Title: Study Supports Broader Access to Lap-Band Weight-Loss Surgery
Category: Health News
Created: 5/2/2013 10:35:00 AM
Last Editorial Review: 5/2/2013 12:00:00 AM

Title: Training Programs Protect Young Athletes From ACL Tears: Report
Category: Health News
Created: 4/28/2014 9:35:00 AM
Last Editorial Review: 4/28/2014 12:00:00 AM

Title: Health Tip: Gear up for Sports
Category: Health News
Created: 4/28/2015 12:00:00 AM
Last Editorial Review: 4/28/2015 12:00:00 AM

Title: 4 in 10 Americans Breathe Unhealthy Air: Report
Category: Health News
Created: 4/29/2015 12:00:00 AM
Last Editorial Review: 4/30/2015 12:00:00 AM

Title: Temporarily Turning Blue Sometimes Normal for Babies, Doctors Say
Category: Health News
Created: 4/25/2016 12:00:00 AM
Last Editorial Review: 4/25/2016 12:00:00 AM

Title: U.S. Health Report Card Finds Racial, Ethnic Disparities Persist
Category: Health News
Created: 4/27/2016 12:00:00 AM
Last Editorial Review: 4/27/2016 12:00:00 AM

Title: Early Emotional Support May Help Kids Manage Feelings Later
Category: Health News
Created: 4/26/2016 12:00:00 AM
Last Editorial Review: 4/27/2016 12:00:00 AM

Title: Births of Triplets, Quadruplets on Decline in U.S.: Report
Category: Health News
Created: 4/28/2016 12:00:00 AM
Last Editorial Review: 4/28/2016 12:00:00 AM

Title: Building Muscle Could Boost the Body's Most Important Muscle
Category: Health News
Created: 4/28/2016 12:00:00 AM
Last Editorial Review: 4/29/2016 12:00:00 AM

Title: Strong Support for NIH in New Budget From Congress
Category: Health News
Created: 5/2/2017 12:00:00 AM
Last Editorial Review: 5/3/2017 12:00:00 AM

Title: Loving, Supportive Kids May Help Lower Seniors' Dementia Risk
Category: Health News
Created: 5/2/2017 12:00:00 AM
Last Editorial Review: 5/3/2017 12:00:00 AM

Title: Health Tip: Protect Your Eyes During Sports
Category: Health News
Created: 5/1/2018 12:00:00 AM
Last Editorial Review: 5/1/2018 12:00:00 AM

Title: No One-Size-Fits-All for Hydrating During Sports
Category: Health News
Created: 4/30/2018 12:00:00 AM
Last Editorial Review: 5/1/2018 12:00:00 AM

Title: Most U.S. Adults Support More Mental Health Services for Kids
Category: Health News
Created: 5/1/2018 12:00:00 AM
Last Editorial Review: 5/2/2018 12:00:00 AM

Title: First Death Reported in E. Coli Outbreak Tied to Romaine Lettuce
Category: Health News
Created: 5/2/2018 12:00:00 AM
Last Editorial Review: 5/3/2018 12:00:00 AM

Title: Care at VA Hospitals as Good or Better Than Elsewhere in U.S.: Report
Category: Health News
Created: 5/4/2018 12:00:00 AM
Last Editorial Review: 5/4/2018 12:00:00 AM

Title: Study Supports Radiation for Early, Hormone-Driven Breast Cancer
Category: Health News
Created: 4/26/2019 12:00:00 AM
Last Editorial Review: 4/29/2019 12:00:00 AM

Title: Researchers Report First U.S. Dog With Coronavirus
Category: Health News
Created: 4/29/2020 12:00:00 AM
Last Editorial Review: 4/30/2020 12:00:00 AM

Title: Will Remdesivir Help COVID-19 Patients? Two Reports Provide Different Answers
Category: Health News
Created: 4/29/2020 12:00:00 AM
Last Editorial Review: 4/30/2020 12:00:00 AM

Title: Temporomandibular Joint Syndrome (TMJ)
Category: Diseases and Conditions
Created: 3/26/1998 12:00:00 AM
Last Editorial Review: 3/27/2020 12:00:00 AM

PMC is happy to announce the addition of a citation exporter feature. This feature makes it easy to retrieve either styled citations that you can copy/paste into your manuscripts, or to download them into a format compatible with your bibliographic reference manager software.

