Keratocystoma: A Distinctive Salivary Gland Neoplasm Characterized by RUNX2 Rearrangements.

Keratocystoma is a rare salivary gland lesion that has been reported primarily in children and young adults. Because of a scarcity of reported cases, very little is known about it, including its molecular underpinnings, biological potential, and histologic spectrum. Purported to be a benign neoplasm, keratocystoma bears a striking histologic resemblance to benign lesions like metaplastic Warthin tumor on one end of the spectrum and squamous cell carcinoma on the other end. This overlap can cause diagnostic confusion, and it raises questions about the boundaries and definition of keratocystoma as an entity. This study seeks to utilize molecular tools to evaluate the pathogenesis of keratocystoma as well as its relationship with its histologic mimics. On the basis of targeted RNA sequencing (RNA-seq) results on a sentinel case, RUNX2 break-apart fluorescence in situ hybridization (FISH) was successfully performed on 4 cases diagnosed as keratocystoma, as well as 13 cases originally diagnosed as tumors that morphologically resemble keratocystoma: 6 primary squamous cell carcinomas, 3 metaplastic/dysplastic Warthin tumors, 2 atypical squamous cysts, 1 proliferating trichilemmal tumor, and 1 cystadenoma. RNA-seq and/or reverse transcriptase-PCR were attempted on all FISH-positive cases. Seven cases were positive for RUNX2 rearrangement, including 3 of 4 tumors originally called keratocystoma, 2 of 2 called atypical squamous cyst, 1 of 1 called proliferating trichilemmal tumor, and 1 of 6 called squamous cell carcinoma. RNA-seq and/or reverse transcriptase-PCR identified IRF2BP2::RUNX2 in 6 of 7 cases; for the remaining case, the partner remains unknown. The cases positive for RUNX2 rearrangement arose in the parotid glands of 4 females and 3 males, ranging from 8 to 63 years old (mean, 25.4 years; median, 15 years). The RUNX2 -rearranged cases had a consistent histologic appearance: variably sized cysts lined by keratinizing squamous epithelium, plus scattered irregular squamous nests, with essentially no cellular atypia or mitotic activity. The background was fibrotic, often with patchy chronic inflammation and/or giant cell reaction. One case originally called squamous cell carcinoma was virtually identical to the other cases, except for a single focus of small nerve invasion. The FISH-negative case that was originally called keratocystoma had focal cuboidal and mucinous epithelium, which was not found in any FISH-positive cases. The tumors with RUNX2 rearrangement were all treated with surgery only, and for the 5 patients with follow-up, there were no recurrences or metastases (1 to 120 months), even for the case with perineural invasion. Our findings solidify that keratocystoma is a cystic neoplastic entity, one which appears to consistently harbor RUNX2 rearrangements, particularly IRF2BP2::RUNX2 . Having a diagnostic genetic marker now allows for a complete understanding of this rare tumor. They arise in the parotid gland and affect a wide age range. Keratocystoma has a consistent morphologic appearance, which includes large squamous-lined cysts that mimic benign processes like metaplastic Warthin tumor and also small, irregular nests that mimic squamous cell carcinoma. Indeed, RUNX2 analysis has considerable promise for resolving these differential diagnoses. Given that one RUNX2 -rearranged tumor had focal perineural invasion, it is unclear whether that finding is within the spectrum of keratocystoma or whether it could represent malignant transformation. Most important, all RUNX2 -rearranged cases behaved in a benign manner.

In situ production and secretion of proteins endow therapeutic benefit against psoriasiform dermatitis and melanoma.

Genetic medicines have the potential to treat various diseases; however, certain ailments including inflammatory diseases and cancer would benefit from control over extracellular localization of therapeutic proteins. A critical gap therefore remains the need to develop and incorporate methodologies that allow for posttranslational control over expression dynamics, localization, and stability of nucleic acid-generated protein therapeutics. To address this, we explored how the body's endogenous machinery controls protein localization through signal peptides (SPs), including how these motifs could be incorporated modularly into therapeutics. SPs serve as a virtual zip code for mRNA transcripts that direct the cell where to send completed proteins within the cell and the body. Utilizing this signaling biology, we incorporated secretory SP sequences upstream of mRNA transcripts coding for reporter, natural, and therapeutic proteins to induce secretion of the proteins into systemic circulation. SP sequences generated secretion of various engineered proteins into the bloodstream following intravenous, intramuscular, and subcutaneous SP mRNA delivery by lipid, polymer, and ionizable phospholipid delivery carriers. SP-engineered etanercept/TNF-α inhibitor proteins demonstrated therapeutic efficacy in an imiquimod-induced psoriasis model by reducing hyperkeratosis and inflammation. An SP-engineered anti-PD-L1 construct mediated mRNA encoded proteins with longer serum half-lives that reduced tumor burden and extended survival in MC38 and B16F10 cancer models. The modular nature of SP platform should enable intracellular and extracellular localization control of various functional proteins for diverse therapeutic applications.

