Altered skin microbiome, inflammation, and JAK/STAT signaling in Southeast Asian ichthyosis patients.

BACKGROUND: Congenital ichthyosis (CI) is a collective group of rare hereditary skin disorders. Patients present with epidermal scaling, fissuring, chronic inflammation, and increased susceptibility to infections. Recently, there is increased interest in the skin microbiome; therefore, we hypothesized that CI patients likely exhibit an abnormal profile of epidermal microbes because of their various underlying skin barrier defects. Among recruited individuals of Southeast Asian ethnicity, we performed skin meta-genomics (i.e., whole-exome sequencing to capture the entire multi-kingdom profile, including fungi, protists, archaea, bacteria, and viruses), comparing 36 CI patients (representing seven subtypes) with that of 15 CI age-and gender-matched controls who had no family history of CI. RESULTS: This case-control study revealed 20 novel and 31 recurrent pathogenic variants. Microbiome meta-analysis showed distinct microbial populations, decreases in commensal microbiota, and higher colonization by pathogenic species associated with CI; these were correlated with increased production of inflammatory cytokines and Th17- and JAK/STAT-signaling pathways in peripheral blood mononuclear cells. In the wounds of CI patients, we identified specific changes in microbiota and alterations in inflammatory pathways, which are likely responsible for impaired wound healing. CONCLUSIONS: Together, this research enhances our understanding of the microbiological, immunological, and molecular properties of CI and should provide critical information for improving therapeutic management of CI patients.

Impact of baseline SARS-CoV-2 load in plasma and upper airways on the incidence of acute extrapulmonary complications of COVID-19: a multicentric, prospective, cohort study.

BACKGROUND: Extrapulmonary complications (EPCs) are common in patients hospitalized for COVID-19, but data on their clinical consequences and association with viral replication and systemic viral dissemination is lacking. METHODS: Patients hospitalized for COVID-19 and enrolled in the TICO (Therapeutics for Inpatients with COVID-19) platform trial at 114 international sites between August 2020 and November 2021 were included in a prospective cohort study. We categorized EPCs into 39 event types within 9 categories and estimated their frequency through day 28 and their association with clinical outcomes through day 90. We analyzed the association between baseline viral burden (plasma nucleocapsid antigen [N-Ag] and upper airway viral load [VL]) and EPCs, adjusting for other baseline factors. RESULTS: 2,625 trial participants were included in the study. The median age was 57 years (IQR 46-68), 57.7% were male, and 537 (20.5%) had at least one EPC. EPCs were associated with higher day-90 all-cause mortality (HR 9.6, 95% CI 7.3, 12.7) after adjustment for other risk factors. The risk of EPCs increased with increasing baseline plasma N-Ag (HR 1.21 per log10 ng/L increase, 95% CI 1.09, 1.34), and upper airway VL (HR 1.12 per log10 copies/mL increase, 95% CI 1.04, 1.19), after adjusting for comorbidities, disease severity, inflammatory markers, and other baseline factors. Trial treatment allocation had no effect on EPC risk. CONCLUSIONS: Systemic viral dissemination as evidenced by high plasma N-Ag and high respiratory viral burden are associated with development of EPCs in COVID-19, which in turn are associated with higher 90-day mortality.

A Boron-Dependent Antibiotic Derived from a Calcium-Dependent Antibiotic.

The prevalence of drug-resistant bacterial pathogens foreshadows a healthcare crisis. Calcium-dependent antibiotics (CDAs) are promising candidates to combat infectious diseases as many of them show modes of action (MOA) orthogonal to widespread resistance mechanisms. The calcium dependence is nonetheless one of the hurdles toward realizing their full potential. Using laspartomycin C (LspC) as a model, we explored the possibility of reducing, or even eliminating, its calcium dependence. We report herein a synthetic LspC analogue (B1) whose activity no longer depends on calcium and is instead induced by phenylboronic acid (PBA). In LspC, Asp1 and Asp7 coordinate to calcium to anchor it in the active conformation; these residues are replaced by serine in B1 and condense with PBA to form a boronic ester with the same anchoring effect. Using thin-layer chromatography, MS, NMR, and complementation assays, we demonstrate that B1 inhibits bacterial growth via the same MOA as LspC, i.e., sequestering the cell wall biosynthetic intermediate undecaprenyl phosphate. B1 is as potent and effective as LspC against several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. Our success in converting a CDA to a boron-dependent antibiotic opens a new avenue in the design and functional control of drug molecules.

