SARS-CoV-2 strains bearing Omicron BA.1 spike replicate in C57BL/6 mice.

Introduction: SARS-CoV-2, the cause of the COVID pandemic, is an RNA virus with a high propensity to mutate. Successive virus variants, including variants of concern (VOC), have emerged with increased transmission or immune escape. The original pandemic virus and early variants replicated poorly, if at all, in mice at least partly due to a mismatch between the receptor binding domain on the viral spike protein and the murine angiotensin converting enzyme 2 (ACE2). Omicron VOC emerged in late 2021 harboring > 50 new mutations, 35 of them in the spike protein. This variant resulted in a very large wave of infections, even in the face of prior immunity, albeit being inherently less severe than earlier variants. Reflecting the lower severity reported in humans, Omicron displayed attenuated infection in hamsters and also in the K18-hACE2 mouse model. K18-hACE2 mice express both the human ACE2 as well as the endogenous mouse ACE2. Methods: Here we infected hACE2 knock-in mice that express only human ACE2 and no murine ACE2, or C57BL/6 wildtype mice with SARS-CoV-2 D614G (first-wave isolate), Delta or Omicron BA.1 variants and assessed infectivity and downstream innate immune responses. Results: While replication of SARS-CoV-2 Omicron was lower in the lungs of hACE2 knock-in mice compared with SARS-CoV-2 D614G and VOC Delta, it replicated more efficiently than the earlier variants in C57BL/6 wildtype mice. This opens the opportunity to test the effect of host genetics on SARS-CoV-2 infections in wildtype mice. As a proof of principle, we tested Omicron infection in mice lacking expression of the interferon-alpha receptor-1 (IFNAR1). In these mice we found that loss of type I IFN receptor signaling resulted in higher viral loads in the lungs were detected. Finally, using a chimeric virus of first wave SARS-CoV-2 harboring the Omicron spike protein, we show that Omicron spike increase infection of C57BL/6 wildtype mice, but non-spike genes of Omicron confer attenuation of viral replication. Discussion: Since this chimeric virus efficiently infected C57BL/6 wildtype mice, and replicated in their lungs, our findings illustrate a pathway for genetic mapping of virushost interactions during SARS-CoV-2 infection.

TASL mediates keratinocyte differentiation by regulating intracellular calcium levels and lysosomal function.

Maintaining epidermal homeostasis relies on a tightly organized process of proliferation and differentiation of keratinocytes. While past studies have primarily focused on calcium regulation in keratinocyte differentiation, recent research has shed light on the crucial role of lysosome dysfunction in this process. TLR adaptor interacting with SLC15A4 on the lysosome (TASL) plays a role in regulating pH within the endo-lysosome. However, the specific role of TASL in keratinocyte differentiation and its potential impact on proliferation remains elusive. In our study, we discovered that TASL deficiency hinders the proliferation and migration of keratinocytes by inducing G1/S cell cycle arrest. Also, TASL deficiency disrupts proper differentiation process in TASL knockout human keratinocyte cell line (HaCaT) by affecting lysosomal function. Additionally, our research into calcium-induced differentiation showed that TASL deficiency affects calcium modulation, which is essential for keratinocyte regulation. These findings unveil a novel role of TASL in the proliferation and differentiation of keratinocytes, providing new insights into the intricate regulatory mechanisms of keratinocyte biology.

HSPA9 reduction exacerbates symptoms and cell death in DSS-Induced inflammatory colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that is influenced by various factors, including environmental factors, immune responses, and genetic elements. Among the factors that influence IBD progression, macrophages play a significant role in generating inflammatory mediators, and an increase in the number of activated macrophages contributes to cellular damage, thereby exacerbating the overall inflammatory conditions. HSPA9, a member of the heat shock protein 70 family, plays a crucial role in regulating mitochondrial processes and responding to oxidative stress. HSPA9 deficiency disrupts mitochondrial dynamics, increasing mitochondrial fission and the production of reactive oxygen species. Based on the known functions of HSPA9, we considered the possibility that HSPA9 reduction may contribute to the exacerbation of colitis and investigated its relevance. In a dextran sodium sulfate-induced colitis mouse model, the downregulated HSPA9 exacerbates colitis symptoms, including increased immune cell infiltration, elevated proinflammatory cytokines, decreased tight junctions, and altered macrophage polarization. Moreover, along with the increased mitochondrial fission, we found that the reduction in HSPA9 significantly affected the superoxide dismutase 1 levels and contributed to cellular death. These findings enhance our understanding of the intricate mechanisms underlying colitis and contribute to the development of novel therapeutic approaches for this challenging condition.

