Injury prevents Ras mutant cell expansion in mosaic skin.

Healthy skin is a mosaic of wild-type and mutant clones1,2. Although injury can cooperate with mutated Ras family proteins to promote tumorigenesis3-12, the consequences in genetically mosaic skin are unknown. Here we show that after injury, wild-type cells suppress aberrant growth induced by oncogenic Ras. HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue but their expansion is prevented after injury owing to an increase in the fraction of proliferating wild-type cells. Mechanistically, we show that, unlike HrasG12V/+ cells, wild-type cells respond to autocrine and paracrine secretion of EGFR ligands, and this differential activation of the EGFR pathway explains the competitive switch during injury repair. Inhibition of EGFR signalling via drug or genetic approaches diminishes the proportion of dividing wild-type cells after injury, leading to the expansion of HrasG12V/+ cells. Increased proliferation of wild-type cells via constitutive loss of the cell cycle inhibitor p21 counteracts the expansion of HrasG12V/+ cells even in the absence of injury. Thus, injury has a role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.

Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo.

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

Effect of Sevoflurane on the Proliferation of A549 Lung Cancer Cells.

Background and Objectives: Sevoflurane has opposing effects on cancer progression, depending on its concentration and the cancer type. This study investigated the effects of sevoflurane on the proliferation of A549 lung cancer cells. Materials and Methods: In vitro, the number of A549 cells exposed to different concentrations of sevoflurane was counted. The size and weight of tumors from a xenograft mouse model exposed to air or sevoflurane were measured in vivo experiments. Additionally, hematoxylin and eosin staining and immunohistochemical detection of Ki-67 in the harvested tumor tissues were performed. Results: A total of 72 culture dishes were used and 24 dishes were assigned to each group: Air group; 2% Sevo group (air + 2% sevoflurane); and 4% Sevo group (air + 4% sevoflurane). The number of A549 cells in the 2% Sevo group was less than that in the Air and 4% Sevo groups (Air: 7.9 ± 0.5; 0.5, 2% Sevo: 6.8 ± 0.4, 4% Sevo: 8.1 ± 0.3; p = 0.000). The tumor size was not significantly different between the two groups (Air: 1.5 ± 0.7, 2% Sevo: 2.4 ± 1.9; p = 0.380). Conclusions: The in vitro data showed that sevoflurane inhibited the proliferation of A549 lung cancer cells in a concentration-specific manner. However, the in vivo data showed no correlation between sevoflurane exposure and A549 cell proliferation. Thus, further research is required to understand fully the effects of sevoflurane on cancer progression and to reconcile differences between the in vitro and in vivo experimental results.

Double knockin mice show NF-κB trajectories in immune signaling and aging.

In vitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins in vivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled. Overcoming hurdles in simultaneous live-cell imaging of RelA and c-Rel, we show that quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells, and shift toward c-Rel in microglia from aged brains. RelA:c-Rel heterodimer is unexpectedly depleted in the nuclei of stimulated cells. Trajectories of subunit co-expression in immune lineages reveal a reduction at key cell maturation stages. These results demonstrate the power of these reporters in gaining deeper insights into NF-κB biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications.

Association between Sagittal Cervical Spinal Alignment and Degenerative Cervical Spondylosis: A Retrospective Study Using a New Scoring System.

(1) Background: Prolonged neck flexion is thought to cause harmful loading on the cervical spine. Along with the degenerative process, cervical alignment tends to change toward lordotic curvature. The association between cervical alignment and cervical spondylosis remains unclear. (2) Methods: Three raters retrospectively assessed cervical radiographies of outpatients at a tertiary center in 2019 using degenerative cervical spondylosis score (DCS score; a newly developed scoring system), C2-7 absolute rotational angle (ARA), and C2-7 sagittal vertical axis (SVA). (3) Results: A total of 561 patients were included in the analysis. Multiple regression analysis with adjustments for age and sex revealed that C2-7 ARA, rather than SVA, was a significant parameter for degenerative spondylosis. The interaction between age and C2-7 ARA was significant, indicating that the increase in DCS score with increasing age was more pronounced in patients with kyphotic cervical alignment. The direct effect of age on DCS score was 0.349 (95% CI 0.319 to 0.380, p < 0.001) and the proportion of the mediation effect of C2-7 ARA was −0.125 (p < 0.001). (4) Conclusions: C2-7 ARA was significantly associated with DCS after adjustment for both age and sex. Subjects with more kyphotic cervical alignment showed a greater correlation between increased DCS score and older age.

