bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species
Authors: Zadravec Petra , Štrukelj Borut , Berlec Aleš 
Published: January 2015

ABSTRACT

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of
Lactococcus lactis
by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus
Lactobacillus
, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control.
Lactobacillus salivarius
ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of
Lb. salivarius
as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on
L. lactis
was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting
Lactobacillus salivarius
ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on
L. lactis
. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.
Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters
Authors: Ahmed W. , Harwood V. J. , Gyawali P. , Sidhu J. P. S. , Toze S. 
Published: January 2015

ABSTRACT

Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl
2
(methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ∼10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg
2+
) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters.
Piliation of Lactobacillus rhamnosus GG Promotes Adhesion, Phagocytosis, and Cytokine Modulation in Macrophages
Authors: Vargas García Cynthia E. , Petrova Mariya , Claes Ingmar J. J. , De Boeck Ilke , Verhoeven Tine L. A. , Dilissen Ellen , von Ossowski Ingemar , Palva Airi , Bullens Dominique M. , Vanderleyden Jos , Lebeer Sarah 
Published: January 2015

ABSTRACT

Recently,
spaCBA
-encoded pili on the cell surface of
Lactobacillus rhamnosus
GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of
L. rhamnosus
GG in the interaction with macrophages
in vitro
by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.
Temporal Stability of the Microbial Community in Sewage-Polluted Seawater Exposed to Natural Sunlight Cycles and Marine Microbiota
Authors: Sassoubre Lauren M. , Yamahara Kevan M. , Boehm Alexandria B. 
Published: January 2015

ABSTRACTBillions of gallons of untreated wastewater enter the coastal ocean each year. Once sewage microorganisms are in the marine environment, they are exposed to environmental stressors, such as sunlight and predation. Previous research has investigated the fate of individual sewage microorganisms in seawater but not the entire sewage microbial community. The present study used next-generation sequencing (NGS) to examine how the microbial community in sewage-impacted seawater changes over 48 h when exposed to natural sunlight cycles and marine microbiota. We compared the results from microcosms composed of unfiltered seawater (containing naturally occurring marine microbiota) and filtered seawater (containing no marine microbiota) to investigate the effect of marine microbiota. We also compared the results from microcosms that were exposed to natural sunlight cycles with those from microcosms kept in the dark to investigate the effect of sunlight. The microbial community composition and the relative abundance of operational taxonomic units (OTUs) changed over 48 h in all microcosms. Exposure to sunlight had a significant effect on both community composition and OTU abundance. The effect of marine microbiota, however, was minimal. The proportion of sewage-derived microorganisms present in the microcosms decreased rapidly within 48 h, and the decrease was the most pronounced in the presence of both sunlight and marine microbiota, where the proportion decreased from 85% to 3% of the total microbial community. The results from this study demonstrate the strong effect that sunlight has on microbial community composition, as measured by NGS, and the importance of considering temporal effects in future applications of NGS to identify microbial pollution sources.
A Halophilic Bacterium Inhabiting the Warm, CaCl
2
-Rich Brine of the Perennially Ice-Covered Lake Vanda, McMurdo Dry Valleys, Antarctica
Authors: Tregoning George S. , Kempher Megan L. , Jung Deborah O. , Samarkin Vladimir A. , Joye Samantha B. , Madigan Michael T. 
Published: January 2015

ABSTRACT

Lake Vanda is a perennially ice-covered and stratified lake in the McMurdo Dry Valleys, Antarctica. The lake develops a distinct chemocline at about a 50-m depth, where the waters transition from cool, oxic, and fresh to warm, sulfidic, and hypersaline. The bottom water brine is unique, as the highly chaotropic salts CaCl
2
and MgCl
2
predominate, and CaCl
2
levels are the highest of those in any known microbial habitat. Enrichment techniques were used to isolate 15 strains of heterotrophic bacteria from the Lake Vanda brine. Despite direct supplementation of the brine samples with different organic substrates in primary enrichments, the same organism, a relative of the halophilic bacterium
Halomonas
(
Gammaproteobacteria
), was isolated from all depths sampled. The Lake Vanda (VAN) strains were obligate aerobes and showed broad pH, salinity, and temperature ranges for growth, consistent with the physicochemical properties of the brine. VAN strains were halophilic and quite CaCl
2
tolerant but did not require CaCl
2
for growth. The fact that only VAN strain-like organisms appeared in our enrichments hints that the highly chaotropic nature of the Lake Vanda brine may place unusual physiological constraints on the bacterial community that inhabits it.
Physical Covering for Control of Escherichia coli O157:H7 and Salmonella spp. in Static and Windrow Composting Processes
Authors: Patel Jitendra R. , Yossa Irene , Macarisin Dumitru , Millner Patricia 
Published: January 2015

