Molecular determinants and signaling effects of PKA RIα phase separation.

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

Acute Necrotizing Myelitis Associated with COVID-19.

Acute ascending hemorrhagic longitudinally extensive transverse myelitis is a rare inflammatory demyelinating disorder, which invades several vertebral segments and progresses rapidly and manifests severe symptoms. We present a case of acute necrotizing myelitis associated with COVID-19 infection. A 10-year-old female, with no previous medical history and no prior administration of COVID-19 vaccination, contracted COVID-19 in early April 2022. Two weeks later, she suffered from severe posterior neck pain and also presented with motor weakness and numbness in both lower extremities, making it difficult to walk independently and spontaneously void urine. Initial spinal cord MR showed longitudinally segmental extensive T2 hyperintensities. Cerebrospinal fluid (CSF) analysis revealed elevated red blood cell, normal white blood cell, and elevated protein levels and absence of oligoclonal bands. CSF culture and viral polymerase chain reaction were negative. Autoimmune work-up was negative. She was started on intravenous methylprednisolone 1g/day for 5 days and immunoglobulin (Ig) 2 g/kg for 5 days. She was also treated with six courses of therapeutic plasma exchange. Nevertheless, her pain and motor weakness persisted. She eventually developed respiratory failure. Follow-up MR presented a newly noted small hemorrhagic component. She was consequently treated with two additional courses of methylprednisolone and Ig. At 6-months follow-up, neurological examination showed improvement with normal sensory function and motor grade IV function in both upper extremities. We present the case of acute necrotizing myelitis associated with COVID-19 infection. Multiple courses of methylprednisolone and Ig showed mild improvement in motor and sensory function. However, poor prognosis was unavoidable due to rapid progression of the disease.

Clinical features of chronic fungal rhinosinusitis in Korean geriatric and non-geriatric patients.

PURPOSE: Worldwide, the incidence of chronic fungal rhinosinusitis (CFRS) has increased. Although ageing leads to weakening of the immune system, which increases susceptibility to CFRS, the CFRS characteristics in geriatric patients are unclear. Therefore, we comparatively analysed the clinical characteristics of CFRS in geriatric and non-geriatric patients. METHODS: This retrospective analysis compared the demographics, rhinologic symptoms, multiple allergen simultaneous tests, olfactory function tests, paranasal sinus computed tomography findings, and outcomes of 131 patients with CFRS who underwent functional endoscopic sinus surgery and 131 enrolled patients were divided in geriatric (> 65 years) and non-geriatric (≤ 65 years) groups. RESULTS: Among the geriatric and non-geriatric participants (n = 65, 49.6% and n = 66, 50.4%, respectively), hypertension and diabetes mellitus were more common in the geriatric group. Demographics, including symptoms, showed no significant intergroup differences. Normosmia and hyposmia were significantly less prevalent, whereas phantosmia and parosmia were more prevalent in the geriatric group than in the non-geriatric group (p = 0.03 and p = 0.01, respectively). Sphenoidal sinus involvement was significantly higher in geriatric patients than in non-geriatric patients (p = 0.02). CONCLUSION: Based on greater sphenoidal sinus involvement, a deeper anatomical area is more vulnerable to fungal infection in the geriatric group than in the non-geriatric group. Increasing clinicians' awareness of CFRS in geriatric patients with olfactory dysfunction, including phantosmia and parosmia, is important for early intervention.

Molecular Determinants and Signaling Effects of PKA RIα Phase Separation.

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures the specific execution of various cellular functions. Liquid-liquid phase separation (LLPS) of the ubiquitously expressed PKA regulatory subunit RIα was recently identified as a major driver of cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces combined with the cAMP-induced release of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence are required to drive RIα condensate formation in cytosol, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in the formation of a non-canonical R:C complex, which serves to maintain low basal PKA activity in the cytosol by enabling the recruitment of active PKA-C to RIα condensates. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

Non-canonical ß-adrenergic activation of ERK at endosomes.

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized ß2-adrenergic receptor (ß2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that ß2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated ß2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal ß2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.

Quantification and visualization of metastatic lung tumors in mice.

