Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome.

Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

Optimizing Neurology Inpatient Documentation: A Pilot Study of a Novel Discharge Documentation EHR Tool.

Background and Purpose: Clinical documentation of patient acuity is a major determinant of payer reimbursement. This project aimed to improve case mix index (CMI) by incorporating a novel electronic health record (EHR) discharge documentation tool into the inpatient general neurology service at the University of California, Los Angeles (UCLA) Medical Center. Methods: We used data from Vizient AMC Hospital: Risk Model Summary for Clinical Data Base (CBD) 2017 to create a discharge diagnosis documentation tool consisting of dropdown menus to better capture relevant secondary diagnoses and comorbidities. After implementation of this tool, we compared pre- (July 2017-June 2019) and post-intervention (July 2019-June 2021) time periods on mean expected length of stay (LOS) and mean CMI with two sample T-tests and the percentage of encounters classified as having Major Complications/Comorbidities (MCC), with Complication/Comorbidity (CC), and without CC/MCC with tests of proportions. Results: Mean CMI increased significantly from 1.2 pre-intervention to 1.4 post-intervention implementation (P < .01). There was a pattern of increased MCC percentages for "Bacterial infections," "Other Disorders of Nervous System", "Multiple Sclerosis," and "Nervous System Neoplasms" diagnosis related groups post-intervention. Conclusions: This pilot study describes the creation of an innovative EHR discharge diagnosis documentation tool in collaboration with neurology healthcare providers, the clinical documentation improvement team, and neuro-informaticists. This novel discharge diagnosis documentation tool demonstrates promise in increasing CMI, shifting diagnosis related groups to a greater proportion of those with MCC, and improving the quality of clinical documentation.

Manifestations and Management of Trimethoprim/Sulfamethoxazole-Resistant Nocardia otitidiscaviarum Infection.

Nocardia can cause systemic infections with varying manifestations. Resistance patterns vary by species. We describe N. otitidiscavarium infection with pulmonary and cutaneous manifestations in a man in the United States. He received multidrug treatment that included trimethoprim/sulfamethoxazole but died. Our case highlights the need to treat with combination therapy until drug susceptibilities are known.

A Chemical Characterization Workflow for Nontargeted Analysis of Complex Extracts from Polymer Based Medical Device Using High Resolution LC/MS.

The chemical characterization of extractables and leachables (E&Ls) is an important aspect of biosafety and biocompatibility assessment in medical device industry. The advent of the body-contact use of medical devices in patient treatment has introduced a potential source for extractables and leachables as these medical devices are comprised of various polymeric materials. Several industry working groups, the FDA and USP, have recognized the guidance for chemical characterizations and nontargeted analysis of medical device extracts, such as ISO 10993-18:2020. The MS application of nontargeted analysis has played a critical role in understanding the E&Ls from medical device extracts. However, there have been very few reports about the MS based workflow with nontargeted analysis for medical device extracts and there is little guidance about the exact methodologies which should be used, even though there is an urgent need for a clearly defined process for the identification of medical device extracts. In this study, we demonstrated an analytical LC/MS (liquid chromatography/mass spectrometry) workflow using high resolution Exploris120 Orbitrap instrument for data acquisition and Compound Discoverer 3.3 intelligent software for data processing to profile the polymer related E&Ls from a balloon dilation catheter device extracted with 40% ethanol. An E&L ID workflow combining LC separation, data-informed MS acquisition strategy, MS information mining (including adduct ions, MS information from both electrospray ionization (ESI) (+) and ESI (-), in-source fragmentation, common fragment ions (CFIs), common neutral losses (CNLs), and in silico MS simulation was described with intelligent software processing and manual data interpretation. The workflow developed in this study was proven to be effective to provide a comprehensive profile of polymer related degradation products, polymer impurities and additives including surfactants, UV curing agent, antioxidants, and plasticizers for the device analyzed. The classification of E&L compounds using CFIs and CNLs was very effective to facilitate the identification of polymer related impurities and extract the polymer related impurities with common structures in a large data result set.

Mechanistic studies in Drosophila and chicken give new insights into functions of DVL1 in dominant Robinow syndrome.

