Function and Structure of FlaK, a Master Regulator of the Polar Flagellar Genes in Marine Vibrio.

Vibrio alginolyticus has a flagellum at the cell pole, and the fla genes, involved in its formation, are hierarchically regulated in several classes. FlaK (also called FlrA) is an ortholog of Pseudomonas aeruginosa FleQ, an AAA+ ATPase that functions as a master regulator for all later fla genes. In this study, we conducted mutational analysis of FlaK to examine its ATPase activity, ability to form a multimeric structure, and function in flagellation. We cloned flaK and confirmed that its deletion caused a nonflagellated phenotype. We substituted amino acids at the ATP binding/hydrolysis site and at the putative subunit interfaces in a multimeric structure. Mutations in these sites abolished both ATPase activity and the ability of FlaK to induce downstream flagellar gene expression. The L371E mutation, at the putative subunit interface, abolished flagellar gene expression but retained ATPase activity, suggesting that ATP hydrolysis is not sufficient for flagellar gene expression. We also found that FlhG, a negative flagellar biogenesis regulator, suppressed the ATPase activity of FlaK. The 20 FlhG C-terminal residues are critical for reducing FlaK ATPase activity. Chemical cross-linking and size exclusion chromatography revealed that FlaK mostly exists as a dimer in solution and can form multimers, independent of ATP. However, ATP induced the interaction between FlhG and FlaK to form a large complex. The in vivo effects of FlhG on FlaK, such as multimer formation and/or DNA binding, are important for gene regulation. IMPORTANCE FlaK is an NtrC-type activator of the AAA+ ATPase subfamily of σ54-dependent promoters of flagellar genes. FlhG, a MinD-like ATPase, negatively regulates the polar flagellar number by collaborating with FlhF, an FtsY-like GTPase. We found that FlaK and FlhG interact in the presence of ATP to form a large complex. Mutational analysis revealed the importance of FlaK ATPase activity in flagellar gene expression and provided a model of the Vibrio molecular mechanism that regulates the flagellar number.

Femtosecond Lasers in Cornea & Refractive Surgery.

Since the introduction of femtosecond laser (FS) systems for corneal flap creation in laser-assisted in-situ keratomileusis there have been numerous applications for FS laser in corneal surgery. This manuscript details the utility of FS lasers in corneal surgical procedures including refractive laser surgeries, intracorneal ring segment tunnels, presbyopic treatments, and FS-assisted keratoplasty. We also review the role of FS lasers in diagnostic procedures such as two photon excitation fluorescence and second harmonic generation.

Effect of changes at the conserved + 3 position of mature archaellins on in vitro cleavage by the pre-archaellin peptidase FlaK of Methanococcus maripaludis.

Archaea swim using archaella that are domain-specific rotary type IV pilus-like appendages. The structural components of the archaellum filament are archaellins, initially made as preproteins with type IV pilin-like signal peptides which are removed by signal peptidases that are homologues of prepilin peptidases that remove signal peptides from type IV pilins. N-terminal sequences of archaellins, including the signal peptide cleavage site, are conserved and various positions have been previously shown to be critical for signal peptide removal. Archaellins have an absolute conservation of glycine at the + 3 position from the signal peptide cleavage site. To investigate its role in signal peptide cleavage, I used archaellin variants in which the + 3 glycine was mutated to all other possibilities in in vitro cleavage reactions. Cleavage was observed with ten different amino acids at the + 3 position, indicating that the observed glycine conservation is not required for this essential processing step.

Refractive Astigmatism Outcomes of Femtosecond Laser-Assisted Arcuate Keratotomies Combined with Femtosecond Laser-Assisted Cataract Surgery: Two-Year Results.