Histopathological examination is important for the diagnosis of various diseases. Conventional histopathology provides a two-dimensional view of the tissues, and requires the tissue to be extracted, fixed, and processed using histotechnology techniques. However, there is an increasing need for three-dimensional (3D) images of structures in biomedical research. The objective of this study was to develop reliable, objective tools for visualizing and quantifying metastatic tumors in mouse lung using micro-computed tomography (micro-CT), optical coherence tomography (OCT), and field emission-scanning electron microscopy (FE-SEM). Melanoma cells were intravenously injected into the tail vein of 8-week-old C57BL/6 mice. The mice were euthanized at 2 or 4 weeks after injection. Lungs were fixed and examined by micro-CT, OCT, FE-SEM, and histopathological observation. Micro-CT clearly distinguished between tumor and normal cells in surface and deep lesions, thereby allowing 3D quantification of the tumor volume. OCT showed a clear difference between the tumor and surrounding normal tissues. FE-SEM clearly showed round tumor cells, mainly located in the alveolar wall and growing inside the alveoli. Therefore, whole-tumor 3D imaging successfully visualized the metastatic tumor and quantified its volume. This promising approach will allow for fast and label-free 3D phenotyping of diverse tissue structures.

Nasopharyngeal Tuberculosis with Concomitant Middle Ear Tuberculosis: A Case Report and Literature Review.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (MTB), can affect the lungs (pulmonary TB) as well as other sites (extrapulmonary TB). Nasopharyngeal tuberculosis (NPTB) is a rare type of extrapulmonary TB. Since NPTB has nonspecific clinical presentation with low index of suspicion, it is difficult for clinicians to make an early diagnosis and proper treatment. We recently encountered a 42-year-old woman with NPTB concomitant with middle ear TB, which strongly mimicked nasopharyngeal carcinoma. Since the diagnosis of NPTB was difficult to confirm based on endoscopic findings and imaging studies, this patient underwent nasopharyngeal biopsy, and finally, polymerase chain reaction (PCR) confirmed NPTB. This report describes our NPTB case as well as summarizes all cases of NPTB reported in South Korea. We highlight that active tissue biopsy with antibacterial smear and additional PCR or specific TB blood test should be considered for cases with high suspicion of NPTB.

Classification of Mouse Lung Metastatic Tumor with Deep Learning.

Traditionally, pathologists microscopically examine tissue sections to detect pathological lesions; the many slides that must be evaluated impose severe work burdens. Also, diagnostic accuracy varies by pathologist training and experience; better diagnostic tools are required. Given the rapid development of computer vision, automated deep learning is now used to classify microscopic images, including medical images. Here, we used a Inception-v3 deep learning model to detect mouse lung metastatic tumors via whole slide imaging (WSI); we cropped the images to 151 by 151 pixels. The images were divided into training (53.8%) and test (46.2%) sets (21,017 and 18,016 images, respectively). When images from lung tissue containing tumor tissues were evaluated, the model accuracy was 98.76%. When images from normal lung tissue were evaluated, the model accuracy ("no tumor") was 99.87%. Thus, the deep learning model distinguished metastatic lesions from normal lung tissue. Our approach will allow the rapid and accurate analysis of various tissues.

Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell-based cartilage tissue engineering.

Despite great progress in biomaterial design strategies for replacing damaged articular cartilage, prevention of stem cell-derived chondrocyte hypertrophy and resulting inferior tissue formation is still a critical challenge. Here, by using engineered biomaterials and a high-throughput system for screening of combinatorial cues in cartilage microenvironments, we demonstrate that biomaterial cross-linking density that regulates matrix degradation and stiffness-together with defined presentation of growth factors, mechanical stimulation, and arginine-glycine-aspartic acid (RGD) peptides-can guide human mesenchymal stem cell (hMSC) differentiation into articular or hypertrophic cartilage phenotypes. Faster-degrading, soft matrices promoted articular cartilage tissue formation of hMSCs by inducing their proliferation and maturation, while slower-degrading, stiff matrices promoted cells to differentiate into hypertrophic chondrocytes through Yes-associated protein (YAP)-dependent mechanotransduction. in vitro and in vivo chondrogenesis studies also suggest that down-regulation of the Wingless and INT-1 (WNT) signaling pathway is required for better quality articular cartilage-like tissue production.

