bluuebunny/crossref_metadata_2025_split huggingface dataset:
Initial study of α1,3‐galactosyltransferase gene‐knockout/CD46 pig full‐thickness corneal xenografts in rhesus monkeys
Authors: Dong Xiaojuan , Hara Hidetaka , Wang Ying , Wang Li , Zhang Yingnan , Cooper David K.C. , Dai Yifan , Pan Zhiqiang 
Published: January 2017

AbstractBackgroundTo investigate graft survival after full‐thickness corneal xenotransplantation from α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs expressing a human complement regulatory protein (GTKO/CD46 pigs) in rhesus monkeys.MethodsRhesus monkeys (n=10) were transplanted with full‐thickness corneas from wild‐type (WT; n=4) and GTKO/CD46 (n=4) pigs or from monkeys (n=2). All recipient monkeys received post‐transplant subconjunctival injections of betamethasone. Corneal grafts were evaluated by slit‐lamp. Histopathology, immunohistochemistry of the grafts, and cytokine concentrations in the aqueous humor were tested 6 months after transplantation. Anti‐pig IgM/IgG and anti‐galactose‐α1,3‐galactose (Gal) antibodies were determined by flow cytometry and ELISA, respectively.ResultsThe longest graft survival of WT and GTKO/CD46 xenografts was 157 and 171 days, respectively. There was no significant difference in graft survival between WT and GTKO pig corneas. Anterior synechiae occurred in two recipients of WT and all recipients of GTKO/CD46 grafts. All xenograft recipients developed a retrocorneal membrane, inflammatory cells (CD3+ T lymphocytes) infiltrated the corneal stroma, and in the aqueous humor, IL‐6 was increased 6 months after transplantation. Induced antibody responses were documented against Gal and/or non‐Gal pig antigens. In contrast, allografts survived >180 days without any rejection, with no increase in cytokines in the aqueous humor, and no elicited serum anti‐pig antibodies were detected.Conclusionsα1,3‐galactosyltransferase gene‐knockout/CD46 pig corneas were not associated with prolonged graft survival or a reduced antibody response compared with WT pig corneas. The prevention of the development of anterior synechiae and a retrocorneal membrane after corneal xenotransplantation would appear to be important if prolonged corneal xenograft survival is to be achieved.
Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species
Authors: Szymczak Paula , Janzen Thomas , Neves Ana Rute , Kot Witold , Hansen Lars H. , Lametsch René , Neve Horst , Franz Charles M. A. P. , Vogensen Finn K. 
Published: December 2016

ABSTRACT

Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of
Streptococcus thermophilus
phages can be divided into either
cos
- or
pac
-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of
S. thermophilus
phages from the Chr. Hansen A/S collection, using PCR specific for the
cos
- or
pac
-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional
S. thermophilus
phages. The third phage, CHPC1151, had a tail size similar to those of the
cos
- or
pac
-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of
Lactococcus lactis
P335 phages. Phage CHPC1151 was closely related to the atypical
S. thermophilus
phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of
S. thermophilus
and
L. lactis
strains, we revealed the possibility of cross-interactions. Our data indicated that the use of
S. thermophilus
together with
L. lactis
, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among
S. thermophilus
phage populations requires that a new classification of the group be proposed.

IMPORTANCE
Streptococcus thermophilus
is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.
Laboratory Surveillance of Polio and Other Enteroviruses in High-Risk Populations and Environmental Samples
Authors: Pogka Vasiliki , Labropoulou Stavroula , Emmanouil Mary , Voulgari-Kokota Androniki , Vernardaki Alexandra , Georgakopoulou Theano , Mentis Andreas F. 
Published: December 2016

