bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of bacterial translocation in calves with atresia coli
Authors: Altan Semih , Koç Yılmaz , Alkan Fahrettin , Sayın Zafer , Erol Muharrem 
Published: April 2018

AbstractObjectiveTo identify whether enteric bacteria pass into the mesenteric lymph nodes (MLNs) and peritoneal cavity in calves with atresia coli and to evaluate whether the presence of bacterial translocation (BT) has an impact on the success of surgical treatment.DesignProspective clinical study.AnimalsTwenty‐six client‐owned calves.InterventionsDuring laparotomy, swab samples were collected from the peritoneal cavity and MLNs using a sterile swab stick and were submitted for microbiological analysis.Measurements and Main ResultsBacterial cultures of swab samples revealed that 65% (n= 17) of the calves experienced BT. Of these, 14 calves experienced BT to the MLNs, 9 to the peritoneal cavity, and 5 to both regions. Of the bacteria isolated from the MLNs, 72% (n = 10) wereEscherichia coli. Of the samples isolated from the peritoneal fluid, 33% (n = 3) containedE. coliand 33% (n = 3) containedE. coli+ coagulase‐negativeStaphylococcus(CNS). In calves with BT that were discharged (n = 13) and without BT that were discharged (n = 7), the median survival was 30 days; these data were found to be similar in the 2 groups.ConclusionsThis study revealed that BT is observed in the majority of atresia coli cases.E. coliis more common in BT, and translocation occurs primarily through the lymphatic route. These results suggest that the presence of BT is closely related to the success of the operation for correction of atresia coli.
Regulation of muscle protein synthesis in an in vitro cell model using ex vivo human serum
Authors: Carson Brian P. , Patel Bijal , Amigo‐Benavent Miryam , Pauk Martina , Kumar Gujulla Sunil , Murphy Sylvia M. , Kiely Patrick A. , Jakeman Philip M. 
Published: April 2018

New Findings
What is the central question of this study?
Can medium conditioned by ex vivo human serum regulate muscle protein synthesis in skeletal muscle cells in vitro?

What is the main finding and its importance?
This study demonstrates that medium conditioned by ex vivo human serum can regulate muscle protein synthesis in skeletal muscle cells in vitro via the mammalian Target of Rapomycin (mTOR) pathway, and this can be regulated differentially by fed and fasted ex vivo human serum.

AbstractHuman serum embodies the integrated systemic response to any condition or perturbation, which may regulate muscle protein synthesis (MPS). Conditioning of medium with human serum represents a physiologically relevant method of regulating MPS in vitro. The primary purpose of the study was the development of a model using ex vivo human serum to condition medium and regulate MPS in in vitro skeletal muscle cells. Four young healthy men reported to the laboratory after an overnight fast and were fed with 0.33 g (kg body mass)−1 whey protein. Blood samples were taken before (Fasted) and 60 min postprandial (Fed). Fully differentiated C2C12 skeletal muscle cells were nutrient and serum deprived for 1 h and subsequently treated with medium conditioned with Fasted or Fed ex vivo human serum (20%) for 4 h. The MPS was measured using the surface sensing of translation technique and activation of mTOR, P70S6K and 4EBP1 by Western blot. Fasted and fed ex vivo human serum increased MPS (P < 0.05). Although a strong effect (ƞ2 = 0.36) for increased MPS in Fed relative to Fasted was observed, this was not statistically significant (P > 0.05). Activation of mTOR, P70S6K and 4EBP1 was significantly increased after treatment with Fed compared with Fasted ex vivo human serum (P < 0.05). Here, we developed and optimized the conditions for culture of C2C12 skeletal muscle cells, measurement of MPS and signalling in medium conditioned by ex vivo human serum. Furthermore, the functionality of the model was demonstrated by comparison of the response to medium conditioned by Fasted and Fed ex vivo human serum.
Evaluation of the Antifungal Activity of the Novel Oral Glucan Synthase Inhibitor SCY-078, Singly and in Combination, for the Treatment of Invasive Aspergillosis
Authors: Ghannoum M. , Long L. , Larkin E. L. , Isham N. , Sherif R. , Borroto-Esoda K. , Barat S. , Angulo D. 
Published: April 2018

