Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport.

The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to microtubule plus-ends). Although LIS1 binding is required for dynein activity, its unbinding prior to initiation of cargo transport is equally important, since preventing dissociation leads to dynein dysfunction. To understand whether and how dynein-LIS1 binding is modulated, we engineered dynein mutants locked in a microtubule-bound (MT-B) or microtubule-unbound (MT-U) state. Whereas the MT-B mutant exhibits low LIS1 affinity, the MT-U mutant binds LIS1 with high affinity, and as a consequence remains almost irreversibly associated with microtubule plus-ends. We find that a monomeric motor domain is sufficient to exhibit these opposing LIS1 affinities, and that this is evolutionarily conserved between yeast and humans. Three cryo-EM structures of human dynein with and without LIS1 reveal microtubule-binding induced conformational changes responsible for this regulation. Our work reveals key biochemical and structural insight into LIS1-mediated dynein activation.

Exploring the knowledge, attitude, and practices of over-the-counter medical sellers in Ghana.

Background: Rural areas in sub-Saharan Africa face a high prevalence and morbidity of skin disease while also lacking access to dermatologists. In Ghana, where approximately 25 licensed dermatologists are available for 25 million people, community pharmacies, called over-the-counter medical sellers (OTCMS), were established to respond to accessibility inequities, albeit without equitable training. Objective: Our study evaluates the dermatologic knowledge, attitudes, and practices (KAP) of OTCMS in Ghana's Ashanti Region. Methods: To assess dermatologic KAP, we created a standardized questionnaire and recorded 13 interviews with OTCMS in seven communities. Interviews were completed with help from Ghanaian translators and transcripts were transcribed verbatim, then analyzed qualitatively to determine common themes for analysis. Results: This analysis identified six major themes: i) prescriber qualifications; ii) diagnostics; iii) therapeutics; iv) economics; v) health systems integration; vi) care-seeking behavior. Analysis of these themes outlined many cultural roles and challenges of OTCMS, including serving as the primary contact for dermatologic conditions in rural communities. While possibly necessary due to the lack of accessible dermatologists, this raises concerns for potential harm in diagnostic error and misuse of therapeutics due to the lack of formal dermatology training. Conclusion: In rural parts of Ghana, the KAP of OTCMS play a pivotal role in assessing and treating skin disease for those who might otherwise lack access to adequate dermatologic management. Furthermore, although our study identifies potential issues related to the roles played by OTCMS, it also suggests strategies to improve the dermatologic health of many Ghanaians by enhancing education and healthcare delivery in rural areas.

Patient-Reported Outcome Measures and Their Clinical Applications in Dermatology.

With more disease- and symptom-specific measures available and research pointing to increased usefulness, patient-reported outcome measures (PROMs) can be routinely used in clinical care. PROMs increase efficiency in healthcare, improve the clinician-patient relationship, and increase patient satisfaction with their care. PROMs can be administered before, during, and after clinic visits using paper-and-pencil, mobile phones, tablets, and computers. Herein, we combine available literature with expert views to discuss overcoming barriers and helping dermatologists incorporate PROMs into routine patient-centered care. We believe dermatology patients will benefit from broader PROM implementation and routine clinical use. However, a few major barriers exist: (1) cost to implement the technology, (2) selecting the right PROMs for each disease, and (3) helping both patients and clinicians understand how PROMs add to and complement their current clinical experience. We provide recommendations to assist dermatologists when considering whether to implement PROMs in their practices.

Incomplete Staining Artifact: A Confounding Frozen Section Pathology Artifact Encountered During Mohs Micrographic Surgery.

The intent of this brief communication is to describe a unique incomplete staining frozen section pathology artifact encountered during Mohs Micrographic Surgery. At the authors’ institution, an amorphous, eosinophilic artifact that obscured cellular architecture was observed multiple times during histological interpretation. It was determined that incomplete tissue staining was likely caused by weak staining, possibly related to an interaction between hematoxylin dye solution and acetone. We adjusted our SLS stain line protocol by adding a 15 second water rinse between the acetone and hematoxylin pots and then compared the old fixation protocol with our new fixation protocol. This artifact, which was regularly found intraoperatively at five separate MMS laboratories has sustainably resolved. Mohs Micrographic Surgery (MMS) is a dermatologic procedure that includes tumor extirpation, tissue grossing, slide preparation, and microscopic histologic interpretation. Tissue grossing and slide preparation are vital components of the MMS procedure. There are many steps throughout tissue processing that can result in frozen section pathology artifacts. Frequently encountered frozen section pathology artifacts include vacuolation of cytoplasm or “freeze artifact,” overstaining and understaining with hematoxylin and eosin, incomplete dehydration, and splaying of collagen in the dermis.1-3 We describe a unique incomplete staining frozen section pathology artifact. J Drugs Dermatol. 2022;21(5):542-544. doi:10.36849/JDD.6722.

Micromagnetic and morphological characterization of heteropolymer human ferritin cores.