Human keratin 1/10-1B tetramer structures reveal a knob-pocket mechanism in intermediate filament assembly.

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314AK1, but not L318AK1, abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 Å structure of K1/10-1B containing a S233LK1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1S233L/K10-1B in solution without affecting secondary structure. The K1S233L/K10-1B octamer structure revealed S233LK1 causes aberrant hydrophobic interactions between 1B tetramers.

Update of the keratin gene family: evolution, tissue-specific expression patterns, and relevance to clinical disorders.

Intermediate filament (IntFil) genes arose during early metazoan evolution, to provide mechanical support for plasma membranes contacting/interacting with other cells and the extracellular matrix. Keratin genes comprise the largest subset of IntFil genes. Whereas the first keratin gene appeared in sponge, and three genes in arthropods, more rapid increases in keratin genes occurred in lungfish and amphibian genomes, concomitant with land animal-sea animal divergence (~ 440 to 410 million years ago). Human, mouse and zebrafish genomes contain 18, 17 and 24 non-keratin IntFil genes, respectively. Human has 27 of 28 type I "acidic" keratin genes clustered at chromosome (Chr) 17q21.2, and all 26 type II "basic" keratin genes clustered at Chr 12q13.13. Mouse has 27 of 28 type I keratin genes clustered on Chr 11, and all 26 type II clustered on Chr 15. Zebrafish has 18 type I keratin genes scattered on five chromosomes, and 3 type II keratin genes on two chromosomes. Types I and II keratin clusters-reflecting evolutionary blooms of keratin genes along one chromosomal segment-are found in all land animal genomes examined, but not fishes; such rapid gene expansions likely reflect sudden requirements for many novel paralogous proteins having divergent functions to enhance species survival following sea-to-land transition. Using data from the Genotype-Tissue Expression (GTEx) project, tissue-specific keratin expression throughout the human body was reconstructed. Clustering of gene expression patterns revealed similarities in tissue-specific expression patterns for previously described "keratin pairs" (i.e., KRT1/KRT10, KRT8/KRT18, KRT5/KRT14, KRT6/KRT16 and KRT6/KRT17 proteins). The ClinVar database currently lists 26 human disease-causing variants within the various domains of keratin proteins.

Harm reduction stories: leveraging graphic medicine to engage veterans in substance use services within the VA.

BACKGROUND: Harm reduction strategies can decrease morbidity and mortality associated with substance use. Various barriers limit conversation around substance use between clinicians and patients. Graphic medicine techniques can inform and encourage patient-centered conversations about substance use. We describe the co-development of a harm reduction-focused graphic medicine comic that depicts the infectious risks associated with injection drug use and patient-centered approaches to providing education about potential risk mitigation strategies. METHODS: We formed a co-design group of veterans with lived experience with substance use, physicians, health services researchers, and community-based harm reduction leaders. Over the course of ten sessions, the co-design team developed a storyline and key messages, reviewed draft content and worked with a graphic designer to develop a comic incorporating the veterans' input. During each session, co-design leads presented drafts of the comic and invited feedback from the group. The comic was edited and adapted via this iterative process. RESULTS: The comic depicts a fictionalized clinical vignette in which a patient develops an injection-related abscess and presents to their primary care provider. The dialogue highlights key healthcare principles, including patient autonomy and agency, and highlights strategies for safer use, rather than emphasizing abstinence. Feedback from co-design group participants highlights lessons learned during the development process. DISCUSSION: Graphic medicine is ideally suited for a patient-centered curriculum about harm reduction. This project is one of several interventions that will be integrated into VA facilities nationally to support incorporation of harm reduction principles into the care of persons who inject drugs.

