Elevated IOP following a bladder filling protocol: A case report.

Purpose: We describe a patient with elevated intraocular pressure (IOP) secondary to an oral water bolus and examine the utility of the water-drinking test. Observations: A 66-year-old male with a history of hypertension presented with headache, bilateral retro-orbital ache, and blurry vision. Symptoms began shortly after his radiation treatment for prostate cancer, for which he consumed a water bolus to fill his bladder 30 minutes prior to treatment initiation. On exam, he had bilateral elevated IOP that responded to topical IOP-lowering medications. Gonioscopy demonstrated open angles and fundus exam showed non-glaucomatous optic nerves with pronounced retinal venous tortuosity. The water-drinking test showed a peak intraocular pressure of 20 mmHg in the right eye (5 mmHg increase from baseline) and 23 mmHg in the left eye (8 mmHg increase from baseline), suggesting impairment of the outflow system in the left compared to the right eye. He was started on topical IOP-lowering therapy and followed in our clinic as a glaucoma suspect. Conclusions: Consumption of a water bolus can be associated with IOP elevation and may be a risk factor in patients with otherwise normal IOPs at risk for glaucoma. The water-drinking test was historically used as provocative testing for open-angle glaucoma and may have an updated role in evaluating at-risk patients without ocular hypertension.

Electron cryo-microscopy reveals the structure of the archaeal thread filament.

Pili are filamentous surface extensions that play roles in bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer. The model archaeaon Sulfolobus acidocaldarius assembles three filaments of the type-IV pilus superfamily (archaella, archaeal adhesion pili and UV-inducible pili), as well as a so-far uncharacterised fourth filament, named "thread". Here, we report on the cryo-EM structure of the archaeal thread. The filament is highly glycosylated and consists of subunits of the protein Saci_0406, arranged in a head-to-tail manner. Saci_0406 displays structural similarity, but low sequence homology, to bacterial type-I pilins. Thread subunits are interconnected via donor strand complementation, a feature reminiscent of bacterial chaperone-usher pili. However, despite these similarities in overall architecture, archaeal threads appear to have evolved independently and are likely assembled by a distinct mechanism.

Propulsive nanomachines: the convergent evolution of archaella, flagella and cilia.

Echoing the repeated convergent evolution of flight and vision in large eukaryotes, propulsive swimming motility has evolved independently in microbes in each of the three domains of life. Filamentous appendages - archaella in Archaea, flagella in Bacteria and cilia in Eukaryotes - wave, whip or rotate to propel microbes, overcoming diffusion and enabling colonization of new environments. The implementations of the three propulsive nanomachines are distinct, however: archaella and flagella rotate, while cilia beat or wave; flagella and cilia assemble at their tips, while archaella assemble at their base; archaella and cilia use ATP for motility, while flagella use ion-motive force. These underlying differences reflect the tinkering required to evolve a molecular machine, in which pre-existing machines in the appropriate contexts were iteratively co-opted for new functions and whose origins are reflected in their resultant mechanisms. Contemporary homologies suggest that archaella evolved from a non-rotary pilus, flagella from a non-rotary appendage or secretion system, and cilia from a passive sensory structure. Here, we review the structure, assembly, mechanism and homologies of the three distinct solutions as a foundation to better understand how propulsive nanomachines evolved three times independently and to highlight principles of molecular evolution.

The structure of the periplasmic FlaG-FlaF complex and its essential role for archaellar swimming motility.

