Imaging S-Adenosyl Methionine Dynamics in Living Cells Using an RNA-Based Fluorescent Sensor.

S-Adenosyl methionine (SAM) is a critical metabolite involved in numerous cellular processes, including DNA methylation and gene expression regulation. Understanding the spatiotemporal dynamics of SAM within living cells is essential for deciphering its roles in maintaining cell homeostasis and in disease development. Here, we describe a protocol based on a recently reported SAM sensor exploiting a fluorogenic RNA and an RNA three-way junction for visualizing SAM dynamics in cultured mammalian cells.

Demographics and Characteristics of US-Based Minimally Invasive Gynecologic Surgery Fellowship Program Directors: A 2023 Descriptive Analysis.

STUDY OBJECTIVE: Fellowship program directors (FPDs) play an important role in the development of fellows and learners, but little is known about their demographics. This cross-sectional study aims to examine the characteristics of minimally invasive gynecologic surgery (MIGS) FPDs. DESIGN: A retrospective cross-sectional study. SETTING: Data obtained from publicly available information on official websites of the program directors studied. SUBJECTS: MIGS fellowship program directors. INTERVENTIONS: All US-based MIGS programs affiliated with the AAGL in 2023 were included. Information about FPD gender, medical school attended and graduation year, residency program attended and graduation year, any additional graduate degrees earned, fellowship programs completed, and the year of their appointment as FPD was collected through publicly available sources. Scholarly activity was measured by peer-reviewed articles and the Hirsch index. MEASUREMENTS AND MAIN RESULTS: Of the 54 FPDs, 28 (51.85%) were female and 26 (48.15%) were male. Male FPDs were significantly older (54.6 ± 8.7 years) than female FPDs (46.2 ± 5.0 years), p <.05. Average age at appointment was 43.1 ± 6.7 years, with female FPDs being appointed at significantly younger ages (39.4 ± 5.1 years) compared to male FPDs (44.5 ± 6.8 years), p <.05. Male FPDs had statistically significant higher Hirsch indices (14 ± 11.4) compared to female FPDs (8 ± 5.8), p <.05. Of the FPDs who completed a fellowship, 27 (50%) did so in MIGS, eight (14.81%) in Gynecologic-Oncology, 6 (11.11%) in Urogynecology, and 4 (7.41%) in Reproductive Endocrinology/Infertility. CONCLUSIONS: MIGS fellowships have a uniquely equal representation of male and female FPDs, as surgical subspecialties historically tend to be male dominant. Notably, there is diversity in the type of fellowship pursued by MIGS FPDs, with nearly half of FPDs completing a fellowship outside of MIGS. The reasons for differences in scholarly contributions, indicated by Hirsch index, of male versus female FPDs is unclear.

Analysis of Reapplicants to Ophthalmology Residency: Factors Associated with Successful Matching.

BACKGROUND: Ophthalmology residency programs are highly competitive and each year there are many unmatched reapplicants who must make time-sensitive decisions on how to prepare for their reapplication. Our analysis of reapplication factors will be the first evidence-based guide to reapplying ophthalmology. OBJECTIVE: To determine the components of a reapplicants application that contribute and that do not contribute to ophthalmology residency match success. METHODS: Eighty-eight total reapplicants to Dell Medical School's Department of Ophthalmology residency program for the year of 2022 and 2023 were analyzed in a retrospective review in 2023 based on their San Francisco match applications. We assessed match success in the context of demographics, academic performance, cognitive measures, letters of recommendation, personal statements, and extracurricular activities. RESULTS: Of 84 reapplicants who completed the San Francisco Match, 41 matched successfully (48.8%). Factors that significantly affected match success include quantity of LORs written by ophthalmologists (P = .0143), choice of interim year activity (p = .0019), and Step 2 Clinical Knowledge score (P = .0017). Our findings demonstrate that noncontributory factors to reapplicant match rate include number of publications during research years, demographics, clinical grades, Alpha Omega Alpha (AOA) membership, LOR qualities, and Step 1 score. CONCLUSIONS: Ophthalmology residency reapplicants should focus their efforts on finding a research year or established pre-ophthalmology residency program for their interim year and bolstering their letters of recommendations within the field. The quality of in-specialty relationships and letters of recommendation are more impactful than the number of publications during a research gap year. Step 2 CK score is a significant factor but would have already been determined by the time of reapplication. Metrics such as Step 1 scores, AOA status, number of volunteering experiences, and LOR and personal statement qualities had no effect on a reapplicant's chance of matching.

