Formulation of Solid Lipid Nanoparticles Loaded with Rosiglitazone and Probiotic: Optimization and In-vitro Characterization.

INTRODUCTION: In the present study, solid lipid nanoparticles loaded with Rosiglitazone and probiotics were prepared via solvent emulsification diffusion method which is patented. As a lipid and surfactant, Gleceryl monostearate and Pluronic -68 were used in the formulation process. METHODS: During characterization, it was determined that ingredient quantity variations significantly impacted Rosiglitazone loading capacity, particle size, polydispersity index, etc. In an optimized formulation of RSG-PB loaded SLNs, spherical particles with a mean particle size of 147.66 ± 1.52 nm, PDI of 0.42 ± 0.02, and loading capacity of 45.36 ± 0.20 were identified. RESULTS: Moreover, the developed SLNs had the potential to discharge the drug for up to 24 hours, as predicted by Higuchi's pharmacokinetic model. The SLNs were stable at 25°C/60%RH for up to 60 days. There was little to no change in particle size, PDI, or loading capacity. In addition, the number of probiotic bacteria was determined using the standard plate count procedure. Further, the antioxidant effect of the prepared formulation is evaluated using the DPPH assay method. CONCLUSION: This study concludes that the method used to fabricate RSG-probiotic-loaded SLNs is straightforward and yields favorable results regarding various parameters, including sustained release property, particle size, PDI, and percent drug loading stability. Furthermore, DPPH radical scavenging activity shows the high antioxidant potential of RSG-PB SLNs when compared to RSG and probiotics alone.

BODIPY-fused uracil: synthesis, photophysical properties, and applications.

Fluorescent nucleobase and nucleic acid analogs are important tools in chemical and molecular biology as fluorescent labelling of nucleobases has applications in cellular imaging and anti-tumor activity. Boron-dipyrromethene (BODIPY) dyes exhibiting high brightness and good photostability are extensively used as fluorescent labelling agents and as type II photosensitizers for photodynamic therapy. Thus, the combination of nucleobases and BODIPY to obtain new compounds with both anti-tumor activity and fluorescent imaging functions is the focus of our research. We synthesized two new nucleobase analogs 1 and 2 by fusing the BODIPY core directly with uracil which resulted in favorable photophysical properties and high emission quantum efficiencies particularly in organic solvents. Further, we explored the newly synthesized derivatives, which possessed good singlet oxygen generation efficiencies and bio-compatibility, as potential PDT agents and our results show that they exhibit in vitro anti-tumor activities.

Reduction in postoperative sore throat by preoperative nebulization with dexmedetomidine, ketamine or saline: A prospective, randomized-controlled trial.

Background and Aims: Postoperative sore throat (POST) is a minor but distressing complication following general anesthesia. The current literature on the effect of preoperative nebulization with dexmedetomidine, or ketamine on POST is, however, sparse. So, we compared the effect of preoperative nebulization with these drugs on POST. Material and Methods: One hundred and thirty-two American Society of Anaesthesiology (ASA) grade I-II patients undergoing elective laparoscopic surgeries under general anesthesia were randomized into three equal groups: D, K, or C to receive dexmedetomidine, ketamine, or saline as preoperative nebulization, respectively. The primary objective was to compare the incidence and severity of POST, as inferred from the patient interviews at 2, 6, 12, 24-h postoperatively. Results: Group D had a significantly lower incidence (29.5%) and severity (12: mild; 1: moderate) of POST compared to group K (54.5% [21: mild; 3: moderate]) and group C (56.8% [19: mild; 6: moderate]), at 2-h postoperatively. The same trend was observed at 6-h postoperatively (group D: 22.7% [9: mild; 1: moderate]); group K: (40.9% [17: mild; 1: moderate]); group C (50% [17: mild; 5: moderate]). The mean arterial pressure was significantly lower in group D at 15 min intraoperatively (84.09 mmHg, P = 0.018) and immediate postoperatively (97.60 mmHg, P = 0.034). The postoperative sedation, nausea, and vomiting was not statistically significant. Conclusion: Preoperative nebulization with dexmedetomidine is effective in the reduction of the incidence and severity of early POST.