ABSTRACT

This study investigated the effect of a 30-cm covering of finished compost (FC) on survival of
Escherichia coli
O157:H7 and
Salmonella
spp. in active static and windrow composting systems. Feedstocks inoculated with
E. coli
O157:H7 (7.41 log CFU/g) and
Salmonella
(6.46 log CFU/g) were placed in biosentry tubes (7.5-cm diameter, 30-cm height) at three locations: (i and ii) two opposing sides at the interface between the FC cover layer (where present) and the feedstock material (each positioned approximately 10 cm below the pile's surface) and (iii) an internal location (top) (approximately 30 cm below the surface). On specific sampling days, surviving populations of inoculated
E. coli
O157:H7 and
Salmonella
, generic
E. coli
, and coliforms in compost samples were determined.
Salmonella
spp. were reduced significantly within 24 h in windrow piles and were below the detection limit after 3 and 7 days at internal locations of windrow and static piles containing FC covering, respectively. Likewise,
E. coli
O157:H7 was undetectable after 1 day in windrow piles covered with finished compost. Use of FC as a covering layer significantly increased the number of days that temperatures in the windrows remained ≥55°C at all locations and in static piles at internal locations. These time-temperature exposures resulted in rapid reduction of inoculated pathogens, and the rate of bacterial reduction was rapid in windrow piles. The sample location significantly influenced the survival of these pathogens at internal locations compared to that at interface locations of piles. Finished compost covering of compost piles aids in the reduction of pathogens during the composting process.
Effect of Temperature and Relative Humidity on the Survival of Foodborne Viruses during Food Storage
Authors: Lee Su Jin , Si Jiyeon , Yun Hyun Sun , Ko GwangPyo 
Published: January 2015

ABSTRACT
Millions of people suffer from foodborne diseases throughout the world every year, and the importance of food safety has grown worldwide in recent years. The aim of this study was to investigate the survival of hepatitis A virus (HAV) and viral surrogates of human norovirus (HuNoV) (bacteriophage MS2 and murine norovirus [MNV]) in food over time. HAV, MNV, and MS2 were inoculated onto either the digestive gland of oysters or the surface of fresh peppers, and their survival on these food matrices was measured under various temperature (4°C, 15°C, 25°C, and 40°C) and relative humidity (RH) (50% and 70%) conditions. Inoculated viruses were recovered from food samples and quantified by a plaque assay at predetermined time points over 2 weeks (0, 1, 3, 7, 10, and 14 days). Virus survival was influenced primarily by temperature. On peppers at 40°C and at 50% RH, >4- and 6-log reductions of MNV and HAV, respectively, occurred within 1 day. All three viruses survived better on oysters. In addition, HAV survived better at 70% RH than at 50% RH. The survival data for HAV, MS2, and MNV were fit to three different mathematical models (linear, Weibull, and biphasic models). Among them, the biphasic model was optimum in terms of goodness of fit. The results of this study suggest that major foodborne viruses such as HAV and HuNoV can survive over prolonged periods of time with a limited reduction in numbers. Because a persistence of foodborne virus on contaminated foods was observed, precautionary preventive measures should be performed.
Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides
Authors: Zhu Zhiwei , Ding Yunfeng , Gong Zhiwei , Yang Li , Zhang Sufang , Zhang Congyan , Lin Xinping , Shen Hongwei , Zou Hanfa , Xie Zhensheng , Yang Fuquan , Zhao Xudong , Liu Pingsheng , Zhao Zongbao K. 
Published: January 2015

ABSTRACT

Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast
Rhodosporidium toruloides
cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in
Saccharomyces cerevisiae
, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.
Functional Complementation Analyses Reveal that the Single PRAT Family Protein of Trypanosoma brucei Is a Divergent Homolog of Tim17 in Saccharomyces cerevisiae
Authors: Weems Ebony , Singha Ujjal K. , Hamilton VaNae , Smith Joseph T. , Waegemann Karin , Mokranjac Dejana , Chaudhuri Minu 
Published: January 2015

ABSTRACT

Trypanosoma brucei
, a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in
Saccharomyces cerevisiae
and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of
S. cerevisiae
. We expressed
S. cerevisiae
Tim23, Tim17, and Tim22 in
T. brucei
. These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the
T. brucei
mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in
T. brucei
was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the
T. brucei
mitochondrion and thus failed to complement the function of TbTim17.
The Anillin-Related Region of Bud4 Is the Major Functional Determinant for Bud4's Function in Septin Organization during Bud Growth and Axial Bud Site Selection in Budding Yeast
Authors: Wu Huan , Guo Jia , Zhou Ya-Ting , Gao Xiang-Dong 
Published: January 2015