The physical properties of in vitro iron-reconstituted and genetically engineered human heteropolymer ferritins were investigated. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), electron energy-loss spectroscopy (EELS), and 57Fe Mössbauer spectroscopy were employed to ascertain (1) the microstructural, electronic, and micromagnetic properties of the nanosized iron cores, and (2) the effect of the H and L ferritin subunit ratios on these properties. Mössbauer spectroscopic signatures indicate that all iron within the core is in the high spin ferric state. Variable temperature Mössbauer spectroscopy for H-rich (H21/L3) and L-rich (H2/L22) ferritins reconstituted at 1000 57Fe/protein indicates superparamagnetic behavior with blocking temperatures of 19 K and 28 K, while HAADF-STEM measurements give average core diameters of (3.7 ± 0.6) nm and (5.9 ± 1.0) nm, respectively. Most significantly, H-rich proteins reveal elongated, dumbbell, and crescent-shaped cores, while L-rich proteins present spherical cores, pointing to a correlation between core shape and protein shell composition. Assuming an attempt time for spin reversal of τ 0 = 10-11 s, the Néel-Brown formula for spin-relaxation time predicts effective magnetic anisotropy energy densities of 6.83 × 104 J m-3 and 2.75 × 104 J m-3 for H-rich and L-rich proteins, respectively, due to differences in surface and shape contributions to magnetic anisotropy in the two heteropolymers. The observed differences in shape, size, and effective magnetic anisotropies of the derived biomineral cores are discussed in terms of the iron nucleation sites within the interior surface of the heteropolymer shells for H-rich and L-rich proteins. Overall, our results imply that site-directed nucleation and core growth within the protein cavity play a determinant role in the resulting core morphology. Our findings have relevance to iron biomineralization processes in nature and the growth of designer's magnetic nanoparticles within recombinant apoferritin nano-templates for nanotechnology.

The continued inappropriate use and overuse of combination topical clotrimazole-betamethasone.

OBJECTIVE: Combination topical clotrimazole/ betamethasone dipropionate (C-BM) contains a high-potency topical corticosteroid and is not infrequently prescribed for inappropriate patient groups and body sites. Use of C-BM can lead to inadequate clearance or exacerbation of fungal infections as well as cutaneous atrophy, striae, and other skin maladies. METHODS: We performed a retrospective chart review of 1,978 clinical visits where C-BM was prescribed within the University of Utah Health system between 2014 and 2018 to better understand current prescribing patterns. RESULTS: 1,974 prescriptions were written for C-BM. 91.6% of patients were at least the recommended age of 17 years. C-BM was most commonly prescribed for rashes of an inflammatory (42.2%) or fungal nature (38.1%). Clotrimazole/betamethasone dipropionate was prescribed for sensitive areas (face, axillae, groin or diaper region) in 48.9% of patients. Family medicine clinicians prescribed 58.3% of C-BM prescriptions, whereas dermatology clinicians accounted for 3.4%. CONCLUSION: We strongly recommend clinicians use alternative treatments for rashes or refer to dermatologists.

Low accuracy of self-reported family history of melanoma in high-risk patients.

Family history of melanoma is a major melanoma risk factor. However, self-reported family histories for some cancers, including melanoma, are commonly inaccurate. We used a unique database, the Utah Population Database (UPDB), as well as the Utah Cancer Registry to determine the accuracy of self-reported family history of melanoma in a large cohort of high-risk patients. Patient charts were reviewed and compared to records in the UPDB and the UCR to confirm personal and family history of melanoma in 1780 patients enrolled in a total body photography monitoring program. Self-reported family history of melanoma in first-degree relatives had an overall sensitivity of 71%, specificity of 79%, PPV of 31%, and NPV of 95%, with decreased accuracy (PPV) for second-degree relatives. A personal history of melanoma was the only factor significantly associated with accuracy in self-reported family history of melanoma. Patient age, sex, estimated nevus count, and number of prior personal melanomas were not significant predictors. Dermatologists should educate patients on the differences between melanomas, keratinocyte carcinomas, and pre-cancers. Confirming self-reported family history of melanoma may improve risk assessment for patients undergoing screening.

Administrative Burden and Costs of Prior Authorizations in a Dermatology Department.

Importance: Insurance companies use prior authorizations (PAs) to address inappropriate prescribing or unnecessary variations in care, most often for expensive medications. Prior authorizations negatively affect patient care and add costs and administrative burden to dermatology offices. Objective: To quantify the administrative burden and costs of dermatology PAs. Design, Setting, and Participants: The University of Utah Department of Dermatology employs 2 full-time and 8 part-time PA staff. In this cross-sectional study at a large academic department spanning 11 clinical locations, these staff itemized all PA-related encounters over a 30-day period in September 2016. Staff salary and benefits were publicly available. Data were analyzed between December 2018 and August 2019. Main Outcomes and Measures: Proportion of visits requiring PAs, median administrative time to finalize a PA (either approval or denial after appeal), and median cost per PA type. Results: In September 2016, 626 PAs were generated from 9512 patient encounters. Staff spent 169.7 hours directly handling PAs, costing a median of $6.72 per PA. Biologic PAs cost a median of $15.80 each and took as long as 31 business days to complete. The costliest PA equaled 106% of the associated visit's Medicare reimbursement rate. Approval rates were 99.6% for procedures, 78.9% for biologics, and 58.2% for other medications. After appeal, 5 of 23 (21.7%) previously denied PAs were subsequently approved. Conclusions and Relevance: Prior authorizations are costly to dermatology practices and their value appears limited for some requests. Fewer unnecessary PAs and appeals might increase practice efficiency and improve patient outcomes.