Implementing syringe services programs within the Veterans Health Administration: facility experiences and next steps.

Syringe services programs are community-based prevention programs that provide evidence-based, lifesaving services for people who use illicit drugs, including access to syringes, naloxone, fentanyl test strips, infection screening, and linkage to treatment. Historically, syringe services programs did not exist within the Veterans Health Administration owing to many factors, including lack of clarity regarding legality for federal agency-purchased syringes. Three champions at Veterans Affairs facilities in Danville, IL, Orlando, FL, and San Francisco, CA, worked to clarify legal considerations, address barriers, and implement syringe services programs that are integrated in the health care systems. Since 2017, these 3 programs have engaged approximately 400 Veterans and distributed nearly 10,000 syringes, 2500 fentanyl test strips, 50 wound care kits, and 45 safer sex kits. These programs, both led by and in collaboration with clinical pharmacist practitioners, paved the way for nationwide implementation within the Veterans Health Administration. This commentary describes successes, challenges, and proposed next steps to increase Veteran access to syringe services programs, written from the perspective of 3 facility-based champions.

Polyurethane/polycaprolactone membrane grafted with conjugated linoleic acid for artificial vascular graft application.

Constructing satisfied small-diameter vascular graft (diameter less than 6 mm) remains an unsolvable challenge in vascular tissue engineering. This study described the fabrication of electrospun polyurethane/polycaprolactone (PU/PCL) membranes chemically grafted with various densities of conjugated linoleic acid (CLA) - an antithrombotic fatty acid - for making small-diameter blood vessel. Differences in mechanical, antithrombotic properties and biocompatibility of the membranes resulting from the CLA-grafting procedure were the focus of the study. Investigation of mechanical properties relevant to vascular graft application revealed that these properties of the membranes remained unaffected and satisfied clinical criteria following the CLA graft. Blood-membrane interaction assays showed that the CLA-grafted membranes mitigated the adhesion of blood cells, as well as preventing blood coagulation. These effects were also commensurate with increasing density of CLA, suggesting an effective approach to improve antithromboticity. Cellular tests suggested that CLA has an optimal density at which it promoted cell proliferation on the surface of the membranes; however, excessive presence of CLA might cause undesirable inhibition on cells. In conclusion, PU/PCL membrane grafted with CLA could be a prospective material for vascular tissue engineering with further development and investigation.

Molecular Modeling of Pathogenic Mutations in the Keratin 1B Domain.

Keratin intermediate filaments constitute the primary cytoskeletal component of epithelial cells. Numerous human disease phenotypes related to keratin mutation remain mechanistically elusive. Our recent crystal structures of the helix 1B heterotetramer from keratin 1/10 enabled further investigation of the effect of pathologic 1B domain mutations on keratin structure. We used our highest resolution keratin 1B structure as a template for homology-modeling the 1B heterotetramers of keratin 5/14 (associated with blistering skin disorders), keratin 8/18 (associated with liver disease), and keratin 74/28 (associated with hair disorder). Each structure was examined for the molecular alterations caused by incorporating pathogenic 1B keratin mutations. Structural modeling indicated keratin 1B mutations can harm the heterodimer interface (R265PK5, L311RK5, R211PK14, I150VK18), the tetramer interface (F231LK1, F274SK74), or higher-order interactions needed for mature filament formation (S233LK1, L311RK5, Q169EK8, H128LK18). The biochemical changes included altered hydrophobic and electrostatic interactions, and altered surface charge, hydrophobicity or contour. Together, these findings advance the genotype-structurotype-phenotype correlation for keratin-based human diseases.

Structural Basis for How Biologic Medicines Bind their Targets in Psoriasis Therapy.

