Variant of uncertain significance Arg866Cys enhances disorderedness of h-BRCA1 (759-1064) region.

High structural flexibility has been reported in the central region of BRCA1, which hinders the structural and functional evaluations of mutations identified in the domain. Additionally, the need to categorize variants of unknown significance (VUS) has increased due to the growth in the number of variants reported in clinical settings. Therefore, unraveling the disease-causing mechanism of VUS identified in different functional domains of BRCA1 is still challenging. The current study uses a multidisciplinary approach to assess the structural impact of BRCA1 Arg866Cys mutation discovered in the central domain of BRCA1. The structural alterations have been characterized using Circular-Dichroism spectroscopy, nano-DSF, and molecular-dynamics simulations. BRCA1 Arg866Cys mutant demonstrated more flexibility and lesser affinity to DNA than the wild-type protein. The BRCA1(759-1064) wild-type protein was shown to be a ßII-rich protein with an induced D-O transition in the presence of DNA and 2,2,2-Trifluoroethanol (TFE). The protein's alpha-helical composition did not significantly change in the presence of TFE, besides an increase in ß-turns and loops. Under Transmission Electron Microscopes (TEM), amyloid-like fibrils structure was detected for Arg866Cys mutant whereas the wild-type protein showed amorphous aggregates. An increased ThT fluorescence indicated ß-rich composition and aggregation-prone behaviour for BRCA1 wild-type protein, while the fluorescence intensity was significantly quenched in the Arg866Cys mutant. Furthermore, increased conformational flexibility in the Arg866Cys variant was observed by principal component analysis. This work aims to comprehend the inherently disordered region of BRCA1 as well as the impact of missense mutations on folding patterns and binding to DNA for functional aspects.

Structural implications of amyloidogenic rare variants Ser282Leu and Gln356Arg identified in h-BRCA1.

Preliminary studies have shown BRCA1 (170-1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h-BRCA1(260-553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small-angle X-ray scattering analysis revealed WT scores Rg -32 Å, Dmax -93 Å, and Rflex-51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder-to-order transition in the presence of cruciform DNA and 2,2,2-Trifluoroethanol (TFE). An increased alpha-helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT-DNA complex compared to the Gln356Arg and Ser282Leu protein-DNA complex. Moreover, mature amyloid-like fibrils were observed with 30 µM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher-order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

Mutations at proximal cysteine residues in PML impair ATO binding by destabilizing the RBCC domain.

Acute promyelocytic leukemia (APL) is characterized by the fusion gene promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) and is conventionally treated with arsenic trioxide (ATO). ATO binds directly to the RING finger, B-box, coiled-coil (RBCC) domain of PML and initiates degradation of the fusion oncoprotein PML-RARA. However, the mutational hotspot at C212-S220 disrupts ATO binding, leading to drug resistance in APL. Therefore, structural consequences of these point mutations in PML that remain uncertain require comprehensive analysis. In this study, we investigated the structure-based ensemble properties of the promyelocytic leukemia-RING-B-box-coiled-coil (PML-RBCC) domains and ATO-resistant mutations. Oligomeric studies reveal that PML-RBCC wild-type and mutants C212R, S214L, A216T, L217F, and S220G predominantly form tetramers, whereas mutants C213R, A216V, L218P, and D219H tend to form dimers. The stability of the dimeric mutants was lower, exhibiting a melting temperature (Tm) reduction of 30 °C compared with the tetrameric mutants and wild-type PML protein. Furthermore, the exposed surface of the C213R mutation rendered it more prone to protease digestion than that of the C212R mutation. The spectroscopic analysis highlighted ATO-induced structural alterations in S214L, A216V, and D219H mutants, in contrast to C213R, L217F, and L218P mutations. Moreover, the computational analysis revealed that the ATO-resistant mutations C213R, A216V, L217F, and L218P caused changes in the size, shape, and flexibility of the PML-RBCC wild-type protein. The mutations C213R, A216V, L217F, and L218P destabilize the wild-type protein structure due to the adaptation of distinct conformational changes. In addition, these mutations disrupt several hydrogen bonds, including interactions involving C212, C213, and C215, which are essential for ATO binding. The local and global structural features induced by these mutations provide mechanistic insight into ATO resistance and APL pathogenesis.

Multimodal approach to characterize the tetrameric form of human PML-RBCC domain and ATO-mediated conformational changes.

