

Setting

GP specialty training in the Netherlands.

Participants

All 82 third year GP trainees who started their final third year in 2011 were approached for inclusion, of whom 79 (96%) participated: 39 in the intervention group and 40 in the control group.

Intervention

Integrated EBM training, in which EBM is embedded closely within the clinical context by joint assignments for the trainee and supervisor in daily practice, and teaching sessions based on dilemmas from actual patient consultations.

Comparison

Stand-alone EBM training at the institute only.

Primary and secondary outcomes

Our primary outcome was EBM behaviour, assessed by measuring guideline adherence (incorporating rational, motivated deviation) and information-seeking behaviour. Our secondary outcomes were EBM attitude and EBM knowledge. Data were acquired using logbooks and questionnaires, respectively. Analyses were performed using mixed models.

Results

Logbook data were available from 76 (96%) of the participating trainees at baseline (7614 consultations), 60 (76%) at the end of the third year (T1, 4973 consultations) and 53 (67%) 1 year after graduation (T2, 3307 consultations). We found no significant differences in outcomes between the 2 groups, with relative risks for guideline adherence varying between 0.96 and 0.99 (95% CI 0.86 to 1.11) at T1, and 0.99 and 1.10 (95% CI 0.92 to 1.25) at T2, and for information-seeking behaviour between 0.97 and 1.16 (95% CI 0.70 to 1.91) and 0.90 and 1.10 (95% CI 0.70 to 1.32), respectively.

Conclusions

Integrated EBM training compared with stand-alone EBM training does not improve EBM behaviour, attitude or knowledge of (future) GPs.