Cutaneous manifestations of cystic fibrosis.

Cystic fibrosis (CF) is caused by a mutation in the Cystic fibrosis transmembrane conductance regulator (CFTR) gene, and features recurrent sinus and pulmonary infections, steatorrhea, and malnutrition. CF is associated with diverse cutaneous manifestations, including transient reactive papulotranslucent acrokeratoderma of the palms, nutrient deficiency dermatoses, and vasculitis. Rarely these are presenting symptoms of CF, prior to pulmonary or gastrointestinal sequelae. Cutaneous drug eruptions are also highly common in patients with CF (PwCF) given frequent antibiotic exposure. Finally, CFTR modulating therapy, which has revolutionized CF management, is associated with cutaneous side effects ranging from acute urticaria to toxic epidermal necrolysis. Recognition of dermatologic clinical manifestations of CF is important to appropriately care for PwCF. Dermatologists may play a significant role in the diagnosis and management of CF and associated skin complications.

Metabolism-driven in vitro/in vivo disconnect of an oral ERɑ VHL-PROTAC.

Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.

Challenges with the Synthesis of a Macrocyclic Thioether Peptide: From Milligram to Multigram Using Solid Phase Peptide Synthesis (SPPS).

We describe an optimization and scale-up of the 45-membered macrocyclic thioether peptide BMS-986189 utilizing solid-phase peptide synthesis (SPPS). Improvements to linear peptide isolation, macrocyclization, and peptide purification were demonstrated to increase the throughput and purification of material on scale and enabled the synthesis and purification of >60 g of target peptide. Taken together, not only these improvements resulted in a 28-fold yield increase from the original SPPS approach, but also the generality of this newly developed SPPS purification sequence has found application in the synthesis and purification of other macrocyclic thioether peptides.

Systematic analysis of the transcriptional landscape of melanoma reveals drug-target expression plasticity.

Metastatic melanoma originates from melanocytes of the skin. Melanoma metastasis results in poor treatment prognosis for patients and is associated with epigenetic and transcriptional changes that reflect the developmental program of melanocyte differentiation from neural crest stem cells. Several studies have explored melanoma transcriptional heterogeneity using microarray, bulk and single-cell RNA-sequencing technologies to derive data-driven models of the transcriptional-state change which occurs during melanoma progression. No study has systematically examined how different models of melanoma progression derived from different data types, technologies and biological conditions compare. Here, we perform a cross-sectional study to identify averaging effects of bulk-based studies that mask and distort apparent melanoma transcriptional heterogeneity; we describe new transcriptionally distinct melanoma cell states, identify differential co-expression of genes between studies and examine the effects of predicted drug susceptibilities of different cell states between studies. Importantly, we observe considerable variability in drug-target gene expression between studies, indicating potential transcriptional plasticity of melanoma to down-regulate these drug targets and thereby circumvent treatment. Overall, observed differences in gene co-expression and predicted drug susceptibility between studies suggest bulk-based transcriptional measurements do not reliably gauge heterogeneity and that melanoma transcriptional plasticity is greater than described when studies are considered in isolation.

Analysis of iron status after initiation of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis.

BACKGROUND: Iron deficiency is highly prevalent in people with cystic fibrosis (PwCF). While elexacaftor/tezacaftor/ivacaftor (ETI) has shown remarkable improvements in respiratory symptoms in PwCF, the effect of ETI on iron status remains unknown. This study aims to identify the effect of ETI on iron status in PwCF. METHODS: A single-center retrospective cohort study of 127 adult PwCF was conducted to assess the impact of ETI on iron, ferritin, transferrin levels, and percent saturation of transferrin (PSAT). Data were collected from the electronic medical record from January 2017 to September 2022, encompassing 2 years before and after ETI initiation. The primary outcome was serum iron parameters: iron, ferritin, transferrin, and PSAT levels following ETI treatment. Secondary outcomes analyzed iron supplementation. Univariate and multivariate mixed-effects models were used for the analysis of ETI. RESULTS: After adjusting for covariates, following ETI initiation, the mean iron level increased by 20.24 µg/dL (p < .001), ferritin levels were 31.4% (p < .001) higher, PSAT showed a 5.09 percentage point increase (p < .001), and transferrin levels increased by 2.71 mg/dL (p = .439). Patients with and without iron supplementation experienced a significant increase in iron after ETI (p < .001). CONCLUSIONS: ETI is associated with a significant increase in iron, ferritin, and PSAT levels. Patients with and without iron supplementation demonstrated a significant increase in iron. This study shows the benefits of ETI on iron status in PwCF. However, further translational studies are required to understand the impact of ETI on iron absorption and metabolism in PwCF.