ABSTRACTIn the context of poliomyelitis eradication, a reinforced supplementary laboratory surveillance of enteroviruses was implemented in Greece. Between 2008 and 2014, the Hellenic Polioviruses/Enteroviruses Reference Laboratory performed detailed supplementary surveillance of circulating enteroviruses among healthy individuals in high-risk population groups, among immigrants from countries in which poliovirus is endemic, and in environmental samples. In total, 722 stool samples and 179 sewage water samples were included in the study. No wild-type polioviruses were isolated during these 7 years of surveillance, although two imported vaccine polioviruses were detected. Enterovirus presence was recorded in 25.3 and 25.1% of stool and sewage water samples, respectively. Nonpolio enteroviruses isolated from stool samples belonged to species A, B, or C; coxsackievirus A24 was the most frequently identified serotype. Only enteroviruses of species B were identified in sewage water samples, including four serotypes of echoviruses and four serotypes of coxsackie B viruses. Phylogenetic analysis revealed close genetic relationships among virus isolates from sewage water samples and stool samples, which in most cases fell into the same cluster. To the best of our knowledge, this is the first study to compare enterovirus serotypes circulating in fecal specimens of healthy individuals and environmental samples, emphasizing the burden of enterovirus circulation in asymptomatic individuals at high risk. Given that Greece continues to receive a large number of short-term arrivals, students, migrants, and refugees from countries in which poliovirus is endemic, it is important to guarantee high-quality surveillance in order to maintain its polio-free status until global eradication is achieved.IMPORTANCEThis article summarizes the results of supplementary poliovirus surveillance in Greece and the subsequent characterization of enteroviral circulation in human feces and the environment. The examination of stool samples from healthy refugees and other individuals in “high-risk” groups for poliovirus enables the identification of enterovirus cases and forms the basis for further investigation of the community-level risk of viral transmission. In addition, the examination of composite human fecal samples through environmental surveillance links poliovirus and nonpoliovirus isolates from unknown individuals to populations served by the sewage or wastewater system. Supplementary surveillance is necessary to comply with the prerequisites imposed by the World Health Organization for monitoring the emergence of vaccine-derived polioviruses, reemergence of wild polioviruses, or disappearance of all vaccine-related strains in order for countries such as Greece to maintain their polio-free status and contribute to global poliovirus eradication.
An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S
Authors: Argov Tal , Rabinovich Lev , Sigal Nadejda , Herskovits Anat A. 
Published: December 2016

ABSTRACT

Construction of
Listeria monocytogenes
mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (
pheS*
). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic
p
-chloro-phenylalanine analog (
p
-Cl-phe) as a substrate. When
pheS*
is introduced into
L. monocytogenes
and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to
p
-Cl-phe supplemented in the medium. This enabled us to utilize
pheS*
as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in
L. monocytogenes
. We used this vector to investigate the monocin genomic region in
L. monocytogenes
strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on
L. monocytogenes
.

IMPORTANCE
L. monocytogenes
is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in
Listeria
research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool for
L. monocytogenes
will allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism.
Cryptosporidium Attenuation across the Wastewater Treatment Train: Recycled Water Fit for Purpose
Authors: King Brendon , Fanok Stella , Phillips Renae , Lau Melody , van den Akker Ben , Monis Paul 
Published: December 2016

ABSTRACT

Compliance with guideline removal targets for
Cryptosporidium
which do not provide any credit for the inactivation of oocysts through wastewater treatment processes can considerably increase the cost of providing recycled water. Here we present the application of an integrated assay to quantify both oocyst numbers and infectivity levels after various treatment stages at three Victorian and two South Australian (SA) wastewater treatment plants (WWTPs). Oocyst density in the raw sewage was commensurate with community disease burden, with early rounds of sampling capturing a widespread cryptosporidiosis outbreak in Victoria. The level of infectivity of oocysts in sewage was stable throughout the year but was significantly lower at the SA WWTPs. Removals across secondary treatment processes were seasonal, with poorer removals associated with inflow variability; however, no decrease in the oocyst infectivity was identified. For SA WWTPs, those oocysts remaining within the secondary treatment-clarified effluent were proportionally more infectious than those in raw sewage. Lagoon systems demonstrated significant inactivation or removal of oocysts, with attenuation being seasonal. Examination of a UV system emphasized its efficacy as a disinfectant barrier but conversely confirmed the importance of a multibarrier approach with the detection of infectious oocysts postdisinfection. The ability to characterize risk from infectious oocysts revealed that the risk from
Cryptosporidium
is significantly lower than previously thought and that its inclusion in quantitative risk assessments of reuse systems will more accurately direct the selection of treatment strategies and capital expenditure, influencing the sustainability of such schemes.