The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. Here, we focus on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (ß-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. We expressed human wild-type DVL1 or DVL1 variants alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. Our data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.

Right Brain: The Purple Scarf and the Cortical Ribbon.

A patient with probable Creutzfeldt Jakob disease (CJD) who was previously a high-functioning financial analyst remains fiercely independent despite her family's concerns about her safety. As a trainee with limited experience in leading these difficult conversations, this neurology resident describes her experience reconciling the viewpoints of herself, the patient, and her family. She reflects on the ability to reframe this delicate conversation from one of loss of independence in a patient with a rapidly progressive, neurodegenerative condition to one of safety, security, and the care of her family.

Hypersensitivity Pneumonitis With and Without Autoimmune Features: A Clinical Comparative Analysis.

BACKGROUND: Hypersensitivity pneumonitis (HP) is an interstitial lung disease (ILD) caused by an immunological reaction to repeated inhalational exposure to antigens. The etiology and exact immunopathology are poorly understood. Autoimmunity overlapping with HP has been described but the role of concomitant autoimmunity in the clinical course and outcome of the HP is not clearly established. In this study, we examined patients diagnosed with HP and compare them to patients with concomitant HP and autoimmunity. METHODS: Patients were retrospectively screened from a single-center ILD registry. Patients > 18 years with an established multidisciplinary diagnosis of HP were included in the study. Patients with HP without autoimmune features and patients with HP with autoimmune features (HPAF) were assessed. We compared the demographics, clinical characteristics, treatment, and outcomes between the two groups. We used a Cox proportional hazards model to compare lung transplant-free survival outcomes of patients with HPAF to those with non-HPAF HP patients. RESULTS: Of 73 patients with HP, 43 were diagnosed with HPAF. Patients with HPAF had a higher echocardiographic probability of pulmonary hypertension as compared to non-HPAF HP patients [48.8 vs 23.3%, p = 0.028, Crude odds ratio (cOR) = 3.14]. Symptomatically, those with HPAF reported a higher prevalence of arthritis as compared to non-HPAF HP (20.9 vs 3.3%, p = 0.040, cOR = 7.68). No significant differences between pulmonary function tests, oxygen requirements, mortality, and lung transplantation rates were found between the two groups. There was no statistically significant difference in transplant-free survival (p = 0.836). CONCLUSION: Patients with HPAF had a higher echocardiographic probability of pulmonary hypertension as compared to patients with non-HPAF HP. The clinical characteristics and outcomes did not differ between the two groups and concomitant autoimmunity among the HP group did not portend a poorer prognosis.

Comparing the effect of virtual and in-person instruction on students' performance in a design for additive manufacturing learning activity.

The goal of this work is to compare the outcome of a design for additive manufacturing (DfAM) heuristics lesson conducted in a virtual learning environment to the same in an in-person learning environment. Prior work revealed that receiving DfAM heuristics at different points in the design process impacts the quality and novelty of designs produced afterward, but this work may have been limited by the solely virtual format. In this work, an identical experiment was performed in a face-to-face learning environment. Results indicate that neither learning format presents an advantage over the other when it comes to the quality of designs produced during the intervention. Participants across all experimental groups reported an increase in self-efficacy after the intervention, with improved performance on quiz-type questions. However, the novelty and variety of the designs produced by the in-person experimental groups were significantly lower than that of the virtual experimental groups. In addition to validating the effectiveness of virtual instruction as a teaching method, these results also support the authors' hypothesis that the priming effect is stronger in an in-person classroom than in a virtual classroom.

Facial diplegia with paresthesia associated with anti-GD1a antibodies.

A 26-year-old previously healthy man presented with progressive facial diplegia and sensory deficits to pinprick in a stocking-glove distribution. Lumbar puncture revealed cytoalbuminologic dissociation, and a nerve conduction study of the right facial nerve demonstrated a proximal demyelinating process. He was started on intravenous immunoglobulin given concern for a Guillain-Barré syndrome variant, and his symptoms improved over several days. This case illustrates the clinical features of facial diplegia with paresthesias, a rare variant of Guillain-Barré syndrome. Unlike most reported cases of facial diplegia with paresthesias that have demonstrated positive anti-ganglioside M2 antibodies, this case is unique given the positivity of anti-ganglioside D1a IgG/IgM antibodies.