ARTISTRY-7: phase III trial of nemvaleukin alfa plus pembrolizumab vs chemotherapy for platinum-resistant ovarian cancer.

Standard single-agent nonplatinum chemotherapy provides only modest benefit in a small proportion of patients with platinum-resistant/-refractory ovarian cancer, with objective response rates of 6-20% and progression-free survival of ≈3-4 months. Nemvaleukin alfa (nemvaleukin, ALKS 4230) is a novel cytokine designed to capture and expand the therapeutic potential of high-dose interleukin-2 (IL-2) while mitigating its associated toxicity issues. Nemvaleukin preferentially activates cytotoxic CD8+ T cells and natural killer cells with minimal, non-dose-dependent effects on CD4+ regulatory T cells. The global, randomized, open-label, phase III ARTISTRY-7 trial will compare efficacy and safety of nemvaleukin plus pembrolizumab with chemotherapy in patients with platinum-resistant ovarian cancer. The primary end point is investigator-assessed progression-free survival. Clinical Trial Registration: GOG-3063; ENGOT-OV68; NCT05092360 (ClinicalTrials.gov).

In many patients with ovarian cancer who are treated with platinum-based chemotherapy, the tumor comes back after a few months and fails to respond to repeated treatment. This type of disease is called platinum-resistant ovarian cancer (PROC). Researchers are searching for new medicines to help more patients with PROC. One treatment approach that has shown promise in different cancers is called immunotherapy. These medicines work by helping the body's immune system attack cancer cells. One of the immunotherapies being studied is called nemvaleukin. It is designed to trigger specific immune responses that may result in the immune system attacking cancer cells while potentially avoiding other immune responses that can block the attack or cause certain unwanted side effects. Nemvaleukin is being studied in a variety of cancer types. In a worldwide clinical trial called ARTISTRY-7, researchers are investigating how nemvaleukin works in patients with PROC when given with another immunotherapy called pembrolizumab. Patients who participate in this trial will be randomly assigned to one of four treatment groups: the combination of nemvaleukin and pembrolizumab, nemvaleukin by itself, pembrolizumab by itself, or a type of chemotherapy selected by the treating physician. The main purpose of ARTISTRY-7 is to understand whether the combination of nemvaleukin and pembrolizumab helps patients with PROC live longer without their cancer getting worse. At the time of this writing, ARTISTRY-7 is open for new patients to join.

Multifunctional BODIPY embedded non-woven fabric for CO release and singlet oxygen generation.

Materials that can simultaneously release CO and generate singlet oxygen upon visible light irradiation under ambient conditions are highly desirable for therapeutic applications. Furthermore, materials that can sequester the undesirable side products into the matrix without affecting the release of CO and singlet oxygen generation would allow them to be used for practical applications. Focussing on these aspects, we prepared two dipicolylamine appended BODIPY­manganese(I) tricarbonyl complexes wherein the metal core was systematically tethered at 5- and 8- positions of the BODIPY core. The complexes were embedded into a polymer matrix via electrospinning and the resulting non-woven fabrics showed CO release as well as singlet oxygen generation upon irradiation. While the hybrid materials were non-toxic in dark, they were strongly photocytotoxic to c6 cancer cells when exposed to light. Rapid CO release alongside significant singlet oxygen generation, indefinite dark stability, good biocompatibility and negligible dark toxicity makes these fabrics a potent candidate for phototherapeutic applications.

Aggregation-Induced Emission, Mechanofluorochromism, and Selective Fluoride Detection by a Tripodal Salicylaldimine.

Fluoride ions are indispensable in biology and environmental science and hence the selective and sensitive detection of fluoride is important. This work reports the design and synthesis of a tripodal Schiff's base 1 through a simple condensation reaction between a commercially available aldehyde and an amine. Single crystal X-ray crystallography revealed that compound 1 is a planar entity with the three salicylidene derivatives on the three arms of the central phenyl moiety linked by imine groups. Compound 1 forms a molecular dimer that resembles a six-petal flower and is stabilized through multiple intermolecular interactions such as C-H.π and π.π interactions. Compound 1 exhibited moderately good emission in the solid state with aggregation induced emission and reversible mechanofluorochromic properties. Moreover, 1 was observed to selectively detect fluoride among various anions with a limit of detection of ∼9 ppm. Compound 1 was also capable of detecting fluoride under a variety of conditions such as in thin films and under cellular conditions.