PURPOSE: To evaluate the effectiveness and stability of refractive astigmatism reduction after penetrating femtosecond laser-assisted arcuate keratotomy performed at the time of femtosecond laser-assisted cataract surgery. METHODS: Non-randomized retrospective data analysis of all patients that underwent femtosecond laser-assisted cataract surgery with femtosecond laser-assisted arcuate keratotomy over a 4-year period with a non-toric monofocal intraocular lens (2017-2021) at a tertiary care academic center. Postoperative visual acuity, manifest refraction, and predicted residual refractive error were also recorded at 1 month, 3-6 months, 12-18 months, and 2 years postoperatively. Preoperative keratometric astigmatism was compared to postoperative refractive astigmatism using vector calculations and the ASCRS double-angle plot tool. RESULTS: This study comprised 266 eyes (179 patients) that met inclusion criteria. The mean preoperative keratometric astigmatism magnitude was 0.99 ± 0.53 D. At 1 month, 3-6 months, 12-18 months, and 2 years postoperatively, the mean refractive cylinder was 0.49 ± 0.45 D, 0.49 ± 0.45 D, 0.55 ± 0.54 D, and 0.52 ± 0.46 D, respectively. Horizontal against-the-rule astigmatism showed a higher tendency toward undercorrection than vertical with-the-rule astigmatism, which had a slightly higher tendency toward overcorrection. With-the-rule astigmatism had smaller difference vectors between target-induced astigmatism and surgically induced astigmatism. CONCLUSIONS: Femtosecond laser-assisted arcuate keratotomy performed at the time of femtosecond laser-assisted cataract surgery was an effective option for correcting low-to-moderate corneal astigmatism for up to 2 years.

Combined Femtosecond Laser-Assisted Keratotomy and Cataract Surgery for Enhancing Refractive Outcomes. An Indonesian Case Study.

Purpose: We evaluate the reduction of corneal astigmatism and the improvement of visual outcomes of this surgical method in the Indonesian population. We also assess the accuracy and predictability of using femtosecond laser astigmatic keratotomy (FLAK) combined with cataract surgery. Patients and Methods: In a retrospective study, a total of 275 subjects (78 with against-the-rule (ATR) astigmatism, 178 with with-the-rule (WTR) astigmatism, and 19 with oblique (OBL) astigmatism) with preexisting corneal astigmatism ranging from 0.75D to 3.00D underwent FLAK. All subjects completed a 3-month follow-up. The femtosecond laser used for creating paired AK 2.2 mm, primary incision, and paracentesis incision was the FEMTO Z8 NEO from Ziemer Ophthalmic System, Switzerland. The surgical approach was guided by the "NAPA" nomogram. Results: The reduction in postoperative astigmatism was 56.90% for the WTR group, 49.46% for the ATR group, and 47.33% for the oblique group. A significant reduction in astigmatism was observed at the 1-week, 1-month, and 3-month follow-up intervals in both the WTR and ATR groups. The reduction in astigmatism was more favorable in cases of moderate astigmatism within the WTR group, as compared to the ATR and oblique groups. Postoperative astigmatism reduction was found to be more predictable in the right eye than in the left eye. Conclusion: The combination of FLAK can be considered as a potential method for reducing corneal astigmatism ranging from 1.00D to <3.00D. The highest reduction was observed in the WTR group, along with a higher rate of intended correction without astigmatism meridian shift in the right eye for the WTR group. However, factors such as cyclotorsion resulting from the surgical technique, alignment of docking, incision length, and preoperative astigmatism need to be taken into account for further enhancement and predictability of astigmatism reduction with this method.

Regulation of the Single Polar Flagellar Biogenesis.

Some bacterial species, such as the marine bacterium Vibrio alginolyticus, have a single polar flagellum that allows it to swim in liquid environments. Two regulators, FlhF and FlhG, function antagonistically to generate only one flagellum at the cell pole. FlhF, a signal recognition particle (SRP)-type guanosine triphosphate (GTP)ase, works as a positive regulator for flagellar biogenesis and determines the location of flagellar assembly at the pole, whereas FlhG, a MinD-type ATPase, works as a negative regulator that inhibits flagellar formation. FlhF intrinsically localizes at the cell pole, and guanosine triphosphate (GTP) binding to FlhF is critical for its polar localization and flagellation. FlhG also localizes at the cell pole via the polar landmark protein HubP to directly inhibit FlhF function at the cell pole, and this localization depends on ATP binding to FlhG. However, the detailed regulatory mechanisms involved, played by FlhF and FlhG as the major factors, remain largely unknown. This article reviews recent studies that highlight the post-translational regulation mechanism that allows the synthesis of only a single flagellum at the cell pole.