Splicing Busts a Move: Isoform Switching Regulates Migration.

Cell migration is essential for normal development, neural patterning, pathogen eradication, and cancer metastasis. Pre-mRNA processing events such as alternative splicing and alternative polyadenylation result in greater transcript and protein diversity as well as function and activity. A critical role for alternative pre-mRNA processing in cell migration has emerged in axon outgrowth during neuronal development, immune cell migration, and cancer metastasis. These findings suggest that migratory signals result in expression changes of post-translational modifications of splicing or polyadenylation factors, leading to splicing events that generate promigratory isoforms. We summarize this recent progress and suggest emerging technologies that may facilitate a deeper understanding of the role of alternative splicing and polyadenylation in cell migration.

Computed tomography-guided navigation assisted drainage for inaccessible deep neck abscess: A case report.

RATIONALE: Deep neck infections (DNIs) in the head and neck area are difficult to treat due to the anatomical complexity of the cervical region. Since inflammation causes changes in anatomy, it is often difficult to find the exact location of the abscess, which leads to failed surgical drainage. PATIENT CONCERN: A 76-year-old female patient was referred to our clinic with trismus and right-side facial swelling. After extraction of her lower third molar 2 weeks ago, due to chronic periodontitis, her trismus had aggravated and her maximal mouth opening was 20 mm. DIAGNOSES: Computed tomography (CT) revealed an approximately 2.5 cm-sized abscess pocket with cellulitis in the right pterygomandibular space. INTERVENTIONS: Since the first surgical drainage attempt using the intraoral approach under general anesthesia had failed and conservative antibiotic treatment was also ineffective, a second surgical procedure with a CT-guided navigation system was performed and the pus was successfully evacuated. OUTCOMES: After drainage with CT-guided navigation, the clinical symptoms and septic conditions of the patient showed remarkable improvement, and there was no recurrence of infection within a year after the procedure. LESSONS: Drainage with CT-guided navigation can be used as a successful surgical tool to aid in the surgery of patients with DNI when it is difficult to accurately target the abscess due to inflammation.

RECK isoforms differentially regulate fibroblast migration by modulating tubulin post-translational modifications.

Cell migration is essential for proper development and the defense against pathogens. Our previous work detailed a pathway of REversion-inducing-Cysteine-rich protein with Kazal motifs (RECK) isoform-mediated invasion in which a shorter RECK protein competes with MMP9 for interaction with the canonical RECK protein on the cell surface. Here we demonstrate that the mechanism through which RECK isoforms affect cell migration is mediated through changes in the levels of post-translational modifications (PTM) of α-tubulin. We show that both the canonical and short RECK isoforms modulate levels of tubulin acetylation and detyrosination. We demonstrate that these changes are sufficient to modulate the rate of fibroblast migration. If these tubulin PTMs are not altered, the effects of the canonical RECK isoform on cell migration are reversed. In defining the molecular pathway linking RECK and tubulin PTMs, we found that MMP9 and integrin activity both act as upstream regulators of tubulin acetylation and detyrosination. Overall, we propose a mechanism in which RECK isoforms on the cell surface have opposing effects on cell migration through MMP9-modulated changes to integrin-extracellular matrix (ECM) interactions that, in turn, affect microtubule PTMs.

RECK isoforms have opposing effects on cell migration.

Cell migration is a highly conserved process involving cytoskeletal reorganization and restructuring of the surrounding extracellular matrix. Although there are many studies describing mechanisms underlying cell motility, little has been reported about the contribution of alternative isoform use toward cell migration. Here, we investigated whether alternative isoform use can affect cell migration focusing on reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), an established inhibitor of cell migration. We found that a shorter isoform of RECK is more highly expressed in proliferating fibroblasts, in TGF-ß-treated fibroblasts, and in tumors compared with differentiated tissue. Knockdown of this short RECK isoform reduces fibroblast migration through Matrigel. Thus, this short isoform of RECK generated by a combination of alternative splicing and alternative polyadenylation plays an opposing role to the canonical RECK isoform, as knockdown of canonical RECK results in faster cell migration through Matrigel. We show that the short RECK protein competes with matrix metalloprotease 9 (MMP9) for binding to the Kazal motifs of canonical RECK, thus liberating MMP9 from an inactivating interaction with canonical RECK. Our studies provide a new paradigm and a detailed mechanism for how alternative isoform use can regulate cell migration by producing two proteins with opposing effects from the same genetic locus.