Probable ponatinib-induced papilledema in a patient with acute lymphocytic leukemia.

Purpose: Papilledema is a very rare complication of leukemia therapies, and particularly tyrosine kinase inhibitor (TKI) therapy. Targeted oncologic therapies are becoming increasingly popular, so it is increasingly important to report rare adverse effects. We present a case of probable papilledema in the setting of ponatinib therapy for acute lymphoblastic leukemia. Observations: Our patient is a 48-year-old male who was diagnosed with acute lymphocytic leukemia. He underwent stem cell transplantation and shortly after was placed on ponatinib therapy. After initiation of ponatinib, he began to note decreased clarity in the inferonasal visual field of his right eye, corroborated on Humphrey visual field (HVF) testing. Neuroimaging was only notable for a partially empty sella. Lumbar puncture demonstrated opening pressures at the upper limit of normal (23 cm H2O) but with normal cellular constituents and chemistry. Slit lamp exam did not reveal any signs of ocular inflammation. Dilated funduscopic examination (DFE) revealed 360-degree blurring of the right optic disc margin as well as 270-degree blurring of the left optic disc (sparing the temporal border). Optical coherence tomography of the retinal nerve fiber layer (OCT-RNFL) showed increased RNFL thickness of 272 µm in the right eye and 113 µm in the left eye. In the absence of evidence for other possible etiologies of optic disc edema, ponatinib-induced papilledema was suspected. No changes to the ponatinib regimen were made; however, the patient was started on acetazolamide 500 mg twice a day. At three-month follow up, the patient reported resolution of his right eye blurriness and his repeat HVF, OCT-RNFL, and DFE showed resolution of optic disc edema, supporting that his initial bilateral optic disc swelling was likely ponatinib-induced papilledema. Conclusions and importance: This is the first report of probable ponatinib-induced papilledema. This case expands on the literature of TKI induced papilledema and demonstrates successful treatment with an oral acetazolamide regimen.

Intricate 3D architecture of a DNA mimic of GFP.

Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures1-3. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates4 conditional fluorophores derived from GFP. To extend previous structural studies5,6 of fluorogenic RNAs, GFP and other fluorescent proteins7 to DNA, we characterize Lettuce-fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen8 in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding-including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA-and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs.

Readability and Suitability of Online Uveitis Patient Education Materials.

PURPOSE: This study assesses readability, suitability, and accountability of online uveitis patient education materials (PEMs). METHODS: Two uveitis specialists reviewed the top 10 websites on Google for keyword "uveitis," with a PubMed review control. Readability was assessed using an online calculator, suitability using the Suitability Assessment of Materials (SAM) tool, and accountability using JAMA benchmarks. RESULTS: The mean SAM score was 21.05, indicating that websites were on average adequately suitable to educate patients. WebMD Uveitis website scored the highest at 25.5, while allaboutvision.org scored the lowest at 18.0. The average Flesch Reading Ease (FRE) score was 44.0 (95% CI: 34.2, 53.8). The average reading grade score was 11.0 (95% CI: 9.4, 12.6). WebMD Uveitis page scored highest on readability. The average accountability score among sites was 2.36 out of 4. CONCLUSIONS: Uveitis websites are only adequately suitable as PEMs, and all are above the recommended reading level. Uveitis specialists should advise patients on quality of online PEMs.

Directors of accredited pediatric ophthalmology fellowship programs: a descriptive analysis.