Impact of the physical-chemical properties of poly(lactic acid)-poly(ethylene glycol) polymeric nanoparticles on biodistribution.

Nanoparticle (NP) formulations are inherently polydisperse making their structural characterization and justification of specifications complex. It is essential, however, to gain an understanding of the physico-chemical properties that drive performance in vivo. To elucidate these properties, drug-containing poly(lactic acid) (PLA)-poly(ethylene glycol) (PEG) block polymeric NP formulations (or PNPs) were sub-divided into discrete size fractions and analyzed using a combination of advanced techniques, namely cryogenic transmission electron microscopy, small-angle neutron and X-ray scattering, nuclear magnetic resonance, and hard-energy X-ray photoelectron spectroscopy. Together, these techniques revealed a uniquely detailed picture of PNP size, surface structure, internal molecular architecture and the preferred site(s) of incorporation of the hydrophobic drug, AZD5991, properties which cannot be accessed via conventional characterization methodologies. Within the PNP size distribution, it was shown that the smallest PNPs contained significantly less drug than their larger sized counterparts, reducing overall drug loading, while PNP molecular architecture was critical in understanding the nature of in vitro drug release. The effect of PNP size and structure on drug biodistribution was determined by administrating selected PNP size fractions to mice, with the smaller sized NP fractions increasing the total drug-plasma concentration area under the curve and reducing drug concentrations in liver and spleen, due to greater avoidance of the reticuloendothelial system. In contrast, administration of unfractionated PNPs, containing a large population of NPs with extremely low drug load, did not significantly impact the drug's pharmacokinetic behavior - a significant result for nanomedicine development where a uniform formulation is usually an important driver. We also demonstrate how, in this study, it is not practicable to validate the bioanalytical methodology for drug released in vivo due to the NP formulation properties, a process which is applicable for most small molecule-releasing nanomedicines. In conclusion, this work details a strategy for determining the effect of formulation variability on in vivo performance, thereby informing the translation of PNPs, and other NPs, from the laboratory to the clinic.

Head and Neck Ultrasound Utilization Rates: 2012 to 2019.

Objective: We measured utilization of clinician-performed head and neck ultrasound among otolaryngologists, endocrinologists, and general surgeons, using Medicare Provider Utilization and Payment Data. Study Design: Retrospective analysis of Medicare billing database. Setting: University. Methods: For each year, the files were filtered to include 4 provider types: Diagnostic Radiology (DR), Endocrinology (ENDO), General Surgery (GS), and Otolaryngology (OTO). Billable procedures are listed by Healthcare Common Procedure Coding System code and a filter was applied to include 76536 Ultrasound, soft tissues of the head and neck. Results: In 2019, OTOs submitted charges for 2.1% of all head and neck diagnostic ultrasounds (76536) performed on Medicare beneficiaries. For each year 2012 to 2019, DRs submitted the most charges, followed by ENDOs, and then OTO and GS. Charges for all groups increased in a proportional manner across the 8-year period. 14.5% of OTOs submitted more than 100 charges apiece during 2019, that is, "super users." The percentage of super users within each specialty increased from 2012 to 2019. Overall, the data support an ever-increasing use of head and neck ultrasound (HNUS) among all provider types. Conclusion: Even with increased use among OTOs, this specialty only accounted for a small percentage of head & neck diagnostic ultrasounds performed on Medicare beneficiaries in 2019. Changes in volume of nonradiology point-of-care HNUS was not associated with changes in DR volume. A greater proportion of OTOs than DRs are "super users" among the ultrasound users within their specialty, performing more than 100 exams/year. Level of Evidence: V.

Outpatient minimally invasive spine surgeries during the COVID-19 pandemic - A retrospective analysis of 164 consecutive cases.