As biologic therapies become first line treatments for many inflammatory disorders, it becomes increasingly important for the practicing physician to be familiar with how these drugs function at the molecular level. This information is useful in making therapeutic decisions and helping patients understand their treatment options. It is critical to patient safety and clinical response that the molecular differences between these drugs inform prescribing practices. To this end, we present and analyze the available structural biology information about the biologics used in the treatment of psoriasis including inhibitors of tumor necrosis factor alpha (TNFα), interleukin-17 (IL-17), and interleukin-23 (IL-23). We describe and analyze the molecular surface character of known binding epitopes for medications in these classes, showing that significant differences exist in epitope location, hydrophobicity, and charge. Some of these differences can be correlated with clinical data, but our analysis ultimately points to the need for more structural information to allow for a better understanding of the structure-function relationship of biologic therapies.

Crystal Structure of Keratin 1/10(C401A) 2B Heterodimer Demonstrates a Proclivity for the C-Terminus of Helix 2B to Form Higher Order Molecular Contacts.

We previously determined the crystal structure of the wild-type keratin 1/10 helix 2B heterodimer at 3.3 Å resolution. We proposed that the resolution of the diffraction data was limited due to the crystal packing effect from keratin 10 (K10) residue Cys401. Cys401K10 formed a disulfide-linkage with Cys401 from another K1/10 heterodimer, creating an "X-shaped" structure and a loose crystal packing arrangement. We hypothesized that mutation of Cys401K10 to alanine would eliminate the disulfide-linkage and improve crystal packing thereby increasing resolution of diffraction and enabling a more accurate side chain electron density map. Indeed, when a K10 Cys401Ala 2B mutant was paired with its native keratin 1 (K1) 2B heterodimer partner its x-ray crystal structure was determined at 2.07 Å resolution; the structure does not contain a disulfide linkage. Superposition of the K1/K10(Cys401Ala) 2B structure onto the wild-type K1/10 2B heterodimer structure had a root-mean-square-deviation of 1.88 Å; the variability in the atomic positions reflects the dynamic motion expected in this filamentous coiled-coil complex. The electrostatic, hydrophobic, and contour features of the molecular surface are similar to the lower resolution wild-type structure. We postulated that elimination of the disulfide linkage in the K1/K10(Cys401Ala) 2B structure could allow for the 2B heterodimers to bind/pack in the A22 tetramer configuration associated with mature keratin intermediate filament assembly. Analysis of the crystal packing revealed a half-staggered anti-parallel tetrameric complex of 2B heterodimers; however, their register is not consistent with models of the A22 mode of tetrameric alignment or prior biochemical cross-linking studies.

ARID1A-SIN3A drives retinoic acid-induced neuroblastoma differentiation by transcriptional repression of TERT.

Aggressive, high-risk neuroblastoma (NB) exhibits an immature differentiation state, profound epigenetic dysregulation and high telomerase activity. It has been suggested that aggressive NB may be treatable by inducing differentiation whereas therapeutic targeting of telomerase is under investigation for multiple cancer types. While epigenetic regulation of the telomerase reverse transcriptase (TERT) promoter has been described in high-risk NB, the exact molecular mechanisms are still not completely understood. Here we used quantitative real-time polymerase chain reaction (PCR), chromatin immunoprecipitation qPCR, quantitative telomeric repeat amplification protocol, and immunoblot techniques to investigate epigenetic regulation of TERT in wild-type and genetically modified NB cell lines. We demonstrated that TERT expression is reduced during 13-cis retinoic acid-induced NB differentiation and that this inversely correlated with increased expression of AT-rich interaction domain 1A (ARID1A), a subunit of the SWItch/sucrose nonfermentable chromatin remodeling complex. We showed that ARID1A directly caused suppression of TERT and was reliant on DNA binding and co-occupancy of the TERT promoter by the SIN3 transcription regulator family member A (SIN3A) repressor complex allowing NB differentiation to proceed. Finally, using data from NB patient cohorts, we reported a significant correlation between low ARID1A expression, elevated expression of TERT, and poorly differentiated, high-risk NB. These results provide insights into a key epigenetic pathway responsible for modulating TERT-driven NB progression, which could represent a target for therapeutic intervention.