Emerging Subspecialties in Neurology: A Career as a Clinical Trialist in Neurology.

The recent therapeutic advances in the field of neurology highlight the importance of ongoing clinical trials. However, while clinical research in neurology has remained relatively stable over the past 10 years, there has been an interval decrease in neuroscience applicants for NIH funding, which has raised concerns about the pipeline and future of clinical research in neurology. Those interested in such a career can begin by identifying a preclinical neuroscience advance that has yet to be translated into clinical trial work or a clinical area of need based on conversations with patients and families. Once such an area of interest is identified, seeking mentors either at one's own local institution or through networking at conferences is important in developing the necessary skills pertaining to clinical trial conduct and design and in gaining access to the relevant professional networks. There is also a myriad of training opportunities, such as the NINDS Clinical Trials Methodology Course, Masters of Science in Clinical Research, and certificate programs that offer formal training. Additional considerations for advancing in this career include exploring the potential for secondary publications using data available from previous clinical trials or serving as a subinvestigator. Challenges in pursuing such a career include the relatively low rate of positive outcomes compared with other fields and consistent salary support throughout one's career. Overall, a career as a clinical trialist in neurology is rewarding because one is able to participate in advancing the field and offer potentially new treatments to their patients.

Chemical Characterization of Leachables in Catheter Device.

Extractables or leachables of biomaterials or residues of additives used in the manufacturing process that are potentially released from a medical device may have an adverse effect on a patient. Chemical characterization of leachable chemicals and degradation products in a medical device is an important aspect of its overall biocompatibility assessment process, which helps to ensure that the therapeutical benefits exceed the potential biological risks associated with the use of the device or its components or materials. By evaluating the types and amounts of chemicals that may migrate from a device to a patient during clinical use, potential toxicological risks can be assessed. A semipolar solvent, 40% ethanol in water (v/v), an appropriate surrogate for blood and blood related substances, was used as an extraction medium to mimic the body fluid in contact with a medical device. The extraction was conducted at 37 °C for 24 h for limited exposure medical devices per ISO 10993-12:2021. From gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis, leachable chemicals of polylactams, linear polyamides, cyclic polytetramethylene ether (PTME), poly(tetramethylene ether) glycol (PTMEG), cyclic and linear poly(tetramethylene ether) glycol adipate (PTMEGA), cyclic and linear poly(tetramethylene ether) glycol adipamide (PTMEGAA) were structurally elucidated. The workflow presented in this study was proven to be a successful approach for rapid extractable and leachable profiling and identification with confidence.

DNA methylation-based surrogates of plasma proteins are associated with Parkinson's disease risk.

BACKGROUND: The epigenome may reflect Parkinson's disease (PD) risk, which serves as a point of convergence of genetic and environmental risk factors. Here, we investigate whether blood DNA methylation (DNAm) markers are associated with PD risk. METHODS: We selected 12 plasma proteins known as predictors of cardiovascular conditions and mortality to evaluate their effects on PD risk in a case-control study. In lieu of protein level measures, however, we assessed the influence of their DNAm surrogates. Primary analysis was restricted to 569 PD patients and 238 controls with DNAm data available. Using univariate logistic regression, we evaluated associations between the DNAm markers and PD. RESULTS: Of the 12 DNAm surrogates, the most robustly associated were DNAm EFEMP-1 and DNAm CD56, which were associated with PD with and without controlling for blood cell composition. DNAm EFEMP-1 was associated with a decreased risk of PD (OR = 0.83 per SD, 95% CI = 0.70, 0.98) whereas DNAm CD56 was associated with an increased risk of PD (OR = 1.41, 95% CI = 1.11, 1.79). CONCLUSIONS: Several DNAm markers, selected as part of a panel to track cardiovascular outcomes and mortality, were associated with PD risk. DNAm markers may inform of factors that are affected differentially in early PD patients compared with controls.

Tooth Removal in the Leopard Gecko and the de novo Formation of Replacement Teeth.