IMPORTANCE
Here we present the application of a recently developed integrated assay not only to quantify the removal of
Cryptosporidium
oocysts but also to quantify their infectivity across various treatment stages at five wastewater treatment plants (WWTPs), thereby better measuring the “true effect” of the treatment train on oocyst risk reduction. For a number of the WWTPs analyzed in this study the risk, is significantly lower than previously thought. Therefore, the inclusion of oocyst infectivity in guideline values and in quantitative microbial risk assessment (QMRA) has the potential to affect future treatment directions and capital expenditure.
Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency
Authors: Yang Hong , Shi Pengjun , Liu Yun , Xia Wei , Wang Xiaoyu , Cao Huifang , Ma Rui , Luo Huiying , Bai Yingguo , Yao Bin 
Published: December 2016

ABSTRACTGlycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases usingNfEG12A fromNeosartorya fischeriP1 and EG (PDB1KS4) fromAspergillus niger. Acidophilic and thermophilicNfEG12A had the highest catalytic efficiency (kcat/Km, 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced intoNfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (kcat/Km) of these enzymes (in descending order,NfEG12A [100%],NfEG12A-FN [140%], andNfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (kcat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCELoop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the −1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases,NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.
N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function
Authors: Tamir Adi , Eichler Jerry 
Published: December 2016

ABSTRACT

N-Glycosylation, the covalent linkage of glycans to select Asn residues of target proteins, is an almost universal posttranslational modification in archaea. However, whereas roles for N-glycosylation have been defined in eukarya and bacteria, the function of archaeal N-glycosylation remains unclear. Here, the impact of perturbed N-glycosylation on the structure and physiology of the haloarchaeon
Haloferax volcanii
was considered. Cryo-electron microscopy was used to examine right-side-out membrane vesicles prepared from cells of a parent strain and from strains lacking genes encoding glycosyltransferases involved in assembling the N-linked pentasaccharide decorating the surface layer (S-layer) glycoprotein, the sole component of the S-layer surrounding
H. volcanii
cells. Whereas a regularly repeating S-layer covered the entire surface of vesicles prepared from parent strain cells, vesicles from the mutant cells were only partially covered. To determine whether such N-glycosylation-related effects on S-layer assembly also affected cell function, the secretion of a reporter protein was addressed in the parent and N-glycosylation mutant strains. Compromised S-layer glycoprotein N-glycosylation resulted in impaired transfer of the reporter past the S-layer and into the growth medium. Finally, an assessment of S-layer glycoprotein susceptibility to added proteases in the mutants revealed that in cells lacking AglD, which is involved in adding the final pentasaccharide sugar, a distinct S-layer glycoprotein conformation was assumed in which the N-terminal region was readily degraded. Perturbed N-glycosylation thus affects S-layer glycoprotein folding. These findings suggest that
H. volcanii
could adapt to changes in its surroundings by modulating N-glycosylation so as to affect S-layer architecture and function.

IMPORTANCE
Long held to be a process unique to eukaryotes, it is now accepted that bacteria and archaea also perform N-glycosylation, namely, the covalent attachment of sugars to select asparagine residues of target proteins. Yet, while information on the importance of N-glycosylation in eukaryotes and bacteria is available, the role of this posttranslational modification in archaea remains unclear. Here, insight into the purpose of archaeal N-glycosylation was gained by addressing the surface layer (S-layer) surrounding cells of the halophilic species
Haloferax volcanii
. Relying on mutant strains defective in N-glycosylation, such efforts revealed that compromised N-glycosylation affected S-layer integrity and the transfer of a secreted reporter protein across the S-layer into the growth medium, as well as the conformation of the S-layer glycoprotein, the sole component of the S-layer. Thus, by modifying N-glycosylation,
H. volcanii
cells can change how they interact with their surroundings.
Dynamics of Salmonella Shedding and Welfare of Hens in Free-Range Egg Production Systems
Authors: Gole Vaibhav C. , Woodhouse Rebecca , Caraguel Charles , Moyle Talia , Rault Jean-Loup , Sexton Margaret , Chousalkar Kapil 
Published: December 2016

