Prediction of long-term visual outcome of idiopathic full-thickness macular hole surgery using optical coherence tomography parameters that estimate potential preoperative photoreceptor damage.

PURPOSE: To identify optical coherence tomography (OCT) parameters that predict postoperative best corrected visual acuity (BCVA) and are based on recent understanding of the pathomechanism of idiopathic full thickness macular hole (iFTMH) formation and closure. METHODS: A retrospective consecutive case series of patients who had macular hole (MH) surgery at our institution between 2016 and 2022 was performed. 32 eyes of 30 patients were selected with at least 12 months of follow-up, closed MH and good quality OCT at each visit. Univariate correlation analysis, multiple logistic regression with forward stepwise selection, and Akaike's Information Criterion (AIC) were used to identify the best predictors for postoperative BCVA at 6 and 12 months (M), and final (≥ 12 M) visits, and a new OCT index was created. Abilities of best models/indices to predict < 0.30 logMAR (> 20/40) BCVA were compared to macular hole index (MHI) using the area under the receiver operating curve (AU-ROC) analysis. RESULTS: Statistical analysis revealed base diameter (B) (6 M), preoperative BCVA and B (12 M) and smaller ELM-GCL distance (A), and B (final visit) as predictors for postoperative BCVA. AU-ROC analysis indicated greatest AUC at 6 M for MHI and B (0.797, p = 0.004 and 0.836 p = 0.001, respectively) and for the new A/B index at 12 M and final visit (0.844, p = 0.002 and 0.913, p = 0.003, respectively). CONCLUSION: Our study suggests that MHI and B can be useful predictors of short term BCVA while the new A/B index that incorporates OCT parameters indicating potential preoperative photoreceptor damage may be a good predictor for long term postoperative BCVA. Our findings support the theory that initial hole formation mechanisms and photoreceptor damage define visual prognosis.

Using Selective Enzymes to Measure Noncanonical DNA Building Blocks: dUTP, 5-Methyl-dCTP, and 5-Hydroxymethyl-dCTP.

Cells maintain a fine-tuned balance of deoxyribonucleoside 5'-triphosphates (dNTPs), a crucial factor in preserving genomic integrity. Any alterations in the nucleotide pool's composition or chemical modifications to nucleotides before their incorporation into DNA can lead to increased mutation frequency and DNA damage. In addition to the chemical modification of canonical dNTPs, the cellular de novo dNTP metabolism pathways also produce noncanonical dNTPs. To keep their levels low and prevent them from incorporating into the DNA, these noncanonical dNTPs are removed from the dNTP pool by sanitizing enzymes. In this study, we introduce innovative protocols for the high-throughput fluorescence-based quantification of dUTP, 5-methyl-dCTP, and 5-hydroxymethyl-dCTP. To distinguish between noncanonical dNTPs and their canonical counterparts, specific enzymes capable of hydrolyzing either the canonical or noncanonical dNTP analogs are employed. This approach provides a more precise understanding of the composition and noncanonical constituents of dNTP pools, facilitating a deeper comprehension of DNA metabolism and repair. It is also crucial for accurately interpreting mutational patterns generated through the next-generation sequencing of biological samples.

dNTPpoolDB: a manually curated database of experimentally determined dNTP pools and pool changes in biological samples.

Stimulated by the growing interest in the role of dNTP pools in physiological and malignant processes, we established dNTPpoolDB, the database that offers access to quantitative data on dNTP pools from a wide range of species, experimental and developmental conditions (https://dntppool.org/). The database includes measured absolute or relative cellular levels of the four canonical building blocks of DNA and of exotic dNTPs, as well. In addition to the measured quantity, dNTPpoolDB contains ample information on sample source, dNTP quantitation methods and experimental conditions including any treatments and genetic manipulations. Functions such as the advanced search offering multiple choices from custom-built controlled vocabularies in 15 categories in parallel, the pairwise comparison of any chosen pools, and control-treatment correlations provide users with the possibility to quickly recognize and graphically analyse changes in the dNTP pools in function of a chosen parameter. Unbalanced dNTP pools, as well as the balanced accumulation or depletion of all four dNTPs result in genomic instability. Accordingly, key roles of dNTP pool homeostasis have been demonstrated in cancer progression, development, ageing and viral infections among others. dNTPpoolDB is designated to promote research in these fields and fills a longstanding gap in genome metabolism research.