Objective: To share our surgical experiences of minimally invasive cervical and lumbar procedures for patients who suffered from non-fatal motor vehicle accidents (MVAs) in the ambulatory surgery centers (ASCs) during the coronavirus disease 2019 (COVID-19) pandemic. Methods: Anterior cervical discectomy and fusion (ACDF), anterior lumbar interbody fusion (ALIF), minimally invasive laminotomy and discectomy (MILD), percutaneous endoscopic laser-assisted discectomy (PELD) and percutaneous kyphoplasty (PK) were performed on carefully selected patients. Results: From January 2020 to December 2021, our group performed 164 cases on 153 patients involving 249 intervertebral disc (IVD) levels. Of these, 116 cases (70.73%) on 114 patients (74.51%) were cervical, 48 cases (29.27%) were lumbar (including 8 PK cases). Eight patients had both cervical and lumbar procedures in a single anesthetic session (SAS) and were discharged on the same day. One hundred and six ACDF cases (92.17%) were at the C4-C5 and C5-C6 levels, which comprised of 146 (76.04%) IVDs. Of the 40 non-PK lumbar cases, 38 (95.0%) were at L4 to S1 lumbar levels. Six of these cases (15.0%) involved 2 lumbar levels. In contrast, 6 out of 8 kyphoplasties (75.0%) involved lower thoracic/higher lumbar vertebral columns (T11 to L2) and 2 were at the lower lumbar L4 level. Conclusions: We successfully and safely performed various cervical and lumbar spine surgeries in the ASCs amid COVID-19 pandemic and all patients achieved the same-day discharge (SDD). In the non-fatal MVAs, mid-lower cervical (C4 to C6) and lower lumbar (L4 to S1) IVDs were the most affected levels.

Multi-institutional feasibility and safety outcomes of retroperitoneal robot-assisted partial nephrectomy in morbidly obese patients.

Background: Robotic-assisted partial nephrectomy (RAPN) is an established treatment modality for small renal masses. While retroperitoneal RAPN (rRAPN) has the benefit of avoiding the peritoneal cavity and provides more direct access to the renal hilum and posterior kidney, there is concern about the feasibility of rRAPN particularly in morbidly obese [body mass index (BMI) ≥40 kg/m2] patients. We present a large scale multi-institutional study on the outcomes of rRAPN in morbidly obese patients. Methods: A retrospective review of a cohort of morbidly obese patients who underwent rRAPN at two academic institutions was performed. Patient characteristics, operative data, and postoperative complication rates were assessed. Results: A total of 22 morbidly obese patients were included for analysis, with a median follow-up duration of 52 months. Median patient age was 61 years and median BMI was 44.9 kg/m2. Based on nephrometry score, 55% of the masses had low complexity and 32% had intermediate complexity. Median operative time was 186.0 minutes and median warm ischemia time was 23.5 minutes. Median postoperative length of stay was 2 days, and only one patient experienced a high-grade complication within 30 days of surgery. Conclusions: rRAPN in select morbidly obese patients appears to have acceptable operative and postoperative outcomes. Further studies and follow-up are needed to better generalization and understand long-term impacts.

Reconstitution of the Arginyltransferase (ATE1) Iron-Sulfur Cluster.

As global regulators of eukaryotic homeostasis, arginyltransferases (ATE1s) have essential functions within the cell. Thus, the regulation of ATE1 is paramount. It was previously postulated that ATE1 was a hemoprotein and that heme was an operative cofactor responsible for enzymatic regulation and inactivation. However, we have recently shown that ATE1 instead binds an iron-sulfur ([Fe-S]) cluster that appears to function as an oxygen sensor to regulate ATE1 activity. As this cofactor is oxygen-sensitive, purification of ATE1 in the presence of O2 results in cluster decomposition and loss. Here, we describe an anoxic chemical reconstitution protocol to assemble the [Fe-S] cluster cofactor in Saccharomyces cerevisiae ATE1 (ScATE1) and Mus musculus ATE1 isoform 1 (MmATE1-1).

Discovery of the Potent and Selective MC4R Antagonist PF-07258669 for the Potential Treatment of Appetite Loss.

The melanocortin-4 receptor (MC4R) is a centrally expressed, class A GPCR that plays a key role in the regulation of appetite and food intake. Deficiencies in MC4R signaling result in hyperphagia and increased body mass in humans. Antagonism of MC4R signaling has the potential to mitigate decreased appetite and body weight loss in the setting of anorexia or cachexia due to underlying disease. Herein, we report on the identification of a series of orally bioavailable, small-molecule MC4R antagonists using a focused hit identification effort and the optimization of these antagonists to provide clinical candidate 23. Introduction of a spirocyclic conformational constraint allowed for simultaneous optimization of MC4R potency and ADME attributes while avoiding the production of hERG active metabolites observed in early series leads. Compound 23 is a potent and selective MC4R antagonist with robust efficacy in an aged rat model of cachexia and has progressed into clinical trials.