GLUT3 promotes macrophage signaling and function via RAS-mediated endocytosis in atopic dermatitis and wound healing.

The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

Precision Medicine: Disease Subtyping and Tailored Treatment.

The genomics-based concept of precision medicine began to emerge following the completion of the Human Genome Project. In contrast to evidence-based medicine, precision medicine will allow doctors and scientists to tailor the treatment of different subpopulations of patients who differ in their susceptibility to specific diseases or responsiveness to specific therapies. The current precision medicine model was proposed to precisely classify patients into subgroups sharing a common biological basis of diseases for more effective tailored treatment to achieve improved outcomes. Precision medicine has become a term that symbolizes the new age of medicine. In this review, we examine the history, development, and future perspective of precision medicine. We also discuss the concepts, principles, tools, and applications of precision medicine and related fields. In our view, for precision medicine to work, two essential objectives need to be achieved. First, diseases need to be classified into various subtypes. Second, targeted therapies must be available for each specific disease subtype. Therefore, we focused this review on the progress in meeting these two objectives.

A plague passing over: Clinical features of the 2022 mpox outbreak in patients of color living with HIV.

INTRODUCTION: Compared with previous geographically localized outbreaks of monkeypox (MPOX), the scale of the 2022 global mpox outbreak has been unprecedented, yet the clinical features of this outbreak remain incompletely characterized. METHODS: We identified patients diagnosed with mpox by polymerase chain reaction (PCR; n = 36) from July to September 2022 at a single, tertiary care institution in the USA. Demographics, clinical presentation, infection course, and histopathologic features were reviewed. RESULTS AND CONCLUSION: Men who have sex with men (89%) and people living with HIV (97%) were disproportionately affected. While fever and chills (56%) were common, some patients (23%) denied any prodromal symptoms. Skin lesions showed a wide range of morphologies, including papules and pustules, and lesions showed localized, not generalized, spread. Erythema was also less appreciable in skin of colour patients (74%). Atypical clinical features and intercurrent skin diseases masked the clinical recognition of several cases, which were ultimately diagnosed by PCR. Biopsies showed viral cytopathic changes consistent with Orthopoxvirus infections. All patients in this case series recovered without complications, although six patients (17%) with severe symptoms were treated with tecovirimat without complication.

Red blood cells as glucose carriers to the human brain: Modulation of cerebral activity by erythrocyte exchange transfusion in Glut1 deficiency (G1D).

Red blood cells circulating through the brain are briefly but closely apposed to the capillary endothelium. We hypothesized that this contact provides a nearly direct pathway for metabolic substrate transfer to neural cells that complements the better characterized plasma to endothelium transfer. While brain function is considered independent of normal fluctuations in blood glucose concentration, this is not borne out by persons with glucose transporter I (GLUT1) deficiency (G1D). In them, encephalopathy is often ameliorated by meal or carbohydrate administration, and this enabled us to test our hypothesis: Since red blood cells contain glucose, and since the red cells of G1D individuals are also deficient in GLUT1, replacing them with normal donor cells via exchange transfusion could augment erythrocyte to neural cell glucose transport via mass action in the setting of unaltered erythrocyte count or plasma glucose abundance. This motivated us to perform red blood cell exchange in 3 G1D persons. There were rapid, favorable and unprecedented changes in cognitive, electroencephalographic and quality-of-life measures. The hypothesized transfer mechanism was further substantiated by in vitro measurement of direct erythrocyte to endothelial cell glucose flux. The results also indicate that the adult intellect is capable of significant enhancement without deliberate practice. ClinicalTrials.gov registration: NCT04137692 https://clinicaltrials.gov/ct2/show/NCT04137692.

Synchronization of Cultured Cells to G1, S, G2, and M Phases by Double Thymidine Block.

The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase. For many experiments, it is desirable to have a population of cells that are traversing the cell cycle synchronously, as it allows population-wide data to be collected rather than relying solely on single-cell experiments. While there are various drugs that can be used to arrest the cell at each specific phase of the cell cycle, they may cause undesired side effects. Here, we describe a protocol to synchronize cells to each cell cycle phase by using only one chemical: thymidine. Non-synchronized cells are synchronized to early S phase by a double thymidine block. The release of the double thymidine block allows the cells to progress through the cell cycle in a synchronized pace. By collecting the cells at various time intervals following the release of double thymidine block, we are able to harvest cells synchronized to the G2, M, and G1 phases. This synchronization can be assessed by various methods, including flow cytometry to examine the DNA content, Western blotting to examine the expression of various cell phase-specific markers, and microscopy to examine the morphology of the chromosome.

Synchronization of HeLa Cells to Various Interphases Including G1, S, and G2 Phases.