PEGylated PAMAM dendrimers loading oxaliplatin with prolonged release and high payload without burst effect.

Oxaliplatin (OXA) was coupled to PEGylated polyamidoamine dendrimers of fourth generation (G4-PEG@OXA) in the comparison to PEGylated ones of odd generation (G3.5-PEG@OXA). Proton nuclear magnetic resonance and Fourier-transform infrared spectroscopy were used to confirm the successful incorporation of OXA as well as the synthesis of carrier systems. Both two types of carrier systems exhibited in sphere nanoparticle shape with size of less than 100 nm that was in the range being able to cause toxicity on cancer cells. The average drug loading efficiency (DLE) of G4-PEG@OXA was obtained at 84.63% that was higher than DLE of G3.5-PEG of 75.69%. The release kinetic of G4-PEG@OXA and G3.5-PEG@OXA did not show any burst release phenomenon while free OXA was released over 40% at the first hour. The sustainable release of OXA was achieved when it was encapsulated in these carriers, but the G4 generation liberated OXA (3.4%-6.4%) slower than G3.5 one (11.9%-22.8%). The in vitro cytotoxicities of G4-PEG@OXA were evaluated in HeLa cell lines using resazurin assay and live/dead staining test. Although the free OXA showed a rather moderate killing ability, the G4-PEG@OXA still displayed the low viability of HeLa that was better to the result of G3.5-PEG@OXA due to released OXA amount. The benefit of this system was to overcome the burst release phenomenon to minimize OXA toxicity without compromising its efficiency.

Complement Protein C1q Enhances Macrophage Foam Cell Survival and Efferocytosis.

In the atherosclerotic lesion, macrophages ingest high levels of damaged modified low-density lipoproteins (LDLs), generating macrophage foam cells. Foam cells undergo apoptosis and, if not efficiently cleared by efferocytosis, can undergo secondary necrosis, leading to plaque instability and rupture. As a component of the innate immune complement cascade, C1q recognizes and opsonizes modified forms of LDL, such as oxidized or acetylated LDL, and promotes ingestion by macrophages in vitro. C1q was shown to be protective in an atherosclerosis model in vivo. Therefore, this study aimed to investigate whether ingestion of modified LDL in the presence of C1q alters macrophage foam cell survival or function. In an unbiased transcriptome analysis, C1q was shown to modulate expression of clusters of genes involved in cell death and apoptosis pathways in human monocyte-derived macrophages ingesting modified LDL; this was validated by quantitative PCR in human and murine macrophages. C1q downregulated levels and activity of active caspase-3 and PARP-1 in human and mouse macrophages during ingestion of modified LDL. This led to a measurable increase in survival and decrease in cell death, as measured by alamarBlue and propidium iodide assays, respectively. C1q opsonization also increased phagocytosis and efferocytosis in macrophage foam cells. These data suggest that C1q promotes macrophage survival during ingestion of excess cholesterol, as well as improves foam cell efferocytic function. This may be important in slowing disease progression and provides insight into the protective role of C1q in early atherosclerosis.

Modified Carboxyl-Terminated PAMAM Dendrimers as Great Cytocompatible Nano-Based Drug Delivery System.

