Unmet needs in uncomplicated urinary tract infection in the United States and Germany: a physician survey.

BACKGROUND: Uncomplicated urinary tract infections (uUTIs/acute cystitis) are among the most common infections in women worldwide. There are differences in uUTI treatment guidelines between countries and understanding the needs of physicians in diverse healthcare systems is important for developing new treatments. We performed a survey of physicians in the United States (US) and Germany to understand their perceptions of, and management approaches to uUTI. METHODS: This was a cross-sectional online survey of physicians in the US and Germany who were actively treating patients with uUTI (≥ 10 patients/month). Physicians were recruited via a specialist panel and the survey was piloted with 2 physicians (1 US, 1 Germany) prior to study commencement. Data were analyzed with descriptive statistics. RESULTS: A total of 300 physicians were surveyed (n = 200 US, n = 100 Germany). Across countries and specialties, physicians estimated 16-43% of patients did not receive complete relief from initial therapy and 33-37% had recurrent infections. Urine culture and susceptibility testing was more common in the US and among urologists. The most commonly selected first-line therapy was trimethoprim-sulfamethoxazole in the US (76%) and fosfomycin in Germany (61%). Ciprofloxacin was the most selected following multiple treatment failures (51% US, 45% Germany). Overall, 35% of US and 45% of German physicians agreed with the statement "I feel there is a good selection of treatment options" and ≥ 50% felt that current treatments provided good symptom relief. More than 90% of physicians included symptom relief amongst their top 3 treatment goals. The overall impact of symptoms on patients' lives was rated "a great deal" by 51% of US and 38% of German physicians, increasing with each treatment failure. Most physicians (> 80%) agreed that antimicrobial resistance (AMR) is serious, but fewer (56% US, 46% Germany) had a high level of confidence in their knowledge of AMR. CONCLUSIONS: Treatment goals for uUTI were similar in the US and Germany, although with nuances to disease management approaches. Physicians recognized that treatment failures have a significant impact on patients' lives and that AMR is a serious problem, though many did not have confidence in their own knowledge of AMR.

Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high-resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.

Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection.

Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.

Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex.

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures-one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.

Ty3 nuclear entry is initiated by viruslike particle docking on GLFG nucleoporins.

Yeast retrotransposons form intracellular particles within which replication occurs. Because fungal nuclear membranes do not break down during mitosis, similar to retroviruses infecting nondividing cells, the cDNA produced must be translocated through nuclear pore complexes. The Saccharomyces cerevisiae long terminal repeat retrotransposon Ty3 assembles its Gag3 and Gag3-Pol3 precursor polyproteins into viruslike particles in association with perinuclear P-body foci. These perinuclear clusters of Ty3 viruslike particles localized to sites of clustered nuclear pore complexes (NPCs) in a nup120Delta mutant, indicating that Ty3 particles and NPCs interact physically. The NPC channels are lined with nucleoporins (Nups) with extended FG (Phe-Gly) motif repeat domains, further classified as FG, FxFG, or GLFG repeat types. These domains mediate partitioning of proteins between the cytoplasm and the nucleus. Here we have systematically examined the requirements for FG repeat domains in Ty3 nuclear transport. The GLFG domains interacted in vitro with virus-like particle Gag3, and this interaction was disrupted by mutations in the amino-terminal domain of Gag3, which is predicted to lie on the external surface of the particles. Accordingly, Ty3 transposition was decreased in strains with the GLFG repeats deleted. The spacer-nucleocapsid domain of Gag3, which is predicted to be internal to the particle, interacted with GLFG repeats and nucleocapsid localized to the nucleus. We conclude that Ty3 particle docking on nuclear pores is facilitated by interactions between Gag3 and GLFG Nups and that nuclear entry of the preintegration complex is further promoted by nuclear localization signals within the nucleocapsid and integrase.

Flexible gates: dynamic topologies and functions for FG nucleoporins in nucleocytoplasmic transport.

The nuclear envelope is a physical barrier between the nucleus and cytoplasm and, as such, separates the mechanisms of transcription from translation. This compartmentalization of eukaryotic cells allows spatial regulation of gene expression; however, it also necessitates a mechanism for transport between the nucleus and cytoplasm. Macromolecular trafficking of protein and RNA occurs exclusively through nuclear pore complexes (NPCs), specialized channels spanning the nuclear envelope. A novel family of NPC proteins, the FG-nucleoporins (FG-Nups), coordinates and potentially regulates NPC translocation. The extensive repeats of phenylalanine-glycine (FG) in each FG-Nup directly bind to shuttling transport receptors moving through the NPC. In addition, FG-Nups are essential components of the nuclear permeability barrier. In this review, we discuss the structural features, cellular functions, and evolutionary conservation of the FG-Nups.

A Novel Saccharomyces cerevisiae FG Nucleoporin Mutant Collection for Use in Nuclear Pore Complex Functional Experiments.

FG nucleoporins (Nups) are the class of proteins that both generate the permeability barrier and mediate selective transport through the nuclear pore complex (NPC). The FG Nup family has 11 members in Saccharomyces cerevisiae, and the study of mutants lacking different FG domains has been instrumental in testing transport models. To continue analyzing the distinct functional roles of FG Nups in vivo, additional robust genetic tools are required. Here, we describe a novel collection of S. cerevisiae mutant strains in which the FG domains of different groups of Nups are absent (Δ) in the greatest number documented to date. Using this plasmid-based ΔFG strategy, we find that a GLFG domain-only pore is sufficient for viability. The resulting extensive plasmid and strain resources are available to the scientific community for future in-depth in vivo studies of NPC transport.

Nucleoporin FG domains facilitate mRNP remodeling at the cytoplasmic face of the nuclear pore complex.

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG "swaps" demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.

From F = ma to flying squirrels: curricular change in an introductory physics course.

We present outcomes from curricular changes made to an introductory calculus-based physics course whose audience is primarily life sciences majors, the majority of whom plan to pursue postbaccalaureate studies in medical and scientific fields. During the 2011-2012 academic year, we implemented a Physics of the Life Sciences curriculum centered on a draft textbook that takes a novel approach to teaching physics to life sciences majors. In addition, substantial revisions were made to the homework and hands-on components of the course to emphasize the relationship between physics and the life sciences and to help the students learn to apply physical intuition to life sciences-oriented problems. Student learning and attitudinal outcomes were assessed both quantitatively, using standard physics education research instruments, and qualitatively, using student surveys and a series of postsemester interviews. Students experienced high conceptual learning gains, comparable to other active learning-based physics courses. Qualitatively, a substantial fraction of interviewed students reported an increased interest in physics relative to the beginning of the semester. Furthermore, more than half of students self-reported that they could now relate physics topics to their majors and future careers, with interviewed subjects demonstrating a high level of ability to come up with examples of how physics affects living organisms and how it helped them to better understand content presented in courses in their major.

Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport.

Transport of macromolecules between the nucleus and cytoplasm is a critical cellular process for eukaryotes, and the machinery that mediates nucleocytoplasmic exchange is subject to multiple levels of control. Regulation is achieved by modulating the expression or function of single cargoes, transport receptors, or the transport channel. Each of these mechanisms has increasingly broad impacts on transport patterns and capacity, and this hierarchy of control directly affects gene expression, signal transduction, development, and disease.