An assessment of various potentially toxic elements and associated health risks in agricultural soil along the middle Gangetic basin, India.

The present study analysed the levels of potentially toxic elements along with physico-chemical properties of agricultural soil samples (n = 59) collected from fields situated along the path of river Ganga in the middle Gangetic floodplain in two districts, Ballia and Ghazipur. Arsenic (As), chromium (Cr), copper (Cu), nickel (Ni), zinc (Zn), lead (Pb), iron (Fe) and manganese (Mn) levels were analysed by Wavelength Dispersive-X-Ray Fluorescence Spectroscopy (WD-XRF) and the associated health risks along with diverse indices were calculated. The mean concentrations of As, Cu, Cr, Pb, Zn and Ni were found to be 15, 42, 85, 18, 87 and 47 mg kg-1, respectively in Ballia and 13, 31, 73, 22, 77 and 34 mg kg-1, respectively in Ghazipur. Physico-chemical properties like pH, ORP and organic matter were found to be 7.91, 209 and 1.20, respectively in Ballia and 8.51, 155 and 1.25, respectively in Ghazipur. The calculated health quotient (HQ) for all the elements was observed to be within the threshold value of one, however with few exemptions. Therefore, the present study showcases the contamination of potentially toxic elements in agricultural fields and possible health hazards for people.

A pneumococcal pneumonia and influenza vaccination quality improvement program for women receiving chemotherapy for gynecologic cancers at a major tertiary cancer Centre.

OBJECTIVE: International guidelines recommend pneumococcal pneumonia and influenza vaccination for all patients with solid organ malignancies prior to initiating chemotherapy. Baseline vaccination rates (March 2019) for pneumococcal pneumonia and influenza at our tertiary cancer centre were 8% and 40%, respectively. The aim of this study was to increase the number of gynecologic chemotherapy patients receiving pneumococcal and influenza vaccinations to 80% by March 2020. METHODS: We performed an interrupted time series study using structured quality improvement methodology. Three interventions were introduced to address vaccination barriers: an in-house vaccination program, a staff education campaign, and a patient care bundle (pre-printed prescription, information brochure, vaccine record booklet). Process and outcome data were collected by patient survey and pharmacy audit and analyzed on statistical process control charts. RESULTS: We identified 195 eligible patients. Pneumococcal and influenza vaccination rates rose significantly from 5% to a monthly mean of 61% and from 36% to a monthly mean of 67%, respectively. The 80% target was reached for both vaccines during one or more months of study. The in-house vaccination and staff education programs were major contributors to the improvement, whereas the information brochure and record booklet were minor contributors. CONCLUSIONS: Three interventions to promote pneumococcal and influenza vaccination among chemotherapy patients resulted in significantly improved vaccination rates. Lessons learned about promoting vaccine uptake may be generalizable to different populations and vaccine types. In response to the global COVID-19 pandemic, initiatives to expand the program to all chemotherapy patients at our centre are underway.

Unity and diversity among viral kinases.