PURPOSE: To examine the characteristics of pediatric ophthalmology fellowship program directors (FPDs) through cross-sectional analysis. METHODS: All pediatric ophthalmology FPDs whose programs participated in the San Francisco Match in January 2020 were included. Information was collected through publicly available sources. Scholarly activity was measured by peer-reviewed articles and the Hirsch index. RESULTS: Of the 43 FPDs, 22 (51%) were male, and 21 (49%) were female. The mean age of current FPDs was 53.5 ± 8.8 years old. There was a significant difference between the current age of male and female FPDs (57.8 ± 8 vs 49 ± 7.3, resp. [P <0.0001]). The mean term length of female and male FPDs also differed (11.5 ± 4.5 vs 16.1 ± 8.9 [P = 0.042]). Thirty-eight FPDs (88%) attended medical school in the United States. Forty-two FPDs had an MD (98%). Thirty-nine (91%) FPDs completed ophthalmology residency in the United States. Ten (23%) of the FPDs were dual fellowship trained. There was a significantly higher Hirsch-index among male than female FPDs (23.9 ± 15.7 vs 10.3 ± 10.1 [P = 0.0017]). There was a higher number of publications for male (91 ± 89) than female FPDs (31.5 ± 48.6 [P = 0.0099]). CONCLUSIONS: Pediatric ophthalmology fellowship programs have a uniquely equal representation of male and female FPDs as women continue to be underrepresented in ophthalmology. Female FPDs were younger and had spent less time in the position, suggesting a shift toward more female FPD over time.

Control of Gαq signaling dynamics and GPCR cross-talk by GRKs.

Numerous processes contribute to the regulation of G protein-coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gαq both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gαq-dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gαq-dependent GPCR regulation with wide-ranging pharmacological and physiological implications.

Bilateral posterior central corneal steepening with decreased visual acuity.

A 40-year-old woman was referred for the assessment of bilateral corneal opacities with gradual visual decline over the course of the past decade. Her past ocular history is significant for bilateral amblyopia and strabismus surgery in both eyes before age 5. The patient's parents were told by her childhood ophthalmologist that she had a hereditary disorder. Her systemic review was significant for anal fissure and human leukocyte antigen-B27 ankylosing spondylitis. Her past ocular record revealed corrected distance visual acuity (CDVA) of 20/80 in both eyes in 2018 with central corneal haze. On presentation, her uncorrected distance visual acuity was 20/150 in both eyes. Her CDVA was 20/100 in both eyes with manifest refraction of +0.50 -2.50 × 075 in the right eye and +5.00 -2.25 × 094 in the left eye. Corneal topography reflected keratometry of 35.75/38.97 × 171 in the right eye and 36.45/38.35 × 32 in the left eye. Central corneal thickness was 669 µm and 652 µm, respectively. External slitlamp examination revealed a central faint stromal opacity inferior to the visual axis in the right eye and a central faint stromal opacity in the left eye, and both were associated with steep posterior curvature of the cornea (Figure 1). Further findings included 0.5 corneal haze with mild guttata, normal irides, and clear lenses in both eyes. Intraocular pressure was 23 mm Hg and 26 mm Hg, respectively (Figure 2, Supplemental Figures 1 and 2, http://links.lww.com/JRS/A543). Gonioscopy was unremarkable. Dilated fundus examination revealed a 0.15 cup-to-disc ratio bilaterally, but otherwise no pertinent vitreoretinal pathologies were noted. What is the most likely diagnosis? What medical or surgical interventions would you recommend for this patient? What is the prognosis for this patient?

Detection of SARS-CoV-2 RNA Using a DNA Aptamer Mimic of Green Fluorescent Protein.

RNA detection is important in diverse diagnostic and analytical applications. RNAs can be rapidly detected using molecular beacons, which fluoresce upon hybridizing to a target RNA but require oligonucleotides with complex fluorescent dye and quencher conjugations. Here, we describe a simplified method for rapid fluorescence detection of a target RNA using simple unmodified DNA oligonucleotides. To detect RNA, we developed Lettuce, a fluorogenic DNA aptamer that binds and activates the fluorescence of DFHBI-1T, an otherwise nonfluorescent molecule that resembles the chromophore found in green fluorescent protein. Lettuce was selected from a randomized DNA library based on binding to DFHBI-agarose. We further show that Lettuce can be split into two separate oligonucleotide components, which are nonfluorescent on their own but become fluorescent when their proximity is induced by a target RNA. We designed several pairs of split Lettuce fragments that contain an additional 15-20 nucleotides that are complementary to adjacent regions of the SARS-CoV-2 RNA, resulting in Lettuce fluorescence only in the presence of the viral RNA. Overall, these studies describe a simplified RNA detection approach using fully unmodified DNA oligonucleotides that reconstitute the Lettuce aptamer templated by RNA.