ABSTRACT

The current study investigated the effect of environmental stressors (i.e., weather changes) on
Salmonella
shedding in free-range production systems and the correlations with behavioral and physiological measures (i.e., fecal glucocorticoid metabolites). This involved longitudinal and point-in-time surveys of
Salmonella
shedding and environmental contamination on four commercial free-range layer farms. The shedding of
Salmonella
was variable across free-range farms and in different seasons. There was no significant effect of season on the
Salmonella
prevalence during this investigation. In this study, the combined
Salmonella
most probable number (MPN) counts in environmental (including feces, egg belt, dust, nest box, and ramp) samples were highest in samples collected during the summer season (4th sampling, performed in February). The predominant serovars isolated during this study were
Salmonella enterica
serovar Mbandaka and
Salmonella enterica
serovar Typhimurium phage types 135 and 135a. These two phage types were involved in several egg product-related
Salmonella
outbreaks in humans. Multilocus variable-number tandem-repeat analysis (MLVA) results indicated that MLVA types detected from human food poisoning cases exhibited MLVA patterns similar to the strains isolated during this study. All
Salmonella
isolates (
n =
209) were tested for 15 different genes involved in adhesion, invasion, and survival of
Salmonella
spp. We also observed variations for
sopA
,
ironA
, and
misL
. There were no positive correlations between fecal corticosterone metabolite (FCM) and
Salmonella
prevalence and/or shedding in feces. Also, there were no positive correlations between
Salmonella
prevalence and
Salmonella
count (log MPN) and any of the other welfare parameters.

IMPORTANCE
In this study, the welfare of laying hens and
Salmonella
shedding were compared over a prolonged period of time in field conditions. This study investigated the long-term shedding of
Salmonella
serovars in a free-range egg production system. Given that there is increasing demand for free-range eggs, it is essential to understand the risks associated with such a production system.
Mechanisms of ACL injury in professional rugby union: a systematic video analysis of 36 cases
Authors: Montgomery Connor , Blackburn Jeff , Withers Daniel , Tierney Gregory , Moran Cathal , Simms Ciaran 
Published: December 2016

BackgroundThe mechanisms of ACL injury in rugby are not well defined.AimTo describe the mechanisms of ACL injury in male professional rugby players using systematic video analysis.Methods36 cases from games played in top professional leagues and international matches were analysed. 5 analysts independently assessed all videos to record the estimated frame/time of initial ground contact, frame/time of ACL tear and a range of play specific variables. This included contact versus non-contact ACL injuries, injury timing, joint flexion angles and foot contact with the ground. 37 side-stepping manoeuvres from a control game were analysed to allow comparison of non-injury versus injury situations.Results57% of ACL injuries occurred in a contact manner. 2 main scenarios were identified: (1) offensive running and (2) being tackled, indicating that the ball carrier might be at higher risk of ACL injury. The majority of non-contact ACL injuries resulted from a side-stepping manoeuvre. In most non-contact cases, initial ground contact was through heel strike. Statistical assessment of heel strike at initial ground contact versus non-heel strike cases showed a significant difference in injury versus non-injury outcomes, with heel strike associated with higher injury risk. Non-contact ACL injuries had lower median knee flexion angles and a more dorsiflexed ankle when compared with a control group (10° vs 20°, p≤0.001 and 10° vs 0°, p=0.033 respectively).ConclusionsOver half of ACL injuries in rugby in our analysis resulted from a contact mechanism. For non-contact injuries, lower knee flexion angles and heel-first ground contact in a side-stepping manoeuvre were associated with ACL injury.