Does the dose of the culprit drug matter in hypersensitivity reactions? / Számít-e a gyógyszer dózisa a túlérzékenységi reakciókban?

Összefoglaló. Két fiatal nobetegnél a valproátról lamotriginre történo gyógyszerátállítás során a 3-4. héten influenzaszeru prodromalis tüneteket követoen toxikus epidermalis necrolysis (TEN), más néven Lyell-szindróma alakult ki. Mindkét beteg 5 napja kezdodött bor- és nyálkahártyatünetekkel, kiterjedt hámleválást okozó hámnekrózissal került felvételre a Debreceni Egyetem Borgyógyászati Klinikájának Égési Intenzív Osztályára. Multidiszciplináris szupportív terápia mellett nagy dózisú szteroid- és immunglobulin-terápiát alkalmaztunk. A 37 éves nobetegnél 3 hét után a kórkép fatális kimenetellel végzodött. A 19 éves nobeteg tünetei 4 hét intenzív terápia után szövodményekkel gyógyultak. A TEN ritka, gyógyszer által okozott, életet veszélyezteto, késoi hiperszenzitivitási reakció. Patogenezisében a gyógyszermolekula, a humán leukocytaantigén (HLA) I. osztályú molekula és a T-sejt-receptor kóros interakciója szerepel. Kezelésében a legfontosabb a kiváltó gyógyszer elhagyása, valamint az azonnal kezdett komplett szupportív terápia alkalmazása. A specifikus kezelést illetoen nincsenek egységes szakmai irányelvek. A veszélyes gyógyszerek titrált bevezetése csökkentheti a kialakuló hiperszenzitivitás súlyosságát, ezenfelül a beteg szoros követése és az adverz tünetek korai felismerése javíthatja a TEN kimenetelét. Orv Hetil. 2020; 161(46): 1959-1965. Summary. After switching from valproate to lamotrigine, on the 3rd-4th weeks, two young female patients developed flu-like prodromal symptoms, followed by the development of toxic epidermal necrolysis (TEN), also known as Lyell syndrome. Both patients were admitted to the Burn Intensive Care Unit of the Department of Dermatology, University of Debrecen with skin and mucosa symptoms; extensive epithelial death and detachment started 5 days earlier. In addition to multidisciplinary supportive treatment, high-dose corticosteroid and immunoglobulin therapy were administered. In the case of the 37-year-old female patient, the disease resulted in a fatal outcome. The 19-year-old patient healed with some sequelae. TEN is a rare, life-threatening delayed-type hypersensitivity reaction caused by drugs. Its pathogenesis involves an interaction between small-molecule drug, human leukocyte antigen class I molecule and T-cell receptor. The most important treatment is immediate withdrawal of potentially causative drugs and prompt application of supportive therapy. There is no standard guidance on specific treatment. Slow dose escalation of dangerous drugs can be beneficial in avoiding severe reactions, furthermore, close patient follow-up and early detection of the possible adverse reactions contribute to a more favourable outcome of TEN. Orv Hetil. 2020; 161(46): 1959-1965.

A user-friendly, high-throughput tool for the precise fluorescent quantification of deoxyribonucleoside triphosphates from biological samples.

Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5'-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes.

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity.

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition.

Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition.

Alport Patients without Classic Ocular Symptoms Have Smaller Lens Diameter.

BACKGROUND The aim of this study was to present ophthalmological findings regarding Alport syndrome and report refractometry data and to present possible early signs of the syndrome. MATERIAL AND METHODS Seven patients suffering from Alport syndrome were referred to the Department of Ophthalmology at the University of Debrecen between January 1st, 2014, and December 31st, 2015. All patients underwent slit lamp evaluation and dilated fundus biomicroscopy, with special attention paid to lenticonus and retinal changes. IOL Master, Pentacam HR, and ultrasound biomicroscopy were performed to assess keratometry, corneal thickness, anterior chamber depth, lens size, and axial length data. RESULTS One patient out of seven had ocular symptoms. Posterior polymorphous corneal dystrophy (PPMD) and dot-and-fleck retinopathy were seen. Meanwhile, although keratoconus was not proven, remarkable astigmatism with high myopia was detected. The other six patients were found to have a significantly smaller lens diameter (an average of 7.82±0.66 mm, p=0.035) compared to normal controls (an average of 8.65±0.46 mm). Lenses also tended to be thicker in Alport patients (3.48±0.19 mm) compared to controls (3.4±0.2 mm), although the difference was not significant (p=0.394). The power of the lens also showed a significant difference (p=0.026), with Alport patients having lower lens power. CONCLUSIONS Alport syndrome patients without classical ophthalmological findings have smaller crystalline lens diameter and lower lens power. These signs may support the diagnosis of Alport syndrome. Ophthalmologists should not only seek for the known classic signs, but also the parameters of the crystalline lens, especially if genetic testing is not available.