Pyoderma Gangrenosum of the Preauricular Area with Ulcerative Colitis: A Case Report and Review.

Pyoderma gangrenosum (PG) is primarily, a sterile, inflammatory, neutrophilic dermatosis, characterized by recurrent cutaneous ulceration with mucopurulent or hemorrhagic exudate. The incidence of PG is uncertain, but it is estimated to be about 3-10 patients per million per year. It occurs most commonly on the lower legs, but has been reported at other sites of the body as well. The causes of PG are unknown, but about 50-70% of cases are associated with other diseases, mainly inflammatory bowel disease. We hereby report a case of PG in a 21-year-old male, with a history of ulcerative colitis (UC). After appropriate diagnostic methods including biopsy for pathologic confirmation, sigmoidoscopy and computed tomography, we excluded other diseases and the lesion was diagnosed as PG. We then carried out regular dressing of the wound, while UC was treated with steroid and immunosuppressant medication, with inputs from the department of gastroenterology during the hospital stay. There occurred recurrence of the skin lesion, 7 months after discharge, after which they improved. UC has been in the remission state as per the follow-up, since 2 years.

Juvenile Myasthenia Gravis in Korea: Subgroup Analysis According to Sex and Onset Age.

Juvenile myasthenia gravis presents before 18 years of age with different characteristics according to racial background and pubertal development. The authors aimed to determine the clinical characteristics of children and adolescents of Korean ethnicity with myasthenia gravis, and evaluate the presentation and clinical outcomes according to the sex and onset age of the patients. The authors recruited 88 Korean juvenile myasthenia gravis patients between September 2005 and August 2015. Worse clinical severity from presentation, more aggressive treatment strategies, and worse final treatment outcomes were noted in girls with postpubertal onset than in the other patients. The symptoms were milder (pure ocular presentation in 96.6% [85/88]) and the disease course was more benign (94.3% [83/88]) in this study than in the literature. The homogenous racial background might have contributed to these results. These findings highlight the influence of pubertal development and the need for timely and appropriate active treatment, including thymectomy, to improve prognosis.

Effects of early adolescent environmental enrichment on cognitive dysfunction, prefrontal cortex development, and inflammatory cytokines after early life stress.

Early postnatal stress such as maternal separation causes cognitive dysfunction later in life, including working memory deficits that are largely mediated by the prefrontal cortex. Maternal separation in male rats also yields a loss of parvalbumin-containing prefrontal cortex interneurons in adolescence, which may occur via inflammatory or oxidative stress mechanisms. Environmental enrichment can prevent several effects of maternal separation; however, effects of enrichment on prefrontal cortex development are not well understood. Here, we report that enrichment prevented cognitive dysfunction in maternally separated males and females, and prevented elevated circulating pro-inflammatory cytokines that was evident in maternally separated males, but not females. However, enrichment did not prevent parvalbumin loss or adolescent measures of oxidative stress. Significant correlations indicated that adolescents with higher oxidative damage and less prefrontal cortex parvalbumin in adolescence committed more errors on the win-shift task; therefore, maternal separation may affect cognitive dysfunction via aberrant interneuron development. © 2015 Wiley Periodicals, Inc. Dev Psychobiol 58: 482-491, 2016.

Intractable pneumococcal meningoencephalitis associated with a TNF-α antagonist.

A 34-year-old man was treated with a TNF-α antagonist for ankylosing spondylitis, and this subsequently developed a CNS infection. Magnetic resonance imaging showed diffuse subcortical white matter lesions. Streptococcus pneumoniae was cultured from the cerebrospinal fluid and blood. The patient died of multifocal widespread brain damage and subarachnoid hemorrhage, despite intensive antibacterial medication. Pneumococcal meningoencephalitis can occur in association with TNF-α antagonists. Clinicians should be aware of both the risk of fatal bacterial meningoencephalitis associated with TNF-α antagonists and the possibility of an unusual presentation of bacterial meningitis.