Viral kinases are known to undergo autophosphorylation and also phosphorylate viral and host substrates. Viral kinases have been implicated in various diseases and are also known to acquire host kinases for mimicking cellular functions and exhibit virulence. Although substantial analyses have been reported in the literature on diversity of viral kinases, there is a gap in the understanding of sequence and structural similarity among kinases from different classes of viruses. In this study, we performed a comprehensive analysis of protein kinases encoded in viral genomes. Homology search methods have been used to identify kinases from 104,282 viral genomic datasets. Serine/threonine and tyrosine kinases are identified only in 390 viral genomes. Out of seven viral classes that are based on nature of genetic material, only viruses having double-stranded DNA and single-stranded RNA retroviruses are found to encode kinases. The 716 identified protein kinases are classified into 63 subfamilies based on their sequence similarity within each cluster, and sequence signatures have been identified for each subfamily. 11 clusters are well represented with at least 10 members in each of these clusters. Kinases from dsDNA viruses, Phycodnaviridae which infect green algae and Herpesvirales that infect vertebrates including human, form a major group. From our analysis, it has been observed that the protein kinases in viruses belonging to same taxonomic lineages form discrete clusters and the kinases encoded in alphaherpesvirus form host-specific clusters. A comprehensive sequence and structure-based analysis enabled us to identify the conserved residues or motifs in kinase catalytic domain regions across all viral kinases. Conserved sequence regions that are specific to a particular viral kinase cluster and the kinases that show close similarity to eukaryotic kinases were identified by using sequence and three-dimensional structural regions of eukaryotic kinases as reference. The regions specific to each viral kinase cluster can be used as signatures in the future in classifying uncharacterized viral kinases. We note that kinases from giant viruses Marseilleviridae have close similarity to viral oncogenes in the functional regions and in putative substrate binding regions indicating their possible role in cancer.

Adjuvant Trastuzumab Therapy: Can We Balance Efficacy and Safety?

Trastuzumab is an effective treatment for HER2-positive breast cancer. Current guidelines recommend withholding trastuzumab in patients experiencing a significant asymptomatic decline in left ventricular function. In this commentary, we discuss the survival benefits afforded by trastuzumab juxtaposed against the risk of trastuzumab-mediated cardiotoxicity. It is not known whether the net benefit of continuing trastuzumab in the setting of mild cardiotoxicity outweighs the associated risks. We describe a potential approach undertaken by our group, and others, and call for a randomized trial.

Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant.

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T1, T2 and T3 transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

Single versus multifraction radiotherapy for spinal cord compression: A systematic review and meta-analysis.

BACKGROUND: While multifraction radiotherapy (RT) regimens (MFRT) have been considered the standard of care in patients with metastatic epidural spinal cord compression (MESCC) with limited prognosis, recent randomized evidence has demonstrated that single fraction RT (SFRT) may be equivalent in terms of functional and overall outcomes. A systematic review and meta-analysis was conducted to determine the effects of SFRT compared to short course MFRT in patients with MESCC. METHODS: A search of OVID, EMBASE, and the Cochrane Central Register of Controlled Trials from inception to February 2018 was conducted. Randomized and prospective non-randomized trials comparing SFRT and short course MFRT for MESCC were included. Data were analyzed using a random effects model, and relative risks (RR) or hazard ratios (HR) were reported with corresponding 95% confidence intervals (CI). Quality of evidence was assessed using the GRADE criteria. RESULTS: Overall 1717 articles were reviewed. Three randomized trials were eligible for inclusion (n = 712 patients). The pooled treatment effect for SFRT versus MFRT with respect to motor response was RR = 0.96 (95% CI = 0.86-1.07, I2 = 19%), HR = 1.00 (95% CI = 0.88-1.13, I2 = 0%) for OS, and RR = 0.97, (95% CI = 0.85-1.11, I2 = 61%) for bladder function. There was insufficient data to perform a meta-analysis on quality of life, toxicity or pain response, however available information suggests pain response appears similar between SFRT and MFRT. Overall quality of evidence was deemed moderate due to risk of bias. There was no evidence of an observed difference with respect to motor response, bladder dysfunction and OS between SFRT and MFRT for MESCC in patients with a limited prognosis.

Safety of Continuing Trastuzumab Despite Mild Cardiotoxicity: A Phase I Trial.