Motility structures are vital in all three domains of life. In Archaea, motility is mediated by the archaellum, a rotating type IV pilus-like structure that is a unique nanomachine for swimming motility in nature. Whereas periplasmic FlaF binds the surface layer (S-layer), the structure, assembly and roles of other periplasmic components remain enigmatic, limiting our knowledge of the archaellum's functional interactions. Here, we find that the periplasmic protein FlaG and the association with its paralogue FlaF are essential for archaellation and motility. Therefore, we determine the crystal structure of Sulfolobus acidocaldarius soluble FlaG (sFlaG), which reveals a ß-sandwich fold resembling the S-layer-interacting FlaF soluble domain (sFlaF). Furthermore, we solve the sFlaG2-sFlaF2 co-crystal structure, define its heterotetrameric complex in solution by small-angle X-ray scattering and find that mutations that disrupt the complex abolish motility. Interestingly, the sFlaF and sFlaG of Pyrococcus furiosus form a globular complex, whereas sFlaG alone forms a filament, indicating that FlaF can regulate FlaG filament assembly. Strikingly, Sulfolobus cells that lack the S-layer component bound by FlaF assemble archaella but cannot swim. These collective results support a model where a FlaG filament capped by a FlaG-FlaF complex anchors the archaellum to the S-layer to allow motility.

Expression, Purification, and Assembly of Archaellum Subcomplexes of Sulfolobus acidocaldarius.

The archaellum assembly machinery and its filament consist of seven proteins in the crenarchaeon Sulfolobus acidocaldarius. We have so far expressed, purified, and biochemically characterized four of these archaellum subunits, namely, FlaX, FlaH, FlaI, and FlaF. FlaX, FlaH, and FlaI tightly interact and form the archaellum motor complex important for archaellum assembly and rotation. We have previously shown that FlaH forms an inner ring within a very stable FlaX ring, and therefore FlaX is believed to provide the scaffold for the assembly of the archaellum motor complex. Here we describe how to express and purify FlaX and FlaH and how the double ring structure both form can be obtained.

Initiative to reduce bone scans for low-risk prostate cancer patients: A quasi-experimental before-and-after study in a Veterans Affairs hospital.

PURPOSE: Bone scans (BS) are a low-value test for asymptomatic men with low-risk prostate cancer. We performed a quality improvement intervention aimed at reducing BS for these patients. METHODS AND MATERIALS: The intervention was a presentation that leveraged the behavioral science concepts of social comparison and normative appeals. Participants were multidisciplinary stakeholders from the Radiation Oncology and Urology services at a Veterans Affairs hospital. We determined the baseline rate of BS by retrospectively analyzing cases of asymptomatic men with newly diagnosed low-risk prostate cancer. For social comparison, we presented contemporary peer BS rates in the United States-including Veterans Affairs hospitals. For normative appeals, we reviewed guidelines from various professional groups. To analyze the effect of this intervention, we performed a quasi-experimental, uncontrolled, before-and-after study. RESULTS: During the 1-year period before the intervention, 32 of 37 patients with low-risk prostate cancer (86.5%) received a BS. The contemporary peer rate was approximately 30%. All reviewed guidelines recommended against BS. During the 1-year period after the intervention, the rate of BS was reduced to 65.5% (19 of 29 patients; P = .043 by one-sided Fisher's exact test). CONCLUSIONS: We observed a modest reduction in guideline-discordant BS after the quality improvement intervention. BS rates might be influenced by initiatives that combine social comparisons with appeals to professional norms.

FlaF Is a ß-Sandwich Protein that Anchors the Archaellum in the Archaeal Cell Envelope by Binding the S-Layer Protein.

Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand ß-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is a paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.

Insights into subunit interactions in the Sulfolobus acidocaldarius archaellum cytoplasmic complex.