RING-B box-coiled coil (RBCC) domain of promyelocytic leukemia (PML) comprises a zinc finger motif that is targeted by arsenic trioxide (ATO) to treat acute promyelocytic leukemia (APL) pathogenesis. Preliminary evidence suggests that the PML-RBCC has different functional characteristics, but no structural details have been reported despite its importance in differential expression and cell-cycle regulation. Therefore, the recombinant h-PML-RBCC protein was purified to its homogeneity, and characterized for oligomeric behaviour which indicated that RBCC domain exists as a tetramer in solution. Furthermore, nano-DSF and circular-dichroism demonstrated that the tetrameric form preserves its native conformation along with thermal stability (Tm = 83.2 °C). In-silico-based PML-RBCC structure was used to perform the molecular dynamics simulation for 300 ns in the presence of zinc atoms, which demonstrated the differential dynamic of PML-RBCC tetrameric chains. MMPBSA analysis also indicated the role of hydrophobic interactions that favours stable tetrameric structure of PML-RBCC. ATO-induced secondary and tertiary structure changes were observed in PML-RBCC using circular dichroism and fluorescence spectroscopy. Dynamic light scattering and transmission electron microscopy revealed ATO-induced higher-order oligomerization and aggregation of PML-RBCC. The unique oligomeric nature of the h-PML-RBCC protein and its interactions with ATO will help to understand the mechanism of APL pathogenesis and drug resistance.

Patient Perspectives on Biologics for Axial Spondyloarthritis in a Cross-sectional Study in a Predominantly Female Population: Treatment Satisfaction, Wear-off Between Doses, and Use of Supplemental Medication.

INTRODUCTION: There is limited information regarding treatment experience of patients with axial spondyloarthritis/ankylosing spondylitis (axSpA/AS) receiving biological disease-modifying antirheumatic drugs (bDMARDs). Here we characterize patient experiences and perspectives, including satisfaction among those currently treated with bDMARD therapy for axSpA/AS. We also assess the use of supplemental medication during perceived wear-off between doses. METHODS: Adult participants from the United States within the ArthritisPower registry with physician-diagnosed axSpA/AS were invited to complete electronic patient-reported outcome measures and an online survey about their perspectives of treatment. Analysis compared patient characteristics and treatment satisfaction by whether wear-off in axSpA/AS between bDMARD doses was reported. RESULTS: Of 128 patients currently taking a DMARD, the mean age was 46.9 (10.3) years, 82.0% were female, and 93.8% were White. A total of 78 (60.9%) perceived wear-off with their current bDMARD before the next dose, 19 (14.8%) did not experience wear-off and 31 (24.2%) were unsure about wear-off. Mean (standard deviation [SD]) Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score indicated poor disease control in all patients receiving bDMARDs (6.4 [1.8]); worse for those perceiving wear-off between doses versus those who did not perceive wear-off or were unsure (6.8 [1.6] vs. 5.9 [2.0], p = 0.011). Patients experiencing wear-off reported being 'very satisfied' or 'somewhat satisfied' with their treatment less frequently than patients without wear-off (73.1 vs. 89.5%, respectively). Of patients reporting wear-off, 82.1% (n = 64) used supplemental medications during wear-off (non-steroidal anti-inflammatory drugs [68.8%, n = 44], muscle relaxants [42.2%, n = 27], and/or opioids [37.5%, n = 24]). CONCLUSIONS: In a predominantly female sample of bDMARD-treated patients with axSpA/AS and high disease activity, the majority expressed treatment satisfaction. However, most experienced wear-off between doses and relied on supplemental medications, including opioids, to manage symptoms.

Phytochemical characterization and evaluation of anticataract potential of seabuckthorn leaf extract.