ABSTRACT

Invasive aspergillosis remains a major cause of death among the immunocompromised population and those receiving long-term immunosuppressive therapy. In light of increased azole resistance, variable outcomes with existing echinocandin monotherapy and combination therapy, and persistent high mortality rates, new antifungal agents for the treatment of invasive aspergillosis are clearly needed. SCY-078 is the first-in-class triterpenoid antifungal, a novel class of glucan synthase inhibitors with broad
in vitro
and
in vivo
activity against a broad spectrum of
Candida
and
Aspergillus
species.
In vitro
testing of clinical strains of
Aspergillus fumigatus
and non-
fumigatus Aspergillus
strains showed that SCY-078 had potent fungistatic activity (minimum effective concentration for 90% of strains tested = 0.125 μg/ml) compared with the activities of amphotericin B (MIC
90
= 8 μg/ml) and voriconazole (MIC
90
= 2 μg/ml). Testing of SCY-078 in combination with isavuconazole or voriconazole demonstrated synergistic activity against the majority of the azole-susceptible strains tested, and SCY-078 in combination with amphotericin B was synergistic against the azole-susceptible strains, as well as one known resistant
cyp51A
mutant. SCY-078 may be an important additional antifungal for first-line or salvage monotherapy or combination treatment of invasive aspergillosis.
Pyocyanin Inhibits Chlamydia Infection by Disabling Infectivity of the Elementary Body and Disrupting Intracellular Growth
Authors: Li Jian Lin , Yang Ningjing , Huang Lei , Chen Dandan , Zhao Yu , Tang M. Matt , Fan Huizhou , Bao Xiaofeng 
Published: April 2018

ABSTRACTThe obligate intracellular bacteriumChlamydiais a widespread human pathogen that causes serious problems, including (but not limited to) infertility and blindness. Our search for novel antichlamydial metabolites from marine-derived microorganisms led to the isolation of pyocyanin, a small compound fromPseudomonas aeruginosa. Pyocyanin is an effective antichlamydial for all threeChlamydiaspp. tested. It has a 50% inhibitory concentration (IC50) of 0.019 to 0.028 μM, which is comparable to the IC50of tetracycline. At concentrations as low as 0.0039 μM, pyocyanin disables infectivity of the chlamydial elementary body (EB). At 0.5 μM or higher concentrations, the continuous presence of pyocyanin also inhibits chlamydial growth in the inclusion during later stages of the developmental cycle. Oxidative stress, a major known antimicrobial mechanism of pyocyanin, appears to be responsible only for the inhibition of bacterial growth and not for the disinfection of EBs. Pyocyanin is well-tolerated by probiotic vaginalLactobacillusspp. Our findings suggest that pyocyanin is of therapeutic value for chlamydial infections and can serve as a valuable chemical probe for studying chlamydial biology.
The Pyrroloquinoline-Quinone-Dependent Pyranose Dehydrogenase from Coprinopsis cinerea Drives Lytic Polysaccharide Monooxygenase Action
Authors: Várnai Anikó , Umezawa Kiwamu , Yoshida Makoto , Eijsink Vincent G. H. 
Published: April 2018

ABSTRACTFungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs catalyze the oxidative cleavage of glycosidic bonds after activation by an external electron donor. So far, only flavin-dependent oxidoreductases (from the auxiliary activity [AA] family AA3) have been shown to activate LPMOs. Here, we present LPMO activation by a pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase (PDH) fromCoprinopsis cinerea,CcPDH, the founding member of the recently discovered auxiliary activity family AA12.CcPDH contains a C-terminal family 1 carbohydrate binding module (CBM1), an N-terminal family AA8 cytochrome domain, and a central AA12 dehydrogenase domain. We have studied the ability of full-lengthCcPDH and its truncated variants to drive catalysis by twoNeurospora crassaLPMOs. The results show thatCcPDH indeed can activate the C-1-oxidizingN. crassaLPMO 9F (NcLPMO9F) and the C-4-oxidizingNeurospora crassaLPMO 9C (NcLPMO9C), that this activation depends on the cytochrome domain, and that the dehydrogenase and the LPMO reactions are strongly coupled. The two testedCcPDH-LPMO systems showed quite different efficiencies, and this difference disappeared upon the addition of free PQQ acting as a diphenol/quinone redox mediator, showing that LPMOs differ when it comes to their direct interactions with the cytochrome domain. Surprisingly, removal of the CBM domain fromCcPDH had a considerable negative impact on the efficiency of theCcPDH-LPMO systems, suggesting that electron transfer in the vicinity of the substrate is beneficial.CcPDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool for studying cellulose-oxidizing LPMOs.IMPORTANCELytic polysaccharide monooxygenases (LPMOs) are currently receiving increasing attention because of their importance in degrading recalcitrant polysaccharides and their potential roles in biological processes, such as bacterial virulence. LPMO action requires an external electron donor, and fungi growing on biomass secrete various so-called glucose-methanol-choline (GMC) oxidoreductases, including cellobiose dehydrogenase, which can donate electrons to LPMOs. This paper describes how an enzyme not belonging to the GMC oxidoreductase family,CcPDH, can activate LPMOs, and it provides new insights into the activation process by (i) describing the roles of individualCcPDH domains (a dehydrogenase, a cytochrome, and a carbohydrate-binding domain), (ii) showing that the PDH and LPMO enzyme reactions are strongly coupled, (iii) demonstrating that LPMOs differ in terms of their efficiencies of activation by the same activator, and (iv) providing indications that electron transferring close to the substrate surface is beneficial for the overall efficiency of theCcPDH-LPMO system.
Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers
Authors: Castillo-Menendez Luis R. , Witt Kristen , Espy Nicole , Princiotto Amy , Madani Navid , Pacheco Beatriz , Finzi Andrés , Sodroski Joseph 
Published: April 2018

ABSTRACT

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1
AD8
Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.

IMPORTANCE
The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.
Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry
Authors: Wang Bin , Liu Yan , Ji Chun-Miao , Yang Yong-Le , Liang Qi-Zhang , Zhao Pengwei , Xu Ling-Dong , Lei Xi-Mei , Luo Wen-Ting , Qin Pan , Zhou Jiyong , Huang Yao-Wei 
Published: April 2018

ABSTRACT

Identification of cellular receptors used by coronavirus (CoV) entry into the host cells is critical to an understanding of pathogenesis and to development of intervention strategies. The fourth CoV genus,
Deltacoronavirus
, evolutionarily related to the
Gammacoronavirus
, has just been defined recently. In the current study, we demonstrate that porcine aminopeptidase N (pAPN) acts as a cross-genus CoV functional receptor for both enteropathogenic porcine deltacoronovirus (PDCoV) and alphacoronovirus (AlphaCoV) (transmissible gastroenteritis virus [TGEV]) based upon three lines of evidence. First, the soluble S1 protein of PDCoV bound to the surface of target porcine cell lines known to express pAPN as efficiently as TGEV-S1, which could be blocked by soluble pAPN pretreatment. Second, both PDCoV-S1 and TGEV-S1 physically recognized and interacted with pAPN by coimmunoprecipitation in pAPN cDNA-transfected cells and by dot blot hybridization assay. Finally, exogenous expression of pAPN in refractory cells conferred susceptibility to PDCoV-S1 binding and to PDCoV entry and productive infection. PDCoV-S1 appeared to have a lower pAPN-binding affinity and likely consequent lower infection efficiency in pAPN-expressing refractory cells than TGEV-S1, suggesting that there may be differences between these two viruses in the virus-binding regions in pAPN. This study paves the way for dissecting the molecular mechanisms of PDCoV-host interactions and pathogenesis as well as facilitates future vaccine development and intervention strategies against PDCoV infection.