Archaella are the archaeal motility structure that is the functional pendant of the bacterial flagellum but is assembled by a mechanism similar to that for type IV pili. Recently, it was shown by Banerjee et al. that FlaX, a crenarchaeal archaellum subunit from Sulfolobus acidocaldarius, forms a ring-like oligomer, and it was proposed that this ring may act as a static platform for torque generation in archaellum rotation [Banerjee A et al. (2012) J Biol Chem 287, 43322-43330]. Moreover, the hexameric crystal structure of FlaI was solved, and its dual function in the assembly and the rotation of the archaellum was demonstrated [Reindl S et al. (2013) Mol Cell 49, 1069-1082]. In this study, we show by biochemical and biophysical techniques that FlaX from S. acidocaldarius acts as a cytoplasmic scaffold in archaellum assembly, as it interacts with FlaI as well as with the recA family protein FlaH, the only cytoplasmic components of the archaellum. Interaction studies using various truncated versions of FlaI demonstrated that its N- and C-termini interact with FlaX. Moreover, using microscale thermophoresis, we show that FlaI, FlaX and FlaH interact with high affinities in the nanomolar range. Therefore, we propose that these three proteins form the cytoplasmic motor complex of the archaellum.

Elective lymph node irradiation with intensity-modulated radiotherapy: is conventional dose fractionation necessary?

PURPOSE: Intensity-modulated radiation therapy (IMRT) is the standard of care for head-and-neck cancer (HNC). We treated patients with HNC by delivering either a moderate hypofractionation (MHF) schedule (66 Gy at 2.2 Gy per fraction to the gross tumor [primary and nodal]) with standard dose fractionation (54-60 Gy at 1.8-2.0 Gy per fraction) to the elective neck lymphatics or a conventional dose and fractionation (CDF) schedule (70 Gy at 2.0 Gy per fraction) to the gross tumor (primary and nodal) with reduced dose to the elective neck lymphatics. We analyzed these two cohorts for treatment outcomes. METHODS AND MATERIALS: Between November 2001 and February 2009, 89 patients with primary carcinomas of the oral cavity, larynx, oropharynx, hypopharynx, and nasopharynx received definitive IMRT with or without concurrent chemotherapy. Twenty patients were treated using the MHF schedule, while 69 patients were treated with the CDF schedule. Patient characteristics and dosimetry plans were reviewed. Patterns of failure including local recurrence (LR), regional recurrence (RR), distant metastasis (DM), disease-free survival (DFS), overall survival (OS), and toxicities, including rate of feeding tube placement and percentage of weight loss, were reviewed and analyzed. RESULTS: Median follow-up was 31.2 months. Thirty-five percent of patients in the MHF cohort and 77% of patients in the CDF cohort received chemotherapy. No RR was observed in either cohort. OS, DFS, LR, and DM rates for the entire group at 2 years were 89.3%, 81.4%, 7.1%, and 9.4%, respectively. Subgroup analysis showed no significant differences in OS (p = 0.595), DFS (p = 0.863), LR (p = 0.833), or DM (p = 0.917) between these two cohorts. Similarly, no significant differences were observed in rates of feeding tube placement and percentages of weight loss. CONCLUSIONS: Similar treatment outcomes were observed for MHF and CDF cohorts. A dose of 50 Gy at 1.43 Gy per fraction may be sufficient to electively treat low-risk neck lymphatics.

Feedforward inhibition controls the spread of granule cell-induced Purkinje cell activity in the cerebellar cortex.

Synapses associated with the parallel fiber (pf) axons of cerebellar granule cells constitute the largest excitatory input onto Purkinje cells (PCs). Although most theories of cerebellar function assume these synapses produce an excitatory sequential "beamlike" activation of PCs, numerous physiological studies have failed to find such beams. Using a computer model of the cerebellar cortex we predicted that the lack of PCs beams is explained by the concomitant pf activation of feedforward molecular layer inhibition. This prediction was tested, in vivo, by recording PCs sharing a common set of pfs before and after pharmacologically blocking inhibitory inputs. As predicted by the model, pf-induced beams of excitatory PC responses were seen only when inhibition was blocked. Blocking inhibition did not have a significant effect in the excitability of the cerebellar cortex. We conclude that pfs work in concert with feedforward cortical inhibition to regulate the excitability of the PC dendrite without directly influencing PC spiking output. This conclusion requires a significant reassessment of classical interpretations of the functional organization of the cerebellar cortex.