When viewing an Entrez search results page, each result summary will now include a "Citation" link. When, clicked, this will open a pop-up window that you can use to easily copy/paste citations formatted in one of three popular styles: AMA (American Medical Association), MLA (Modern Language Association), or APA (American Psychological Association). In addition, the box has links at the bottom that can be used to download the citation information in one of three machine-readable formats, which most bibliographic reference management software can import.

The same citation box can also be invoked from an individual article, either in classic view (with the "Citation" link among the list of formats) or the PubReader view, by clicking on the citation information just below the article title in the banner.

These human-readable styled citations, and machine-readable formats, will be available through a public API, and we will be providing more details about that in another announcement, on the pmc-utils-announce mailing list. Please subscribe to that list if you are interested.

Machine-readability of scholarly outputs is critical to supporting large-scale analysis of the scientific literature. To that end, PMC’s Tagging Guidelines and internal processes have been updated to support the JATS4R recommendations for tagging clinical trial information. NLM encourages PMC-participating publishers, journals, and data providers to review this guidance. Please contact us at pubmedcentral@ncbi.nlm.nih.gov if you have any questions.

Title: Second HIV Patient Reportedly 'Cured'
Category: Health News
Created: 3/9/2020 12:00:00 AM
Last Editorial Review: 3/10/2020 12:00:00 AM

Title: Skipping Sleep to Watch Sports is The Real March Madness
Category: Health News
Created: 3/6/2020 12:00:00 AM
Last Editorial Review: 3/6/2020 12:00:00 AM

TNF-related apoptosis-inducing ligand (TRAIL) and an agonistic antibody against the death-inducing TRAIL receptor 5, DR5, are thought to selectively induce tumor cell death and therefore, have gained attention as potential therapeutics currently under investigation in several clinical trials. However, some tumor cells are resistant to TRAIL/DR5–induced cell death, even though they express DR5. Previously, we reported that DR5 is transported into the nucleus by importin β1, and knockdown of importin β1 upregulates cell surface expression of DR5 resulting in increased TRAIL sensitivity in vitro. Here, we examined the impact of importin β1 knockdown on agonistic anti-human DR5 (hDR5) antibody therapy. Drug-inducible importin β1 knockdown sensitizes HeLa cells to TRAIL-induced cell death in vitro, and exerts an antitumor effect when combined with agonistic anti-hDR5 antibody administration in vivo. Therapeutic importin β1 knockdown, administered via the atelocollagen delivery system, as well as treatment with the importin β inhibitor, importazole, induced regression and/or eradication of two human TRAIL-resistant tumor cells when combined with agonistic anti-hDR5 antibody treatment. Thus, these findings suggest that the inhibition of importin β1 would be useful to improve the therapeutic effects of agonistic anti-hDR5 antibody against TRAIL-resistant cancers.

Purpose: Oral and craniofacial conditions or diseases can impact an individual's health and quality of life. The purpose of this study was to assess the perceived oral health related quality of life (OHRQoL) of children, and evaluate the reported level of agreement between caregivers and their children.Methods: Purposive sampling was used to recruit children ages 8-15, and their caregivers from a dental clinic in a pediatric hospital for this descriptive, cross-sectional study. A modified version of a validated measure, Child Oral Health Impact Profile-Short Form (COHIP-SF), was used for a 22-item questionnaire encompassing three subscales: oral health, functional well-being, and social emotional well-being. Two additional items were included to assess child/caregiver's level of agreement. A dental chart review was also conducted to assess the child's overbite, overjet, and decayed surfaces. Data were analyzed through descriptive statistics and examined for assumptions of normality and linearity.Results: Sixty child/caregiver pairs (n=120) participated in this study. Overbite, overjet and decayed surfaces were not found to be related to any OHRQoL variable, including child/caregiver ratings and overall agreement (p>.05). Average OHRQoL scores for caregivers found to be more positive those of their children (p=.02). Agreement between caregivers and the child's gender was shown to be significant (p=.01). Female child scores differed significantly from males with respect to their caregiver responses (p=.02). Caregivers rated a higher OHRQoL for female children, thus overestimating their female child's reported OHRQoL.Conclusions: The moderate level of agreement found between children and caregivers reinforces the importance of including the child, as well as the caregiver, when assessing OHRQoL.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

ABSTRACT

Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity.

IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.

ABSTRACT

Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas. Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide.

IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster (Crassostrea gigas), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates.

ABSTRACT

The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host.

IMPORTANCE Toxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

ABSTRACT

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.

IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

ABSTRACT

Mitochondrial Ca²⁺ transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that Trypanosoma brucei MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the TbMCUC subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and P_i transport with ATP synthesis, a process that is stimulated by Ca²⁺ in trypanosomes and human cells.

IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei, the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells.

ABSTRACT

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

IMPORTANCE Candida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans. Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

ABSTRACT

The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a "crankshaft conrod" mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA.

IMPORTANCE The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a "crankshaft conrod" mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.

ABSTRACT

The appressoria that are generated by the rice blast fungus Magnaporthe oryzae in response to surface cues are important for successful colonization. Previous work showed that regulators of G-protein signaling (RGS) and RGS-like proteins play critical roles in appressorium formation. However, the mechanisms by which these proteins orchestrate surface recognition for appressorium induction remain unclear. Here, we performed comparative transcriptomic studies of Morgs mutant and wild-type strains and found that M. oryzae Aa91 (MoAa91), a homolog of the auxiliary activity family 9 protein (Aa9), was required for surface recognition of M. oryzae. We found that MoAA91 was regulated by the MoMsn2 transcription factor and that its disruption resulted in defects in both appressorium formation on the artificial inductive surface and full virulence of the pathogen. We further showed that MoAa91 was secreted into the apoplast space and was capable of competing with the immune receptor chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immune responses. In summary, we have found that MoAa91 is a novel signaling molecule regulated by RGS and RGS-like proteins and that MoAa91 not only governs appressorium development and virulence but also functions as an effector to suppress host immunity.

IMPORTANCE The rice blast fungus Magnaporthe oryzae generates infection structure appressoria in response to surface cues largely due to functions of signaling molecules, including G-proteins, regulators of G-protein signaling (RGS), mitogen-activated protein (MAP) kinase pathways, cAMP signaling, and TOR signaling pathways. M. oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), and MoRgs1, MoRgs3, MoRgs4, and MoRgs7 were found to be particularly important in appressorium development. To explore the mechanisms by which these proteins regulate appressorium development, we have performed a comparative in planta transcriptomic study and identified an auxiliary activity family 9 protein (Aa9) homolog that we named MoAa91. We showed that MoAa91 was secreted from appressoria and that the recombinant MoAa91 could compete with a chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immunity. By identifying MoAa91 as a novel signaling molecule functioning in appressorium development and an effector in suppressing host immunity, our studies revealed a novel mechanism by which RGS and RGS-like proteins regulate pathogen-host interactions.

ABSTRACT

Mobile elements—plasmids and phages—are important components of microbial function and evolution via traits that they encode and their capacity to shuttle genetic material between species. We here report the unusually rich array of mobile elements within the genome of Arsenophonus nasoniae, the son-killer symbiont of the parasitic wasp Nasonia vitripennis. This microbe’s genome has the highest prophage complement reported to date, with over 50 genomic regions that represent either intact or degraded phage material. Moreover, the genome is predicted to include 17 extrachromosomal genetic elements, which carry many genes predicted to be important at the microbe-host interface, derived from a diverse assemblage of insect-associated gammaproteobacteria. In our system, this diversity was previously masked by repetitive mobile elements that broke the assembly derived from short reads. These findings suggest that other complex bacterial genomes will be revealed in the era of long-read sequencing.

IMPORTANCE The biology of many bacteria is critically dependent on genes carried on plasmid and phage mobile elements. These elements shuttle between microbial species, thus providing an important source of biological innovation across taxa. It has recently been recognized that mobile elements are also important in symbiotic bacteria, which form long-lasting interactions with their host. In this study, we report a bacterial symbiont genome that carries a highly complex array of these elements. Arsenophonus nasoniae is the son-killer microbe of the parasitic wasp Nasonia vitripennis and exists with the wasp throughout its life cycle. We completed its genome with the aid of recently developed long-read technology. This assembly contained over 50 chromosomal regions of phage origin and 17 extrachromosomal elements within the genome, encoding many important traits at the host-microbe interface. Thus, the biology of this symbiont is enabled by a complex array of mobile elements.

