Radial Spoke Proteins of \u3cem\u3eChlamydomonas\u3c/em\u3e Flagella

The radial spoke is a ubiquitous component of `9+2\u27 cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii radial spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional radial spoke proteins. Many of the newly identified proteins in the spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the radial spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes

Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella

To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2–GFP, about 10% of flagellar CrPKD2–GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2–GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating

Dynamics of cathode-associated microbial communities and metabolite profiles in a glycerol-fed bioelectrochemical system

Electrical current can be used to supply reducing power to microbial metabolism. This phenomenon is typically studied in pure cultures with added redox mediators to transfer charge. Here, we investigate the development of a current-fed mixed microbial community fermenting glycerol at the cathode of a bioelectrochemical system in the absence of added mediators and identify correlations between microbial diversity and the respective product outcomes. Within 1 week of inoculation, a Citrobacter population represented 95 to 99% of the community and the metabolite profiles were dominated by 1,3-propanediol and ethanol. Over time, the Citrobacter population decreased in abundance while that of a Pectinatus population and the formation of propionate increased. After 6 weeks, several Clostridium populations and the production of valerate increased, which suggests that chain elongation was being performed. Current supply was stopped after 9 weeks and was associated with a decrease in glycerol degradation and alcohol formation. This decrease was reversed by resuming current supply; however, when hydrogen gas was bubbled through the reactor during open-circuit operation (open-circuit potential) as an alternative source of reducing power, glycerol degradation and metabolite production were unaffected. Cyclic voltammetry revealed that the community appeared to catalyze the hydrogen evolution reaction, leading to a +400-mV shift in its onset potential. Our results clearly demonstrate that current supply can alter fermentation profiles; however, further work is needed to determine the mechanisms behind this effect. In addition, operational conditions must be refined to gain greater control over community composition and metabolic outcomes

Longitudinal Assessments Using Validated Instruments In Myasthenia Gravis Outpatients Receiving Long-Term Therapeutic Plasma Exchange

Purpose: In the most recent American Society for Apheresis Guidelines on the Use of Therapeutic Apheresis in Clinical Practice, employing therapeutic plasma exchange (TPE) for long-term treatment of myasthenia gravis (MG) patients is a new indication and carries a category II, grade 2B recommendation. As data for this indication is evolving and subjective assessments of these patients are often uninformative, we sought to better characterize the impacts of long-term TPE in these MG patients using validated instruments. Methods: In this prospective observational study, we used a combination of validated instruments and openended questions clarifying concerns that are routinely applied to MG patients. The two validated instruments were the MG Activities of Daily Living (MG-ADL) and MG Quality of Life 15 (MG-QoL15r) profiles. Based on previous literature, a 2-point change in the MG-ADL and a 10-point change in the MGQoL15r indicates significant improvement or worsening. Over a 3-month period, MG patients receiving long-term TPE were assessed using a single-form questionnaire that integrated all of the MG-ADL and MG-QoL15r elements and was completed at every visit. Patients unable to complete the survey due to their medical condition were exempted. Results: In total, 9 patients were eligible and received long-term TPE at frequencies from 3 times per week to 1 time per month. Five patients (56%) were female, 6 (67%) were white, and mean age was 54 years. Active pharmacotherapy included prednisone, azathioprine, mycophenolate, rituximab, and pyridostigmine. All patients reported that lengthening the interval between successive TPE treatments, even by a few days, resulted in noticeable MG changes. Five patients (56%) were clinically stable during the study period and had no significant changes in MG-ADL or MG-QoL15r profiles, though these patients still documented issues with sight, speech, muscular weakness, and fatigue. During the study period, 4 patients (44%) had significant changes identified by the MG-ADL, a mean of 5.5 times per patient (range 2-8) and 2 (22%) had significant changes identified by the MG-QoL15r, a mean of 2 times per patient (range 1-3). MG-ADL appeared to be more sensitive in correlating with patient-reported clinical changes, with clinical improvements identified a mean of 3.2 times per patient and clinical deteriorations identified a mean of 2.3 times per patient (compared to 1.5 and 1 times per patient, respectively, for the MG-QoL15r; p=0.03 for interaction effect). In the 4 patients with significant changes in profile scores, associated clinical changes in breathing, swallowing, sight, speech, muscular strength, and fatigue were reported. Importantly, for all of 4 of these patients, subjective clinical deteriorations in conjunction with objectively worsening MG-ADL scores were used as evidence to medically justify intensification of TPE therapy. Conclusions: Objective longitudinal assessments in MG patients receiving long-term TPE may be helpful for accurate disease monitoring. A subset of MG patients receiving long-term TPE still has dynamic changes in disease status as assessed by clinical history and two different validated instruments. In all patients with stable MG, both the MD-ADL and MG-QoL15r accurately indicated no significant changes. However, in those with fluctuating disease status, MG-ADL was more sensitive to both clinical improvement and worsening. These findings need to be validated in larger studies

The Versatile Molecular Complex Component LC8 Promotes Several Distinct Steps of Flagellar Assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3’s LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly

Policy Feedback and the Politics of the Affordable Care Act

There is a large body of literature devoted to how “policies create politics” and how feedback effects from existing policy legacies shape potential reforms in a particular area. Although much of this literature focuses on self‐reinforcing feedback effects that increase support for existing policies over time, Kent Weaver and his colleagues have recently drawn our attention to self‐undermining effects that can gradually weaken support for such policies. The following contribution explores both self‐reinforcing and self‐undermining policy feedback in relationship to the Affordable Care Act, the most important health‐care reform enacted in the United States since the mid‐1960s. More specifically, the paper draws on the concept of policy feedback to reflect on the political fate of the ACA since its adoption in 2010. We argue that, due in part to its sheer complexity and fragmentation, the ACA generates both self‐reinforcing and self‐undermining feedback effects that, depending of the aspect of the legislation at hand, can either facilitate or impede conservative retrenchment and restructuring. Simultaneously, through a discussion of partisan effects that shape Republican behavior in Congress, we acknowledge the limits of policy feedback in the explanation of policy stability and change

Delays in Leniency Application: Is There Really a Race to the Enforcer's Door?

This paper studies cartels’ strategic behavior in delaying leniency applications, a take-up decision that has been ignored in the previous literature. Using European Commission decisions issued over a 16-year span, we show, contrary to common beliefs and the existing literature, that conspirators often apply for leniency long after a cartel collapses. We estimate hazard and probit models to study the determinants of leniency-application delays. Statistical tests find that delays are symmetrically affected by antitrust policies and macroeconomic fluctuations. Our results shed light on the design of enforcement programs against cartels and other forms of conspiracy