OBJECTIVES: This study sought to evaluate the safety of continuing trastuzumab in patients with human epidermal growth factor receptor-positive breast cancer who developed mild cardiotoxicity. BACKGROUND: Cardiotoxicity is the most common dose-limiting toxicity associated with trastuzumab. Current standard of care is discontinuation of trastuzumab, which can lead to worse cancer outcomes. It is unknown whether it is safe to continue trastuzumab despite mild cardiotoxicity. METHODS: Patients were eligible for this phase I, prospective, single-arm trial if left ventricular ejection fraction (LVEF) was between 40% and the lower limit of normal or if it fell ≥15% from baseline. Participants were treated with angiotensin-converting enzyme (ACE) inhibitors and/or beta-blockers in a cardio-oncology clinic and were followed clinically and with serial echocardiograms for 1 year. The primary outcome was cardiac dose-limiting toxicity, defined as cardiovascular death, LVEF <40% together with any heart failure symptoms, or LVEF <35%. RESULTS: All 20 participants received ACE inhibitors and/or beta-blockers. A total of 18 participants (90%) received all planned trastuzumab doses. Two (10%) participants developed cardiac dose-limiting toxicity (heart failure with LVEF <40%). Their LVEF and heart failure symptoms improved to nearly normal following permanent trastuzumab discontinuation. There were no deaths. LVEF rose progressively from a mean of 49% at enrollment to 55% at 12 months (p < 0.001). CONCLUSIONS: It may be feasible to continue trastuzumab despite mild cardiotoxicity in the setting of a cardio-oncology clinic, where ACE inhibitors and beta-blockers are administered. Approximately 10% of patients may develop moderate to severe heart failure using this approach. (Safety of Continuing Chemotherapy in Overt Left Ventricular Dysfunction Using Antibodies to Human Epidermal Growth Factor Receptor-2 [SCHOLAR]; NCT02907021).

SIFTS: updated Structure Integration with Function, Taxonomy and Sequences resource allows 40-fold increase in coverage of structure-based annotations for proteins.

The Structure Integration with Function, Taxonomy and Sequences resource (SIFTS; http://pdbe.org/sifts/) was established in 2002 and continues to operate as a collaboration between the Protein Data Bank in Europe (PDBe; http://pdbe.org) and the UniProt Knowledgebase (UniProtKB; http://uniprot.org). The resource is instrumental in the transfer of annotations between protein structure and protein sequence resources through provision of up-to-date residue-level mappings between entries from the PDB and from UniProtKB. SIFTS also incorporates residue-level annotations from other biological resources, currently comprising the NCBI taxonomy database, IntEnz, GO, Pfam, InterPro, SCOP, CATH, PubMed, Ensembl, Homologene and automatic Pfam domain assignments based on HMM profiles. The recently released implementation of SIFTS includes support for multiple cross-references for proteins in the PDB, allowing mappings to UniProtKB isoforms and UniRef90 cluster members. This development makes structure data in the PDB readily available to over 1.8 million UniProtKB accessions.

The design of synthetic superoxide dismutase mimetics: seven-coordinate water soluble manganese(ii) and iron(ii) complexes and their superoxide dismutase-like activity studies.

Bio-inspired manganese [Mn(N5Py)(H2O)(CH3OH)](ClO4)2 (1) and iron [Fe(N5Py)(H2O)(ClO4)]ClO4 (2) complexes derived from a pentadentate ligand (N5Py = 2,6-bis((E)-1-phenyl-2-(pyridin-2-ylmethylene)hydrazinyl)pyridine) framework containing a N5 binding motif were synthesized and characterized using different spectroscopic methods. The molecular structures of complexes 1 and 2 were determined by X-ray crystallography. These complexes were found to be stable under physiological conditions and exhibited an excellent superoxide dismutase (SOD) activity. The SOD activity was determined by a xanthine-xanthine oxidase-nitro blue tetrazolium assay and the IC50 values were determined to be 1.53 and 2.09 µM, respectively.

Mn(II) based T1 and T2 potential MRI contrast agent appended with tryptamine: Recognition moiety for Aß-plaques.

We report the synthesis and characterization of manganese(II) complexes having pentadentate ligands L1 (2,6-bis(1-(2-phenyl-2-(pyridin-2-yl)hydrazono)ethyl)pyridine), L2 (methyl 2,6-bis((E)-1-(2-phenyl-2-(pyridin-2yl)hydrazono)ethyl)isonicotinate), L3 (N-(2-(1H-indol-3-yl)ethyl)-2,6-bis((E)-1-(2-phenyl-2-(pyridin2yl)hydrazono)ethyl)isonicotiamide) and their application as dual contrast agents for simultaneous T1 and T2 weighted magnetic resonance imaging. Single crystal analysis of all the complexes [MnIIL1, MnIIL2 and MnIIL3] confirm the formation of novel seven-coordinate manganese complexes with an inner sphere water and perchlorate ion. The Magnetic Resonance Imaging (MRI) contrast agent [MnL2] was further modified by incorporating tryptamine as a binding moiety specific to Amyloid Beta-fibrils (Aß-fibrils) in Alzhiemer's disease (AD) and it's in vitro evaluation for specific binding with Aß-fibrils indicated as a bio-marker of AD.