Naturally occurring three-way junctions can be repurposed as genetically encoded RNA-based sensors.

Small molecules can be imaged in living cells using biosensors composed of RNA. However, RNA-based devices are difficult to design. Here, we describe a versatile platform for designing RNA-based fluorescent small-molecule sensors using naturally occurring highly stable three-way junction RNAs. We show that ligand-binding aptamers and fluorogenic aptamers can be inserted into three-way junctions and connected in a way that enables the three-way junction to function as a small-molecule-regulated fluorescent sensor in vitro and in cells. The sensors are designed so that the interhelical stabilizing interactions in the three-way junction are only induced upon ligand binding. We use these RNA-based devices to measure the dynamics of S-adenosylmethionine levels in mammalian cells in real time. We show that this strategy is compatible with diverse metabolite-binding RNA aptamers, fluorogenic aptamers, and three-way junctions. Overall, these data demonstrate a versatile method for readily generating RNA devices that function in living cells.

Obesity-Related Foot Pain: Diagnosis and Surgical Planning.

Obese patients are at higher risk for surgical complications and consist of a large portion of podiatric patients. Obese patients are additionally at increased risk of developing specific podiatric conditions, and it is important to be able to identify and appropriately treat these conditions accordingly. Initially, conservative treatment is adequate for a variety of pathologic conditions related to obesity. Occasionally surgical intervention is warranted depending on the severity and lack of response to conservative measures. Arthrodesis-type procedures are often preferable and may be necessary, as opposed to periarticular osteotomy, in obese patients even if the deformity is flexible.

Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.

Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.

Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA-fluorophore complexes would extend the utility of this technology, the identification of novel RNA-fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA-fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.

Low-profile titanium plate construct for early weightbearing with first metatarsophalangeal joint arthrodesis.

First metatarsophalangeal joint arthrodesis is a useful procedure for various first ray pathologic entities. Multiple constructs for fixation have provided successful fusion. A retrospective study of 21 feet (18 patients) was performed after first metatarsophalangeal joint fusion using crossed Kirschner wires or compression fixation with cannulated screws followed by the application of a 2-hole low-profile partially locking titanium plate. The median age was 59 (range 41 to 76) years, and we had 4 smokers and 3 patients with diabetes in our series. Postoperatively, a compression dressing with a posterior splint was applied. The patients then transitioned to a controlled ankle motion walker, and all patients reported full weightbearing by 2 weeks postoperatively. The mean follow-up duration was 11.43 (range 6 to 27) months. The overall primary fusion rate was 95.24% (20 of 21). Two nonunions occurred; one was asymptomatic and successfully consolidated at 12 months. The second nonunion required revisional surgery with an autogenous bone graft to heal successfully. This patient was noncompliant with the postoperative regimen and had a 48-pack year history of tobacco usage. Our results have shown early weightbearing after first metatarsophalangeal joint arthrodesis can be successfully initiated with splintage or lag screw fixation and a 2-hole, low-profile, partially locking titanium plate.

Digital arthrodesis: current fixation techniques.

Several hammertoe implant devices have recently been introduced in an attempt to provide optimal fixation for proximal interphalangeal joint arthrodesis. This article reviews these implants individually, and discusses their advantages and disadvantages. There is a lack of research with long-term follow-up available for these devices. Percutaneous Kirschner-wire fixation persists as a time-honored and effective method of fixation. The buried Kirschner-wire technique is also an effective, cost-conscious option, with many of the same advantages as newer implantable devices.

First metatarsophalangeal joint arthrodesis: current fixation options.

This article reviews the current literature on first metatarsophalangeal joint arthrodesis rates using various forms of fixation, as well as reviewing biomechanical studies comparing the strengths of the different fixation options that are available.