Exploiting a Phage-Bacterium Interaction System as a Molecular Switch to Decipher Macromolecular Interactions in the Living Cell.

Pathogenicity islands of Staphylococcus aureus are under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, may also be exploited as versatile molecular tools. The Stl repressor from this protein family has been recently investigated and although the binding site of Stl on DNA was recently discovered, there is a lack of knowledge on the specific protein segments involved in this interaction. Here, we develop a generally applicable system to reveal the mechanism of the interaction between Stl and its cognate DNA within the cellular environment. Our unbiased approach combines random mutagenesis with high-throughput analysis based on the lac operon to create a well-characterized gene expression system. Our results clearly indicate that, in addition to a previously implicated helix-turn-helix segment, other protein moieties also play decisive roles in the DNA binding capability of Stl. Structural model-based investigations provided a detailed understanding of Stl:DNA complex formation. The robustness and reliability of our novel test system were confirmed by several mutated Stl constructs, as well as by demonstrating the interaction between Stl and dUTPase from the Staphylococcal ϕ11 phage. Our system may be applied to high-throughput studies of protein:DNA and protein:protein interactions.

Differential control of dNTP biosynthesis and genome integrity maintenance by the dUTPase superfamily enzymes.

dUTPase superfamily enzymes generate dUMP, the obligate precursor for de novo dTTP biosynthesis, from either dUTP (monofunctional dUTPase, Dut) or dCTP (bifunctional dCTP deaminase/dUTPase, Dcd:dut). In addition, the elimination of dUTP by these enzymes prevents harmful uracil incorporation into DNA. These two beneficial outcomes have been thought to be related. Here we determined the relationship between dTTP biosynthesis (dTTP/dCTP balance) and the prevention of DNA uracilation in a mycobacterial model that encodes both the Dut and Dcd:dut enzymes, and has no other ways to produce dUMP. We show that, in dut mutant mycobacteria, the dTTP/dCTP balance remained unchanged, but the uracil content of DNA increased in parallel with the in vitro activity-loss of Dut accompanied with a considerable increase in the mutation rate. Conversely, dcd:dut inactivation resulted in perturbed dTTP/dCTP balance and two-fold increased mutation rate, but did not increase the uracil content of DNA. Thus, unexpectedly, the regulation of dNTP balance and the prevention of DNA uracilation are decoupled and separately brought about by the Dcd:dut and Dut enzymes, respectively. Available evidence suggests that the discovered functional separation is conserved in humans and other organisms.

Does beta-ray emitting therapy of ciliary body tumors decrease central corneal endothelial cell density.

PURPOSE: To evaluate the effect of plaque radiation therapy using beta-radioactive isotope for anterior segment tumors on the density and morphology of endothelial cells. 
 METHODS: Endothelial cell density (ECD) and morphometry in 15 eyes of 15 patients with ciliary body tumor was examined prior to and 6 months after radiation therapy. After irradiation, central ECD values were also compared with peripheral (2.0 mm from limbus) ECD values measured around the plaque. The ECD, average cell area, coefficient of variation of cell area, and pachymetry measurements were conducted with contact specular microscopy.
 RESULTS: The mean corrected ECD values prior to irradiation were 2147 ± 128 cells/mm2 and 2050 ± 108 cells/mm2 after the radiation therapy. After irradiation, the mean peripheral ECD values were 2056 ± 101 cells/mm2. A significant decrease in ECD values was observed after radiation (p = 0.007). Peripheral ECD values measured around the plaque showed no significant difference (p = 0.86) as compared to central ECD values.
 CONCLUSIONS: According to our measurements, plaque therapy for tumors in the anterior segment decreases ECD significantly, but not highly, even in case of plaques containing beta-radiation isotope, and the plaques are not placed direct on the cornea surface. The decreased ECD causes no changes in corneal thickness or transparency, but it may have an influence on a subsequent cataract surgery, which generates further endothelial loss.