Structural basis for recognition and methylation of p97 by METTL21D, a valosin-containing protein lysine methyltransferase.

p97 is a human AAA+ (ATPase associated with diverse cellular activities, also known as valosin-containing protein [VCP]) ATPase, which is involved in diverse cellular processes such as membrane fusion and proteolysis. Lysine-specific methyltransferase of p97 (METTL21D) was identified as a class I methyltransferase that catalyzes the trimethylation of Lys315 of p97, a so-called VCP lysine methyltransferase (VCPKMT). Interestingly, VCPKMT disassembles a single hexamer ring consisting of p97-D1 domain and methylates Lys315 residue. Herein, the structures of S-adenosyl-L-methionine-bound VCPKMT and S-adenosyl-L-homocysteine-bound VCPKMT in complex with p97 N/D1 (N21-Q458) were reported at a resolution of 1.8 Å and 2.8 Å, respectively. The structures revealed the molecular details for the recognition and methylation of monomeric p97 by VCPKMT. Using biochemical analysis, we also investigated whether the methylation of full-length p97 could be sufficiently enhanced through cooperation between VCPKMT and the C terminus of alveolar soft part sarcoma locus (ASPL). Our study provides the groundwork for future structural and mechanistic studies of p97 and inhibitors.

Protective Effect of GIP against Monosodium Glutamate-Induced Ferroptosis in Mouse Hippocampal HT-22 Cells through the MAPK Signaling Pathway.

The effect of glucose-dependent insulinotropic polypeptide (GIP) on cells under oxidative stress induced by glutamate, a neurotransmitter, and the underlying molecular mechanisms were assessed in the present study. We found that in the pre-treatment of HT-22 cells with glutamate in a dose-dependent manner, intracellular ROS were excessively generated, and additional cell damage occurred in the form of lipid peroxidation. The neurotoxicity caused by excessive glutamate was found to be ferroptosis and not apoptosis. Other factors (GPx-4, Nrf2, Nox1 and Hspb1) involved in ferroptosis were also identified. In other words, it was confirmed that GIP increased the activity of sub-signalling molecules in the process of suppressing ferroptosis as an antioxidant and maintained a stable cell cycle even under glutamate-induced neurotoxicity. At the same time, in HT-22 cells exposed to ferroptosis as a result of excessive glutamate accumulation, GIP sustained cell viability by activating the mitogen-activated protein kinase (MAPK) signalling pathway. These results suggest that the overexpression of the GIP gene increases cell viability by regulating mechanisms related to cytotoxicity and reactive oxygen species production in hippocampal neuronal cell lines.

Overexpression of Lin28a Aggravates Psoriasis-Like Phenotype by Regulating the Proliferation and Differentiation of Keratinocytes.

PURPOSE: Psoriasis is a common and well-studied autoimmune skin disease, which is characterized by plaques. The formation of psoriasis plaques occurs through the hyperproliferation and abnormal differentiation of keratinocytes, infiltration of numerous immune cells into the dermis, increased subepidermal angiogenesis, and various autoimmune-associated cytokines and chemokines. According to previous research, Lin28 regulates the let-7 family, and let-7b is associated with psoriasis. However, the link between Lin28 and psoriasis is unclear. In this study, an association was identified between Lin28a and psoriasis progression, which promoted the pathological characteristic of psoriasis in epidermal keratinocytes. PATIENTS AND METHODS: This study aims to investigate the role of Lin28a and its underlying mechanism in psoriasis through in vivo and in vitro models, which include the Lin28a-overexpressing transgenic (TG) mice and Lin28a-overexpressing human keratinocyte (HaCaT) cell lines, respectively. RESULTS: In vivo and in vitro results revealed that overexpression of Lin28a downregulated microRNA let-7 expression levels and caused hyperproliferation and abnormal differentiation in keratinocytes. In imiquimod (IMQ)-induced psoriasis-like inflammation, Lin28a overexpressing transgenic (TG) mice exhibited more severe symptoms of psoriasis. CONCLUSION: Mechanistically, Lin28a exacerbated psoriasis-like inflammation through the activation of the extracellular-signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 signaling (STAT 3) by targeting proinflammatory cytokine interleukin-6 (IL-6).