ABSTRACT

The anillin-related protein Bud4 of
Saccharomyces cerevisiae
is required for axial bud site selection by linking the axial landmark to the septins, which localize at the mother bud neck. Recent studies indicate that Bud4 plays a role in septin organization during cytokinesis. Here we show that Bud4 is also involved in septin organization during bud growth prior to cytokinesis, as
bud4
Δ
shs1
Δ cells displayed an elongated bud morphology and defective septin organization at 18°C. Bud4 overexpression also affected septin organization during bud growth in
shs1
Δ cells at 30°C. Bud4 was previously thought to associate with the septins via its central region, while the C-terminal anillin-related region was not involved in septin association. Surprisingly, we found that the central region of Bud4 alone targets to the bud neck throughout the cell cycle, unlike full-length Bud4, which localizes to the bud neck only during G
2
/M phase. We identified the anillin-related region to be a second targeting domain that cooperates with the central region for proper septin association. In addition, the anillin-related region could largely mediate Bud4's function in septin organization during bud growth and bud site selection. We show that this region interacts with the C terminus of Bud3 and the two segments depend on each other for association with the septins. Moreover, like the
bud4
Δ mutant, the
bud3
Δ mutant genetically interacts with
shs1
Δ and
cdc12-6
mutants in septin organization, suggesting that Bud4 and Bud3 may cooperate in septin organization during bud growth. These observations provide new insights into the interaction of Bud4 with the septins and Bud3.
Injury risk factors, screening tests and preventative strategies: a systematic review of the evidence that underpins the perceptions and practices of 44 football (soccer) teams from various premier leagues
Authors: McCall Alan , Carling Chris , Davison Michael , Nedelec Mathieu , Le Gall Franck , Berthoin Serge , Dupont Gregory 
Published: January 2015

PurposeTo systematically review the scientific level of evidence for the ‘Top 3’ risk factors, screening tests and preventative exercises identified by a previously published survey of 44 premier league football (soccer) teams. Also, to provide an overall scientific level of evidence and graded recommendation based on the current research literature.MethodsA systematic literature search (Pubmed [MEDLINE], SportDiscus, PEDRO and Cochrane databases). The quality of the articles was assessed and a level of evidence (1++ to 4) was assigned. Level 1++ corresponded to the highest level of evidence available and 4, the lowest. A graded recommendation (A: strong, B: moderate, C: weak, D: insufficient evidence to assign a specific recommendation) for use in the practical setting was given.ResultsFourteen studies were analysed. The overall level of evidence for the risk factors previous injury, fatigue and muscle imbalance were 2++, 4 and ‘inconclusive’, respectively. The graded recommendation for functional movement screen, psychological questionnaire and isokinetic muscle testing were all ‘D’. Hamstring eccentric had a weak graded ‘C’ recommendation, and eccentric exercise for other body parts was ‘D’. Balance/proprioception exercise to reduce ankle and knee sprain injury was assigned a graded recommendation ‘D’.ConclusionsThe majority of perceptions and practices of premier league teams have a low level of evidence and low graded recommendation. This does not imply that these perceptions and practices are not important or not valid, as it may simply be that they are yet to be sufficiently validated or refuted by research.
Skills, expertise and role of Australian emergency clinicians in caring for people with advanced cancer
Authors: Jelinek G A , Marck C H , Weil J , Lane H , Philip J , Boughey M , Weiland T J 
Published: January 2015

ObjectivesTo explore the views of Australian emergency department (ED) clinicians about their skills, role and expertise in caring for people with advanced cancer.MethodsA cross-sectional electronic survey of doctors and nurses working in Australian EDs was undertaken. Comparisons were made by demographics and whether respondents had received palliative care education.ResultsThe sample comprised 444 doctors (response rate 13.5%), the majority Fellows (emergency medicine specialists) of the Australasian College for Emergency Medicine, and 237 nurses, from all states, territories and regions (metropolitan and regional). A minority (n=123, 20.6%) felt that the ED was not an appropriate place for patients with advanced cancer to present for acute care, while almost two-thirds (n=397, 64.8%) found caring for such patients rewarding, particularly nurses and those who had received palliative care education; very few (n=40, 6.5%) reported feeling uncomfortable talking to the families of dying patients. A minority (n=129, 21.0%) felt that it was not appropriate for junior medical staff to assess these patients, nurses much more than doctors (42.9% vs 8.5%, p<0.001). Over half (n=338, 55.1%) felt sufficiently skilled in managing pain for people with advanced cancer, with Fellows, more experienced doctors, and those who had received palliative care education more likely to feel skilled.ConclusionsED clinicians in Australia, particularly those who have received palliative care education, feel comfortable and adequately skilled in managing people with advanced cancer presenting to EDs, and most find it rewarding. The importance of palliative care education to emergency clinicians’ training should be recognised.