The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase, which allows the study of phase-specific cellular events. While interphase cells can be easily distinguished from mitotic cells by examining their chromosome morphology, it is much more difficult to separate and distinguish the interphases from each other. Here, we describe drug-derived protocols for synchronizing HeLa cells to various interphases of the cell cycle: G1 phase, S phase, and G2 phase. G1 phase synchronization is achieved through serum starvation, S phase synchronization is achieved through a double thymidine block, and G2 phase synchronization is achieved through the release of the double thymidine block followed by roscovitine treatment. Successful synchronization can be assessed using flow cytometry to examine the DNA content and Western blot to examine the expression of various cyclins.

Synchronization of HeLa Cells to Mitotic Subphases.

The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.

Beth Levine's Legacy: From the Discovery of BECN1 to Therapies. A Mentees' Perspective.

With great sadness, the scientific community received the news of the loss of Beth Levine on 15 June 2020. Dr. Levine was a pioneer in the autophagy field and work in her lab led not only to a better understanding of the molecular mechanisms regulating the pathway, but also its implications in multiple physiological and pathological conditions, including its role in development, host defense, tumorigenesis, aging or metabolism. This review does not aim to provide a comprehensive view of autophagy, but rather an outline of some of the discoveries made by the group of Beth Levine, from the perspective of some of her own mentees, hoping to honor her legacy in science.

Human Papillomavirus‒Positive and ‒Negative Vulvar Squamous Cell Carcinoma Are Biologically but Not Clinically Distinct.

Vulvar squamous cell carcinoma pathogenesis is traditionally defined by the presence or absence of human papillomavirus (HPV), but the definition of these groups and their molecular characteristics remain ambiguous across studies. In this study, we present a retrospective cohort analysis of 36 patients with invasive vulvar squamous cell carcinoma where HPV status was determined using RNA in situ hybridization and PCR. Clinical annotation, p16 immunohistochemistry, PD-L1 immunohistochemistry, HPV16 circular E7 RNA detection, and RNA sequencing of the cases were performed. A combination of in situ hybridization and PCR identified 20 cases (55.6%) as HPV positive. HPV status did not impact overall survival (hazard ratio: 1.36, 95% confidence interval = 0.307-6.037, P = 0.6857) or progression-free survival (hazard ratio: 1.12, 95% confidence interval = 0.388-3.22, P = 0.8367), and no significant clinical differences were found between the groups. PD-L1 expression did not correlate with HPV status, but increased expression of PD-L1 correlated with worse overall survival. Transcriptomic analyses (n = 23) revealed distinct groups, defined by HPV status, with multiple differentially expressed genes previously implicated in HPV-induced cancers. HPV-positive tumors showed higher global expression of endogenous circular RNAs, including several circular RNAs that have previously been implicated in the pathogenesis of other cancers.

Research Techniques Made Simple: Studying Circular RNA in Skin Diseases.

Circular RNAs (circRNAs) are a unique class of covalently closed, single-stranded RNAs. High-throughput sequencing has uncovered the abundance and complexity of circRNAs. Changes in levels of circRNAs correlate with diverse disease states, including many skin diseases. CircRNAs can function as microRNA inhibitors, protein interactors, or mRNAs. Although circRNAs do have unique topological features, they share many similarities, including primary sequence, with their linear orthologs, so carefully controlled experiments are required to detect and study them. Here, we summarize some protocols used in the identification, validation, and characterization of circRNAs. We also discuss ways to repress and overexpress specific circRNAs to assess potential unique functions for these molecules. These techniques may be useful in exploring how circRNAs contribute to skin disease.

Biallelic variants in RNU12 cause CDAGS syndrome.

CDAGS Syndrome is a rare congenital disorder characterized by Craniosynostosis, Delayed closure of the fontanelles, cranial defects, clavicular hypoplasia, Anal and Genitourinary malformations, and Skin manifestations. We performed whole exome and Sanger sequencing to identify the underlying molecular cause in five patients with CDAGS syndrome from four distinct families. Whole exome sequencing revealed biallelic rare variants that disrupt highly conserved nucleotides within the RNU12 gene. RNU12 encodes a small nuclear RNA that is a component of the minor spliceosome and is essential for minor intron splicing. Targeted sequencing confirmed allele segregation within the four families. All five patients shared the same rare mutation NC_000022.10:g.43011402C>T, which alters a highly conserved nucleotide within the precursor U12 snRNA 3' extension. Each of them also carried a rare variant on the other allele that either disrupts the secondary structure or the Sm binding site of the RNU12 snRNA. Whole transcriptome sequencing analysis of lymphoblastoid cells identified 120 differentially expressed genes, and differential alternative splicing analysis indicated there was an enrichment of alternative splicing events in the patient. These findings provide evidence of the involvement of RNU12 in craniosynostosis, anal and genitourinary patterning, and cutaneous disease.

Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus.

Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative cells. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and infected tissues and express proteins that have the potential to modulate the infectious and tumorigenic properties of these viruses.