Overexpression of cathepsin S exacerbates lupus pathogenesis through upregulation TLR7 and IFN-α in transgenic mice.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs. Recent studies suggest relevance between cysteine protease cathepsin S (CTSS) expression and SLE. To investigate the mechanism of CTSS in SLE, CTSS-overexpressing transgenic (TG) mice were generated, and induced lupus-like symptoms. Eight months later, the TG mice spontaneously developed typical SLE symptoms regardless of the inducement. Furthermore, we observed increased toll-like receptor 7 (TLR7) expression with increased monocyte and neutrophil populations in the TG mice. In conclusion, overexpression of CTSS in mice influences TLR7 expression, autoantibodies and IFN-α, which leads to an autoimmune reaction and exacerbates lupus-like symptoms.

Click chemistry for improvement in selectivity of quinazoline-based kinase inhibitors for mutant epidermal growth factor receptors.

Discovery of mutant-selective kinase inhibitors is one of the challenges in medicinal chemistry and is a main issue for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. We tried to improve the selectivity of pan-HER inhibitors for mutant EGFRs. Utilizing click chemistry, triazole-tethered quinazoline derivatives were synthesized, based on a quinazoline scaffold showing pan-HER inhibition. The representative compound 5j exhibited 17- and 52-fold improved selectivity for EGFR L858R/T790M over wild-type EGFR and HER2, respectively, and demonstrated 6.7-fold more potent antiproliferative activity against PC9 cells harboring EGFR-activating mutation than gefitinib. Although the described quinazolines did not surpass pyrimidines as 3rd generation EGFR inhibitors in terms of selectivity for mutant EGFRs, our approach might provide information that would help in the identification of mutant-selective compounds among pan-HER inhibitors using the quinazoline scaffold.

Investigation of cognitive circuits using steady-state cerebral blood volume and diffusion tensor imaging in patients with mild cognitive impairment following electrical injury.

PURPOSE: We utilized cerebral blood volume (CBV) magnetic resonance imaging and diffusion tensor imaging (DTI) to investigate changes in cognitive networks in patients experiencing cognitive dysfunction following electrical injury. METHODS: Cognitive function was assessed across various domains, including attention, verbal memory, executive function, and language. Depressive symptoms were also evaluated. CBV maps and DTI measures were obtained from 24 patients (age, 41.8 ± 5.8 years; education, 13.3 ± 1.9 years) and 24 healthy controls (age, 42.3 ± 2.7 years; education, 14.3 ± 1.9 years). CBV maps and DTI measures were compared between patients and controls, and correlations between these measures and each cognitive assessment score were examined. RESULTS: Patients exhibited lower attention, verbal memory, and executive function scores than controls (all p < 0.01). Patients also exhibited higher depression scores than controls (p < 0.01), as well as a predominant increase in CBV in the cerebellar vermis relative to that of controls (height p < uncorrected 0.001, extent p < corrected 0.05). Correlation analyses revealed a strong association between executive function scores and CBV in the bilateral posterior cingulate cortex and left mammillary body in patients (height p < uncorrected 0.001, extent p < corrected 0.05). There were no significant differences in DTI measures between patients and controls. CONCLUSION: The CBV maps showed hypermetabolism in the cerebello-limbic system; DTI did not find any microstructural changes. Our results suggest that patients experiencing cognitive dysfunction following electrical injury may possess a cognitive reserve that protects against deteriorating conditions such as dementia.

Preliminary Investigation of Pain-Related Changes in Cerebral Blood Volume in Patients With Phantom Limb Pain.