OBJECTIVE: This study was undertaken to carry out phytochemical characterization of aqueous extract of Seabuckthorn (SBT, Hippophae rhamnoides L.) leaves and evaluation of its therapeutic role in oxidative stress-induced cataract in isolated goat lenses using Vit. E as reference compound. ANIMAL STUDIED: A total of 42 goat eye lenses were used in the present study. PROCEDURE: Seabuckthorn leaf extract was characterized by total phenol content estimation and HPLC analysis of quercetin and catechin. Further, cataract was induced in goat lenses using hydrogen peroxide (H2O2) and anticataract activity was evaluated using the extract in the dose range of 100, 200, 500, and 1000 µg/mL concentrations through estimation of biochemical markers such as superoxide dismutase (SOD), reduced glutathione (GSH), and malonaldehyde (MDA). RESULTS: The results of the phytochemical characterization showed the total phenol content of the extract to be 365 mg/g in terms of gallic acid equivalents. Quercetin and catechin were estimated to be 0.01 and 0.12% w/w, respectively. In biochemical analysis, H2O2 introduction resulted in a decrease in SOD (approximately 85%) and GSH (approximately 63%) contents and an increase in MDA content (approximately 300%). The decreased levels of SOD and GSH were significantly restored in experimental groups receiving 500 and 1000 1g/mL of SBT extract. All the experimental groups showed significantly reduced MDA level in all the doses. CONCLUSION: Aqueous extract of SBT leaves showed the potential to delay onset and/or progression of cataract, at least during in vitro conditions. Results indicate the possibilities of evaluating this extract for its use as anticataract agent during in vivo conditions.

In vitro anti-cataract evaluation of standardised Abies pindrow leaf extract using isolated goat lenses.

Oxidative stress is known to spark off the pathogenesis of cataract. Antioxidant potential of Abies pindrow Royle leaf extract (APE) is well established in the literature. In this context, standard aqueous leaf extract of this plant was evaluated for its role in hydrogen peroxide-induced cataract in isolated goat lenses using varying concentrations (5, 10, 15 and 20 mg/mL). Total phenol and flavonoidal content was evaluated and found to be high in concentration. Biochemical parameters, namely superoxide dismutase (SOD), glutathione (GSH), total protein content (TPC) and malondialdehyde (MDA), were evaluated. SOD, GSH and TPC formation was found to increase proportionally with increasing concentration. However, MDA level decreased significantly as the concentration of the extract increased. The results suggest that the extract under investigation can delay the onset and/or prevent the progression of cataract. Its anti-cataract potential may be attributed to the presence of high phenolics and flavonoids in APE. Photographic evaluation, further, confirmed the observation.

Effect of standardized fruit extract of Luffa cylindrica on oxidative stress markers in hydrogen peroxide induced cataract.

OBJECTIVE: The ability of Luffa cylindrica Roem fruit extract (LCE) to modulate biochemical parameters was investigated by in vitro studies for its role in hydrogen peroxide induced cataract on isolated goat lenses which were incubated for 72 h at 37°C. MATERIALS AND METHODS: Test groups contained 5, 10, 15, 20, 25, and 30 µg/ml of LCE along with 1 ml of H2O2 (0.5 mM) as cataract inducer. Lenses were examined for morphological variation and transparency periodically during the incubation. Biochemical parameters such as superoxide dismutase (SOD), reduced glutathione (GSH), total protein content (TPC), and malondialdehyde (MDA) were estimated. RESULTS: SOD, GSH, and TPC levels were found to increase proportionally with the concentration of LCE. However, MDA levels were found to be inversely proportional to the concentration of LCE. Opacity was graded as per "lens opacities classification system III." Morphological examination suggested that LCE (25 µg/ml) maintained a vision for 44 h. No lens in LCE dose groups developed dense nuclear opacity after 24 h as opposed to 80% in negative control. CONCLUSION: The results suggest that LCE can delay the onset and/or prevent the progression of cataract which can be attributed to the presence of adequate phenolics, flavonoids, and Vitamin A and its high nutritional value. This preliminary study can be further synergized by testing LCE against other in vivo and in vitro models of cataract.

Phytochemistry, Pharmacology and Toxicology of Spilanthes acmella: A Review.

Spilanthes acmella is an important medicinal plant, found in tropical and subtropical countries mainly India and South America. Popularly, it is known as toothache plant which reduces the pain associated with toothaches and can induce saliva secretion. Various extracts and active metabolites from various parts of this plant possess useful pharmacological activities. Literature survey proposed that it has multiple pharmacological actions, which include antifungal, antipyretic, local anaesthetic, bioinsecticide, anticonvulsant, antioxidant, aphrodisiac, analgesic, pancreatic lipase inhibitor, antimicrobial, antinociception, diuretic, vasorelaxant, anti-human immunodeficiency virus, toothache relieve and anti-inflammatory effects. This review is elaborately describing the traditional uses, phytochemistry, pharmacology, and toxicology of this plant. This review would assist researchers to search scientific information in the future.