IMPORTANCE
The emergence of new human and animal coronaviruses is believed to have occurred through interspecies transmission that is mainly mediated by a species-specific receptor of the host. Among the four genera of the
Coronavirinae
, a couple of functional receptors for the representative members in the genera
Alphacoronavirus
and
Betacoronavirus
have been identified, whereas receptors for
Gammacoronavirus
and
Deltacoronavirus
, which are believed to originate from birds, are still unknown. Porcine coronaviruses, including the newly discovered porcine deltacoronavirus (PDCoV) associated with diarrhea in newborn piglets, have posed a serious threat to the pork industry in Asia and North America. Here, we report that PDCoV employs the alphacoronavirus TGEV functional receptor porcine aminopeptidase N (pAPN) for cellular entry, demonstrating the usage of pAPN as a cross-genus CoV functional receptor. The identification of the PDCoV receptor provides another example of the expanded host range of CoV and paves the way for further investigation of PDCoV-host interaction and pathogenesis.
EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in
Bacillus subtilis
Authors: Witzky Anne , Hummels Katherine R. , Tollerson Rodney , Rajkovic Andrei , Jones Lisa A. , Kearns Daniel B. , Ibba Michael 
Published: April 2018

ABSTRACT

Elongation factor P (EF-P) is a ubiquitous translation factor that facilitates translation of polyproline motifs. In order to perform this function, EF-P generally requires posttranslational modification (PTM) on a conserved residue. Although the position of the modification is highly conserved, the structure can vary widely between organisms. In
Bacillus subtilis
, EF-P is modified at Lys32 with a 5-aminopentanol moiety. Here, we use a forward genetic screen to identify genes involved in 5-aminopentanolylation. Tandem mass spectrometry analysis of the PTM mutant strains indicated that
ynbB
,
gsaB
, and
ymfI
are required for modification and that
yaaO
,
yfkA
, and
ywlG
influence the level of modification. Structural analyses also showed that EF-P can retain unique intermediate modifications, suggesting that 5-aminopentanol is likely directly assembled on EF-P through a novel modification pathway. Phenotypic characterization of these PTM mutants showed that each mutant does not strictly phenocopy the
efp
mutant, as has previously been observed in other organisms. Rather, each mutant displays phenotypic characteristics consistent with those of either the
efp
mutant or wild-type
B. subtilis
depending on the growth condition.
In vivo
polyproline reporter data indicate that the observed phenotypic differences result from variation in both the severity of polyproline translation defects and altered EF-P context dependence in each mutant. Together, these findings establish a new EF-P PTM pathway and also highlight a unique relationship between EF-P modification and polyproline context dependence.

IMPORTANCE
Despite the high level of conservation of EF-P, the posttranslational modification pathway that activates EF-P is highly divergent between species. Here, we have identified and characterized in
B. subtilis
a novel posttranslational modification pathway. This pathway not only broadens the scope of potential EF-P modification strategies, but it also indicates that EF-P modifications can be assembled directly on EF-P. Furthermore, characterization of these PTM mutants has established that an altered modification state can impact both the severity of polyproline translational defects and context dependence.
Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in Enterococcus faecalis
Authors: Rice Louis B. , Desbonnet Charlene , Tait-Kamradt Amelia , Garcia-Solache Monica , Lonks John , Moon Thomas M. , D’Andréa Éverton D. , Page Rebecca , Peti Wolfgang 
Published: April 2018

ABSTRACT

Enterococcus faecalis
strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an
E. faecalis
strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded
E. faecalis
with MICs of penicillins and carbapenems higher than those for wild-type strain
E. faecalis
JH2-2. Sequence analysis of the
pbp4
gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted
pbp4
−35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of
pbp4
in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in
E. faecalis
LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.

IMPORTANCE
Enterococcus faecalis
is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an
E. faecalis
strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to “suppress” infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.
CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis
Authors: Kujoth Gregory C. , Sullivan Thomas D. , Merkhofer Richard , Lee Taek-Jin , Wang Huafeng , Brandhorst Tristan , Wüthrich Marcel , Klein Bruce S. 
Published: April 2018

ABSTRACT

Blastomyces dermatitidis
is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to
Blastomyces
, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into
Blastomyces
via
Agrobacterium
gene transfer. We succeeded in disrupting several fungal genes, including
PRA1
and
ZRT1
, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of
Blastomyces
gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of
PRA1
or
ZRT1
had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of
PRA1
or
ZRT1
resulted in a reduction of the fungal burden in a mouse model of
Blastomyces
infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in
Blastomyces
fitness
in vivo
.

IMPORTANCE
Blastomyces
is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into
Blastomyces
. We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.