Thermodynamic and Kinetic Studies of the Interaction of Nuclear Receptor Coactivator-4 (NCOA4) with Human Ferritin.

Ferritinophagy is a ferritin autophagic degradation process mediated by the selective nuclear receptor coactivator-4 (NCOA4). NCOA4 binds to ferritin and delivers it to nascent autophagosomes, which then merge with the lysosomes for ferritin degradation and iron release. Earlier studies have demonstrated a specific association of NCOA4 with ferritin H-subunits, but not L-subunits. However, neither the thermodynamics of this interaction nor the effect of NCOA4 on iron oxidation, iron mineral core formation, or iron mobilization in ferritin has been explored. Using isothermal titration calorimetry, light absorption spectroscopy, and a soluble fragment (residues 383-522) of human NCOA4 expressed in Escherichia coli, we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of ∼8 NCOA4 molecules per ferritin shell, and Kd values of ∼0.4 and ∼2 µM for homopolymer H-chain ferritin and heteropolymer H-rich ferritin, respectively. The binding reaction was both enthalpically and entropically favored. Whereas the iron oxidation kinetics were not affected by the presence of NCOA4, iron mobilization from ferritin by two different reducing agents (FMN/NADH and sodium dithionite) showed a strong inhibitory effect that was dependent on the concentration of NCOA4 present in solution. Our results suggest that the binding of NCOA4 to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

Ferritin exhibits Michaelis-Menten behavior with oxygen but not with iron during iron oxidation and core mineralization.

The excessively high and inconsistent literature values for Km,Fe and Km,O2 prompted us to examine the iron oxidation kinetics in ferritin, the major iron storage protein in mammals, and to determine whether a traditional Michaelis-Menten enzymatic behavior is obeyed. The kinetics of Fe(ii) oxidation and mineralization catalyzed by three different types of ferritins (recombinant human homopolymer 24H, HuHF, human heteropolymer ∼21H:3L, HL, and horse spleen heteropolymer ∼3.3H:20.7L, HosF) were therefore studied under physiologically relevant O2 concentrations, but also in the presence of excess Fe(ii) and O2 concentrations. The observed iron oxidation kinetics exhibited two distinct phases (phase I and phase II), neither of which obeyed Michaelis-Menten kinetics. While phase I was very rapid and corresponded to the oxidation of approximately 2 Fe(ii) ions per H-subunit, phase II was much slower and varied linearly with the concentration of iron(ii) cations in solution, independent of the size of the iron core. Under low oxygen concentration close to physiological, the iron uptake kinetics revealed a Michaelis-Menten behavior with Km,O2 values in the low µM range (i.e. ∼1-2 µM range). Our experimental Km,O2 values are significantly lower than typical cellular oxygen concentration, indicating that iron oxidation and mineralization in ferritin should not be affected by the oxygenation level of cells, and should proceed even under hypoxic events. A kinetic model is proposed in which the inhibition of the protein's activity is caused by bound iron(iii) cations at the ferroxidase center, with the rate limiting step corresponding to an exchange or a displacement reaction between incoming Fe(ii) cations and bound Fe(iii) cations.

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.

Chemical conversion of human fibroblasts into functional Schwann cells.

Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

Characterization of post-surgical alterations in the bile duct-cannulated rat.

The bile duct-cannulated (BDC) rat is a standard animal model used in ADME experiments. The aim of this study was to investigate post-surgical alterations that are relevant to ADME investigations in BDC rats compared with sham- and non-operated animals. Water and food intake was reduced in the animals' post-surgery. This led to a lower body weight in operated animals. In BDC animals, aspartate aminotransferase (AST) levels in plasma were transiently elevated and total bile acid levels were reduced. Alpha(1)-acid glycoprotein (AGP) in plasma and the concentration of bile components in bile were elevated. Histopathology showed inflammation in the area of the cannulation between the liver and the small intestine. A microarray-based gene expression and RTq-PCR analysis identified altered expression for several genes involved in drug disposition including the down-regulation of cytochrome P450 enzymes. This led to reduced cytochrome P450 content in the liver and lower metabolic activity in microsomes from BDC and sham-operated rats compared with naïve animals. The results of the study suggest that the post-surgical inflammation leads to physiological changes relevant for drug absorption and disposition. These alterations should be accounted for in the interpretation of ADME studies in BDC animals.