OBJECTIVE: To investigate changes in the pain network associated with phantom limb pain, magnetic resonance imaging (MRI) was used to measure cerebral blood volume (CBV) in patients who had undergone unilateral arm amputation after electrical injury. DESIGN: Case-controlled exploratory MRI study of CBV via MRI. SETTING: University hospital. PARTICIPANTS: Participants (N=26) comprised patients with phantom limb pain after unilateral arm amputation (n=10) and healthy, age-matched persons (n=16). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The intensity of phantom limb pain was measured using the visual analog scale (VAS). Depressive mood was assessed using the Hamilton Depression Rating Scale, and cognitive function was assessed using the Korean version of the Mini-Mental State Examination. Voxel-wise comparisons of relative CBV maps were made between amputees and controls over the entire brain volume. The relationship between individual participant CBV (measured in voxels) and VAS score was also examined. RESULTS: Compared with control participants, amputees exhibited greater degrees of depression; significantly higher CBV in the bilateral medial frontal area (orbitofrontal cortex [OFC] and pregenual anterior cingulate cortex [pACC]); and significantly lower CBV in the right midcingulate cortex, posterior cingulate cortex, and primary somatosensory cortex. CBV increased in the contralateral and ipsilateral hemispheres of the amputated arm, regardless of the amputation side. This CBV increase in the OFC and pACC was strongly correlated with pain intensity in all amputees. CONCLUSIONS: We observed increased CBV in regions associated with emotion in the cerebral pain network of patients who had undergone unilateral arm amputation after electrical injury. This study suggests that CBV changes were related to neuroplasticity associated with phantom limb pain.

Correction: Risk Factors for Delirium During Acute and Subacute States of Various Disorders in Patients Admitted to Rehabilitation Units.

[This corrects the article on p. 1082 in vol. 40, PMID: 28119839.].

Risk Factors for Delirium During Acute and Subacute Stages of Various Disorders in Patients Admitted to Rehabilitation Units.

OBJECTIVE: To assess the risk factors for delirium in patients admitted to a rehabilitation unit for acute or subacute neurological or musculoskeletal disorders. METHODS: We reviewed the medical records of 537 patients admitted to a rehabilitation unit and selected 398 patients in the acute or subacute stage of various neurological or musculoskeletal disorders. Among them, patients who had suffered from delirium were categorized into the delirium group (n=65), and the other patients were categorized into the non-delirium group (n=333). As potential risk factors for delirium, the patients' diagnosis, underlying disease, demographic data, hospital stay duration, surgery, and laboratory findings were reviewed, and the differences between the two groups with respect to independent risk factors were analyzed. RESULTS: The average age in the delirium group was higher; the hospital stay and pre-transfer periods were longer. A large proportion of the patients were admitted for musculoskeletal disorders, and many patients had diabetes mellitus, dementia, and depression as underlying diseases. Laboratory tests revealed increases in the white blood cells (WBC), glucose, blood urea nitrogen (BUN), total bilirubin, aspartate transaminase (AST), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels in the delirium group, while the hemoglobin, calcium, phosphorus, protein, albumin, and potassium levels were decreased. Depression, musculoskeletal disorders, traumatic brain injury, elevated WBC, BUN, AST, and CRP levels, and decreased potassium and phosphorus levels were identified as independent risk factors for delirium. CONCLUSION: Risk factors treatable before delirium onset were identified in rehabilitation patients in acute and subacute stages of various disorders. Early diagnosis and prevention of these risk factors could decrease delirium occurrence and increase rehabilitation effectiveness.

Novel information on anatomic factors causing grasp reflex in frontal lobe infarction: a case report.

We report a patient with a severe limitation of function in the right hand resulting from grasp reflex following a stroke affecting the left anterior cerebral artery region. We describe, using diffusion tensor tractography (DTT), a disconnection between the bilateral frontal lobes via the corpus callosum. The patient could not control his right hand at all, even though his bilateral corticospinal tracts were intact. We noted that over the infarcted lesion on DTT, the white matter was invisible from the corpus callosum to the prefrontal cortex. These findings reflected a unique pattern of white-matter disconnection between the ipsilateral medial frontal lobe and ipsilateral and contralateral frontal cortex causing hand function deterioration in the form of severe grasp reflex.