Growth, morphology, structure and characterization of L-histidinium dihydrogen arsenate orthoarsenic acid single crystal.

L-Histidinium dihydrogen arsenate orthoarsenic acid (LHAS) crystals were grown by the slow evaporation method. Single-crystal X-ray diffraction confirms monoclinic structure. The growth rates of various planes of LHAS crystals were estimated by morphological study. Hirshfeld surface and fingerprint plots were analyzed to investigate the intermolecular interactions at 0.002 a.u. present in the crystal structure. The functional groups and phase behavior of the compound are studied by FTIR spectroscopy and differential scanning calorimetry (DSC). A ferroelectric to paraelectric phase transition at 307 K was observed in dielectric studies. The piezoelectric charge coefficients of the grown crystal were found to be 2 pC/N. The values of coercive field (Ec), remnant polarization (Pr) and spontaneous polarization (Ps) in the hysteresis loop are found to be 5.236 kV cm(-1), 0.654 µC cm(-2) and 2.841 µC cm(-2), respectively. Piezoelectricity and ferroelectricity are reported for the first time in LHAS crystals. The mechanical strength was confirmed from microhardness study and void volume. Due to the low value of the dielectric constant, and good piezoelectric and ferroelectric properties, LHAS crystals can be used in microelectronics, sensors and advanced electronic devices.

Expression of a Cystatin Transgene in Eggplant Provides Resistance to Root-knot Nematode, Meloidogyne incognita.

Root-knot nematodes (RKN) cause substantial yield decline in eggplant and sustainable management options to minimize crop damage due to nematodes are still limited. A number of genetic engineering strategies have been developed to disrupt the successful plant-nematode interactions. Among them, delivery of proteinase inhibitors from the plant to perturb nematode development and reproduction is arguably the most effective strategy. In the present study, transgenic eggplant expressing a modified rice cystatin (OC-IΔD86) gene under the control of the root-specific promoter, TUB-1, was generated to evaluate the genetically modified nematode resistance. Five putative transformants were selected through PCR and genomic Southern blot analysis. Expression of the cystatin transgene was confirmed in all the events using western blotting, ELISA and qPCR assay. Upon challenge inoculation, all the transgenic events exhibited a detrimental effect on RKN development and reproduction. The best transgenic line (a single copy event) showed 78.3% inhibition in reproductive success of RKN. Our results suggest that cystatins can play an important role for improving nematode resistance in eggplant and their deployment in gene pyramiding strategies with other proteinase inhibitors could ultimately enhance crop yield.

Integrating population variation and protein structural analysis to improve clinical interpretation of missense variation: application to the WD40 domain.

We present a generic, multidisciplinary approach for improving our understanding of novel missense variants in recently discovered disease genes exhibiting genetic heterogeneity, by combining clinical and population genetics with protein structural analysis. Using six new de novo missense diagnoses in TBL1XR1 from the Deciphering Developmental Disorders study, together with population variation data, we show that the ß-propeller structure of the ubiquitous WD40 domain provides a convincing way to discriminate between pathogenic and benign variation. Children with likely pathogenic mutations in this gene have severely delayed language development, often accompanied by intellectual disability, autism, dysmorphology and gastrointestinal problems. Amino acids affected by likely pathogenic missense mutations are either crucial for the stability of the fold, forming part of a highly conserved symmetrically repeating hydrogen-bonded tetrad, or located at the top face of the ß-propeller, where 'hotspot' residues affect the binding of ß-catenin to the TBLR1 protein. In contrast, those altered by population variation are significantly less likely to be spatially clustered towards the top face or to be at buried or highly conserved residues. This result is useful not only for interpreting benign and pathogenic missense variants in this gene, but also in other WD40 domains, many of which are associated with disease.