Polyamidoamine (PAMAM) dendrimers are extensively researched as potential drug delivery system thanks to their desirable features such as controlled and stable structures, and ease of functionalization onto their surface active groups. However, there have been concerns about the toxicity of full generation dendrimers and risks of premature clearance from circulation, along with other physical drawbacks presented in previous formulations, including large particle sizes and low drug loading efficiency. In our study, carboxyl-terminated PAMAM dendrimer G3.5 was grafted with poly (ethylene glycol) methyl ether (mPEG) to be employed as a nano-based drug delivery system with great cytocompatibility for the delivery of carboplatin (CPT), a widely prescribed anticancer drug with strong side effects so that the drug will be effectively entrapped and not exhibit uncontrolled outflow from the open structure of unmodified PAMAM G3.5. The particles formed were spherical in shape and had the optimal size range (around 36 nm) that accommodates high drug entrapment efficiency. Surface charge was also determined to be almost neutral and the system was cytocompatible. In vitro release patterns over 24 h showed a prolonged CPT release compared to free drug, which correlated to the cytotoxicity assay on malignant cell lines showing the lack of anticancer effect of CPT/mPEG-G3.5 compared with CPT.

Suppression of the Ubiquitin Pathway by Small Molecule Binding to Ubiquitin Enhances Doxorubicin Sensitivity of the Cancer Cells.

Development of inhibitors for ubiquitin pathway has been suggested as a promising strategy to treat several types of cancers, which has been showcased by recent success of a series of novel anticancer drugs based on inhibition of ubiquitin pathways. Although the druggability of enzymes in ubiquitin pathways has been demonstrated, ubiquitin itself, the main agent of the pathway, has not been targeted. Whereas conventional enzyme inhibitors are used to silence the ubiquitination or reverse it, they cannot disrupt the binding activity of ubiquitin. Herein, we report that the scaffolds of sulfonated aryl diazo compounds, particularly Congo red, could disrupt the binding activity of ubiquitin, resulting in the activity equivalent to inhibition of ubiquitination. NMR mapping assay demonstrated that the chemical directly binds to the recognition site for ubiquitin processing enzymes on the surface of ubiquitin, and thereby blocks the binding of ubiquitin to its cognate receptors. As a proof of concept for the druggability of the ubiquitin molecule, we demonstrated that Congo red acted as an intracellular inhibitor of ubiquitin recognition and binding, which led to inhibition of ubiquitination, and thereby, could be used as a sensitizer for conventional anticancer drugs, doxorubicin.

An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors.

The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the ß-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases.

Discrete element modelling of sediment falling in water.

The Discrete Element Method (DEM) is a discrete, particle-based method commonly used in studies involving granular media, e.g. sediment transport, and geomechanics. It is heavily dependent on particle properties, and one important component is the force model, which relates the relative positions and velocities of the simulated particles to the forces they experience. In this paper we model a collection of lightly compacted granular material, released at a short distance above a flat base in a quiescent fluid --similar to the process whereby sediment tailings are released back into the sea during nodule harvesting. We employ different typical force models, and consider how their varying components affect the simulated outcome. The results are compared with a physical experiment of similar dimensions. We find that a realistic simulation is achieved when the force model considers the local solid fraction in the drag force, and incorporates the hydrodynamic effect of neighbouring particles. The added mass effect increases the accuracy of the outcome, but does not contribute significantly in a qualitative sense.

Knowledge and fears among Asian Americans chronically infected with hepatitis B.

Asian Americans are disproportionately affected by hepatitis B (HBV) infection. In the USA, one in ten Asian Americans is infected with HBV. The purpose of the study was to explore knowledge, fears and follow-up care among Asian Americans chronically infected with hepatitis B. A sample of 154 completed an online survey which included demographic information, follow-up treatment information, and HBV knowledge and fears/concerns. Our findings indicate that there are differences by ethnicity and birthplace among Asian Americans in terms of follow-up care and treatment. In addition, our study indicates that there were substantial knowledge gaps regarding contracting HBV, transmission, and prevention. We found that HBV-infected respondents had many fears about their infection. Three primary concerns were identified: (a) concerns related to liver cancer and overall health, (b) transmission to loved ones, and (c) stigma. Our findings indicate that Asian Americans chronically infected with HBV need more education to manage HBV including information on the importance of follow-up treatment and care and education on preventing transmission, risk for liver cancer, and HBV impact on overall health.