A mechanistic study on the anti-cancer activity of ethyl caffeate in human ovarian cancer SKOV-3 cells.

In the present study, we investigated the effect and molecular mechanism of ethyl caffeate (EC), a natural phenolic compound isolated from Ligularia fischeri, on human ovarian cancer cell proliferation and progression. EC-mediated inhibition of cell proliferation in SKOV-3 cells was accompanied by reduced expression of cell cycle-related proteins such as cyclin-dependent kinases and cyclins, resulting in pRb hypophosphorylation and G1 phase cell cycle arrest. Moreover, EC treatment markedly inhibited cell migration and invasion. These regulatory effects of EC on ovarian cancer cell proliferation, migration and invasion were associated with inactivation of mitogenic signaling pathways such as Akt, ERK and p38(MAPK), and down-regulation of cell surface signaling molecules including receptor tyrosine kinases, integrin α3ß1 and N-cadherin. Taken together, these findings suggest further evaluation and development of EC for the treatment and prevention of ovarian cancer.

Regulatory effects of Siegesbeckia glabrescens on non-small cell lung cancer cell proliferation and invasion.

Siegesbeckia glabrescens (SG) Makino (Compositae) has been used as a traditional medicine for the treatment of allergic and inflammatory diseases. In the present study, we examined the effects and molecular mechanism of the ethanol extract of SG on cell proliferation and invasion in p53 wild-type A549 and p53-deficient H1299 non-small cell lung cancer (NSCLC) cells. SG treatment markedly inhibited the proliferation and invasion in both cell lines, independently of p53 expression. The anti-proliferative effect of SG on A549 cells was mediated by the inactivation of Akt and p70(S6K) as evidenced by treatment with LY294002 and rapamycin, respectively. In addition, anti-invasive activity of SG in A549 cells was found to be associated with the inhibition of p70(S6K). In contrast, in H1299 cells the inactivation of p38(MAPK) appeared to be involved in SG-mediated inhibition of cell proliferation and invasion. Collectively, these findings suggest that SG modulates cellular fates such as proliferation and invasion by differential regulation of signaling pathways, depending on the status of p53 expression in NSCLC, and support the development of SG as a potent therapeutic agent for the treatment of NSCLC.

Interleukin-8 reduces post-surgical lymphedema formation by promoting lymphatic vessel regeneration.

Lymphedema is mainly caused by lymphatic obstruction and manifested as tissue swelling, often in the arms and legs. Lymphedema is one of the most common post-surgical complications in breast cancer patients and presents a painful and disfiguring chronic illness that has few treatment options. Here, we evaluated the therapeutic potential of interleukin (IL)-8 in lymphatic regeneration independent of its pro-inflammatory activity. We found that IL-8 promoted proliferation, tube formation, and migration of lymphatic endothelial cells (LECs) without activating the VEGF signaling. Additionally, IL-8 suppressed the major cell cycle inhibitor CDKN1C/p57(KIP2) by downregulating its positive regulator PROX1, which is known as the master regulator of LEC-differentiation. Animal-based studies such as matrigel plug and cornea micropocket assays demonstrated potent efficacy of IL-8 in activating lymphangiogenesis in vivo. Moreover, we have generated a novel transgenic mouse model (K14-hIL8) that expresses human IL-8 in the skin and then crossed with lymphatic-specific fluorescent (Prox1-GFP) mouse. The resulting double transgenic mice showed that a stable expression of IL-8 could promote embryonic lymphangiogenesis. Moreover, an immunodeficient IL-8-expressing mouse line that was established by crossing K14-hIL8 mice with athymic nude mice displayed an enhanced tumor-associated lymphangiogenesis. Finally, when experimental lymphedema was introduced, K14-hIL8 mice showed an improved amelioration of lymphedema with an increased lymphatic regeneration. Together, we report that IL-8 can activate lymphangiogenesis in vitro and in vivo with a therapeutic efficacy in post-surgical lymphedema.