Iron-sulfur clusters are involved in post-translational arginylation.

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

Expression of membrane Hsp90 is a molecular signature of T cell activation.

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells. In cell-based assays, stimulation of human T cells induced a 20-fold increase in mHsp90 expression at the plasma membrane, suggesting trafficking of mHsp90 is regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in reducing the mean clinical score in the CIA arthritis model. Our results suggest mHsp90 expression on T cells is a molecular marker of T cell activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis.

Preclinical Characterization of AZD5305, A Next-Generation, Highly Selective PARP1 Inhibitor and Trapper.

PURPOSE: We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination. EXPERIMENTAL DESIGN: AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination. RESULTS: AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response. CONCLUSIONS: AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB.

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein-protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA-NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

Initial experience and cancer detection rates of office-based transperineal magnetic resonance imaging-ultrasound fusion prostate biopsy under local anesthesia.

INTRODUCTION: We aimed to demonstrate feasibility and cancer detection rates of office-based ultrasound-guided transperineal magnetic resonance imaging-ultrasound (MRI-US) fusion (TFB) prostate biopsy under local anesthesia. METHODS: With institutional review board approval, records of men undergoing TFB in the office setting under local anesthesia were reviewed. Baseline patient characteristics, MRI findings, cancer detection rates, and complications were recorded. The PrecisionPoint Transperineal Access System (Perineologic, Cumberland, MD, U.S.), along with UroNav 3.0 image-fusion system (Invivo International, Best, The Netherlands) were used for all procedures. Following biopsy, men were surveyed to assess patient experience. RESULTS: Between January 2019 and February 2020, 200 TFBs were performed, of which 141 (71%) were positive for prostate cancer, with 117 (83%) Gleason grade group 2 or higher. A total of 259 of 265 MRI lesions were biopsied, with 127 (49%) positive overall. Prostate Imaging-Reporting and Data System (PI-RADS) 4-5 lesions were positive for prostate cancer in 59% of cases. The mean procedural time was 20 minutes, with a patient enter-to-exit room time of 54 minutes. There were no septic complications, no patients required post-procedure hospital admission, and all procedures were successfully completed. Seventy-five percent of patients surveyed reported complete resolution of pain at three days following the procedure. CONCLUSIONS: Office-based TFB represents a viable approach to prostate cancer detection following prostate MRI. Larger-scale assessment is needed to categorize cancer detection rates more accurately by PI-RADs subset, patient selection factors, complication rate, and cost relative to TFB under anesthesia.

Multi-modal molecular imaging maps the correlation between tumor microenvironments and nanomedicine distribution.

Gaining insight into the heterogeneity of nanoparticle drug distribution within tumors would improve both design and clinical translation of nanomedicines. There is little data showing the spatio-temporal behavior of nanomedicines in tissues as current methods are not able to provide a comprehensive view of the nanomedicine distribution, released drug or its effects in the context of a complex tissue microenvironment. Methods: A new experimental approach which integrates the molecular imaging and bioanalytical technologies MSI and IMC was developed to determine the biodistribution of total drug and drug metabolite delivered via PLA-PEG nanoparticles and to overlay this with imaging of the nanomedicine in the context of detailed tumor microenvironment markers. This was used to assess the nanomedicine AZD2811 in animals bearing three different pre-clinical PDX tumors. Results: This new approach delivered new insights into the nanoparticle/drug biodistribution. Mass spectrometry imaging was able to differentiate the tumor distribution of co-dosed deuterated non-nanoparticle-formulated free drug alongside the nanoparticle-formulated drug by directly visualizing both delivery approaches within the same animal or tissue. While the IV delivered free drug was uniformly distributed, the nanomedicine delivered drug was heterogeneous. By staining for multiple biomarkers of the tumor microenvironment on the same tumor sections using imaging mass cytometry, co-registering and integrating data from both imaging modalities it was possible to determine the features in regions with highest nanomedicine distribution. Nanomedicine delivered drug was associated with regions higher in macrophages, as well as more stromal regions of the tumor. Such a comparison of complementary molecular data allows delineation of drug abundance in individual cell types and in stroma. Conclusions: This multi-modal imaging solution offers researchers a better understanding of drug and nanocarrier distribution in complex tissues and enables data-driven drug carrier design.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.