Many reptiles are able to continuously replace their teeth through life, an ability attributed to the existence of epithelial stem cells. Tooth replacement occurs in a spatially and temporally regulated manner, suggesting the involvement of diffusible factors, potentially over long distances. Here, we locally disrupted tooth replacement in the leopard gecko (Eublepharis macularius) and followed the recovery of the dentition. We looked at the effects on local patterning and functionally tested whether putative epithelial stem cells can give rise to multiple cell types in the enamel organs of new teeth. Second generation teeth with enamel and dentine were removed from adult geckos. The dental lamina was either left intact or disrupted in order to interfere with local patterning cues. The dentition began to reform by 1 month and was nearly recovered by 2-3 months as shown in µCT scans and eruption of teeth labeled with fluorescent markers. Microscopic analysis showed that the dental lamina was fully healed by 1 month. The deepest parts of the dental lamina retained odontogenic identity as shown by PITX2 staining. A pulse-chase was carried out to label cells that were stimulated to enter the cell cycle and then would carry BrdU forward into subsequent tooth generations. Initially we labeled 70-78% of PCNA cells with BrdU. After a 1-month chase, the percentage of BrdU + PCNA labeled cells in the dental lamina had dropped to 10%, consistent with the dilution of the label. There was also a population of single, BrdU-labeled cells present up to 2 months post surgery. These BrdU-labeled cells were almost entirely located in the dental lamina and were the likely progenitor/stem cells because they had not entered the cell cycle. In contrast fragmented BrdU was seen in the PCNA-positive, proliferating enamel organs. Homeostasis and recovery of the gecko dentition was therefore mediated by a stable population of epithelial stem cells in the dental lamina.

Symmetry and fluctuation of cell movements in neural crest-derived facial mesenchyme.

In the face, symmetry is established when bilateral streams of neural crest cells leave the neural tube at the same time, follow identical migration routes and then give rise to the facial prominences. However, developmental instability exists, particularly surrounding the steps of lip fusion. The causes of instability are unknown but inability to cope with developmental fluctuations are a likely cause of congenital malformations, such as non-syndromic orofacial clefts. Here, we tracked cell movements over time in the frontonasal mass, which forms the facial midline and participates in lip fusion, using live-cell imaging of chick embryos. Our mathematical examination of cell velocity vectors uncovered temporal fluctuations in several parameters, including order/disorder, symmetry/asymmetry and divergence/convergence. We found that treatment with a Rho GTPase inhibitor completely disrupted the temporal fluctuations in all measures and blocked morphogenesis. Thus, we discovered that genetic control of symmetry extends to mesenchymal cell movements and that these movements are of the type that could be perturbed in asymmetrical malformations, such as non-syndromic cleft lip. This article has an associated 'The people behind the papers' interview.

Robinow syndrome skeletal phenotypes caused by the WNT5AC83S variant are due to dominant interference with chondrogenesis.

Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.

Genetic association study of NLRP1, CARD, and CASP1 inflammasome genes with chronic Chagas cardiomyopathy among Trypanosoma cruzi seropositive patients in Bolivia.

About 20-30% of people infected with Chagas disease present with chronic Chagas cardiomyopathy (CCC), the most serious and frequent manifestation of the disease, while others remain asymptomatic and often do not experience Chagas-specific mortality. It is not currently well understood what causes these differential disease outcomes, but a genetic predisposition within the host could play an important role. This study examined variants in the NLRP1, CARD, and CASP1 inflammasome genes among 62 T. cruzi seropositive patients from Bolivia (38 cases with CCC and 24 asymptomatic controls) to uncover associations with CCC. All subjects underwent a complete medical examination including electrocardiogram (EKG) and echocardiogram. After genotype calling and quality control filtering with exclusion of 3 cases and 3 controls, association analysis was performed across 76 directly genotyped SNPs in NLRP1, CARD, and CASP1 genes, adjusting for age, sex, and population stratification. One SNP (rs11651270; Bonferroni-corrected p = 0.036) corresponding to a missense mutation in NLPR1 was found to be significant after adjustment for multiple testing, and a suggestive association was seen in CARD11 (rs6953573; Bonferroni-corrected p = 0.060). Although limited by sample size, the study results suggest variations in the inflammasome, particularly in NLRP1 and CARD11, may be associated with CCC.