ABSTRACT

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.

IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.

Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

ABSTRACT

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated adcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the adcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated adcAII strains. However, transformation of adcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated adcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of adcAII. Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.

IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the adcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and adcAII, further investigation of which could further characterize mechanisms of capsule regulation.

ABSTRACT

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

ABSTRACT

Staphylococcus aureus can colonize the human host and cause a variety of superficial and invasive infections. The success of S. aureus as a pathogen derives from its ability to modulate its virulence through the release, sensing of and response to cyclic signaling peptides. Here we provide, for the first time, evidence that S. aureus processes and secretes small linear peptides through a specialized pathway that converts a lipoprotein leader into an extracellular peptide signal. We have identified and confirmed the machinery for each step and demonstrate that the putative membrane metalloprotease Eep and the EcsAB transporter are required to complete the processing and secretion of the peptides. In addition, we have identified several linear peptides, including the interspecies signaling molecule staph-cAM373, that are dependent on this processing and secretion pathway. These findings are particularly important because multiple Gram-positive bacteria rely on small linear peptides to control bacterial gene expression and virulence.

IMPORTANCE Here, we provide evidence indicating that S. aureus secretes small linear peptides into the environment via a novel processing and secretion pathway. The discovery of a specialized pathway for the production of small linear peptides and the identification of these peptides leads to several important questions regarding their role in S. aureus biology, most interestingly, their potential to act as signaling molecules. The observations in this study provide a foundation for further in-depth studies into the biological activity of small linear peptides in S. aureus.

ABSTRACT

Temporal dynamics of certain human microbiotas have been described in longitudinal studies; variability often relates to modifiable factors or behaviors. Early studies of the urinary microbiota preferentially used samples obtained by transurethral catheterization to minimize vulvovaginal microbial contributions. Whereas voided specimens are preferred for longitudinal studies, the few studies that reported longitudinal data were limited to women with lower urinary tract (LUT) symptoms, due to ease of accessing a clinical population for sampling and the impracticality and risk of collecting repeated catheterized urine specimens in a nonclinical population. Here, we studied the microbiota of the LUT of nonsymptomatic, premenopausal women using midstream voided urine (MSU) specimens to investigate relationships between microbial dynamics and personal factors. Using 16S rRNA gene sequencing and a metaculturomics method called expanded quantitative urine culture (EQUC), we characterized the microbiotas of MSU and periurethral swab specimens collected daily for approximately 3 months from a small cohort of adult women. Participants were screened for eligibility, including the ability to self-collect paired urogenital specimens prior to enrollment. In this population, we found that measures of microbial dynamics related to specific participant-reported factors, particularly menstruation and vaginal intercourse. Further investigation of the trends revealed differences in the composition and diversity of LUT microbiotas within and across participants. These data, in combination with previous studies showing relationships between the LUT microbiota and LUT symptoms, suggest that personal factors relating to the genitourinary system may be an important consideration in the etiology, prevention, and/or treatment of LUT disorders.

IMPORTANCE Following the discovery of the collective human urinary microbiota, important knowledge gaps remain, including the stability and variability of this microbial niche over time. Initial urinary studies preferentially utilized samples obtained by transurethral catheterization to minimize contributions from vulvovaginal microbes. However, catheterization has the potential to alter the urinary microbiota; therefore, voided specimens are preferred for longitudinal studies. In this report, we describe microbial findings obtained by daily assessment over 3 months in a small cohort of adult women. We found that, similarly to vaginal microbiotas, lower urinary tract (LUT) microbiotas are dynamic, with changes relating to several factors, particularly menstruation and vaginal intercourse. Our study results show that LUT microbiotas are both dynamic and resilient. They also offer novel opportunities to target LUT microbiotas by preventative or therapeutic means, through risk and/or protective factor modification.

Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK)-mediated calcium signaling has been widely reported to function in plant development and various stress responses, particularly in ion homeostasis. Sugars are the most important primary metabolites, and thus sugar homeostasis requires precise regulation. Here, we describe a CBL2-CIPK6-Tonoplast-Localized Sugar Transporter2 (TST2) molecular module in cotton (Gossypium hirsutum) that regulates plant sugar homeostasis, in particular Glc homeostasis. GhCIPK6 is recruited to the tonoplast by GhCBL2 and interacts with the tonoplast-localized sugar transporter GhTST2. Overexpression of either GhCBL2, GhCIPK6, or GhTST2 was sufficient to promote sugar accumulation in transgenic cotton, whereas RNAi-mediated knockdown of GhCIPK6 expression or CRISPR-Cas9-mediated knockout of GhTST2 resulted in significantly decreased Glc content. Moreover, mutation of GhCBL2 or GhTST2 in GhCIPK6-overexpressing cotton reinstated sugar contents comparable to wild-type plants. Heterologous expression of GhCIPK6 in Arabidopsis (Arabidopsis thaliana) also promoted Glc accumulation, whereas mutation of AtTST1/2 in GhCIPK6-overexpressing Arabidopsis similarly reinstated wild-type sugar contents, thus indicating conservation of CBL2-CIPK6-TST2-mediated sugar homeostasis among different plant species. Our characterization of the molecular players behind plant sugar homeostasis may be exploited to improve sugar contents and abiotic stress resistance in plants.

First described in 1967, one of the most vexing problems in the care of preterm infants continues to be bronchopulmonary dysplasia (BPD). The clinical presentation and pathological changes associated with BPD, also referred to as chronic lung disease of prematurity, have changed substantially since that initial description by Northway et al. [1]. The condition described in that seminal report, characterised by marked respiratory distress associated with pulmonary oedema due to shunting across the patent ductus arteriosus, was also specific to preterm infants that had received high inspired oxygen concentrations for at least a week.

The World Health Organization (WHO) has listed moxifloxacin and linezolid among the preferred "group A" drugs in the treatment of multidrug-resistant (MDR)-tuberculosis (TB) [1]. Therapeutic drug monitoring (TDM) could potentially optimise MDR-TB therapy, since moxifloxacin and linezolid show large pharmacokinetic variability [1–4]. TDM of moxifloxacin focuses on identifying patients with low drug exposure who are at risk of treatment failure and acquired fluoroquinolone resistance [5, 6]. Alternatively, TDM of linezolid strives to reduce toxicity while ensuring an adequate drug exposure because of its narrow therapeutic index [1, 3, 7].

Objective

To use the variations in neurology consultations requested by emergency department (ED) physicians to identify opportunities to implement multidisciplinary interventions in an effort to reduce ED overcrowding.

Early recognition of longus colli calcific tendinitis can prevent unnecessary interventions including antibiotics and surgical procedures.

Cystinuria is an autosomal recessive disorder characterized by excessive urinary excretion of cystine, resulting in recurrent cystine kidney stones, often presenting in childhood. Current treatment options for cystinuria include dietary and/or fluid measures and potassium citrate to reduce cystine excretion and/or increase solubility. Tiopronin and D-penicillamine are used in refractory cases to bind cystine in urine, albeit with serious side effects. A recent study revealed efficacy of nutritional supplement α-lipoic acid (ALA) treatment in preventing kidney stones in a mouse model of cystinuria. Here, we report 2 pediatric patients (6 and 15 years old) with cystinuria who received regular doses of ALA in addition to conventional therapy with potassium citrate. Both patients tolerated ALA without any adverse effects and had reduced frequency of symptomatic and asymptomatic kidney stones with disappearance of existing kidney stones in 1 patient after 2 months of ALA therapy. ALA treatment markedly improved laboratory markers of cystine solubility in urine with increased cystine capacity (–223 to –1 mg/L in patient 1 and +140 to +272 mg/L in patient 2) and decreased cystine supersaturation (1.7 to 0.88 in patient 1 and 0.64 to 0.48 in patient 2) without any changes in cystine excretion or urine pH. Our findings suggest that ALA improves solubility of cystine in urine and prevents stone formation in patients with cystinuria who do not respond to diet and citrate therapy.