From start to finish-a molecular link in wound repair.

p53 mediates epithelial cell migration and leader cell elimination during wound repair.

Regional analgesia techniques for video-assisted thoracic surgery: a frequentist network meta-analysis.

BACKGROUND: Various regional analgesia techniques are used to reduce postoperative pain in patients undergoing video-assisted thoracic surgery (VATS). This study aimed to determine the relative efficacy of regional analgesic interventions for VATS using a network meta-analysis (NMA). METHODS: We searched the Medline, EMBASE, Cochrane Controlled Trial Register, Web of Science, and Google Scholar databases to identify all randomized controlled trials (RCTs) that compared the analgesic effects of the following interventions: control, thoracic paravertebral block (TPVB), erector spinae plane block (ESPB), serratus plane block (SPB), and intercostal nerve block (INB). The primary outcome was opioid consumption during the first 24-h postoperative period. Pain scores were also collected during three different postoperative periods: the early (0-6 h), middle (6-18 h), and late (18-24 h) periods. RESULTS: A total of 21 RCTs (1391 patients) were included. TPVB showed the greatest effect on opioid consumption compared with the control (mean difference [MD] = -13.2 mg; 95% CI [-16.2, -10.1]). In terms of pain scores in the early period, ESPB had the greatest effect compared to control (MD = -1.6; 95% CI [-2.3, -0.9]). In the middle and late periods, pain scores showed that TPVB, ESPB and INB had superior analgesic effects compared to controls, while SPB did not. CONCLUSIONS: TPVB had the best analgesic efficacy following VATS, though the analgesic efficacy of ESPBs was comparable. However, further studies are needed to determine the optimal regional analgesia technique to improve postoperative pain control following VATS.

NFAT5 Deficiency Alleviates Formalin-Induced Inflammatory Pain Through mTOR.

Nuclear factor of activated T cells (NFAT5) is a well-known transcription factor that regulates the expression of genes involved in osmotic stress. However, the role of NFAT5 in inflammatory pain remains unknown. Here, we studied the function of NFAT5 in inflammatory pain using NFAT5-heterozygous (Het) mice. To study inflammatory pain, we injected 10 µL of 2% formalin into the right hind paws of mice and monitored pain behaviors, such as licking, lifting, and flinching, for 60 min. After the first 15 min (phase I), there were no significant differences in pain behaviors between wild-type (WT) and NFAT5-Het mice. However, from 15-60 min (phase II), NFAT5-Het mice displayed significantly fewer pain behaviors compared to WT mice. Further, the expression levels of inflammatory-pain-related factors, including c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated n-methyl-D-aspartate receptor subunit 2B (p-NR2B), were significantly elevated in the spinal dorsal neurons of formalin-treated WT mice but was not elevated in NFAT5-Het mice. Similarly, c-Fos, p-ERK, and p-NR2B levels were significantly higher in glutamate-treated PC12 neuronal cells but were not affected by Nfat5 silencing in glutamate-treated PC12 cells. Altogether, our findings suggest that NFAT5 deficiency may mitigate formalin-induced inflammatory pain by upregulating mammalian target of rapamycin (mTOR) expression and downregulating its downstream factors in spinal dorsal neurons. Therefore, NFAT5 is a potential therapeutic target for the treatment of inflammatory pain.

Comparison of continuous and single interscalene block for quality of recovery score following arthroscopic rotator cuff repair.

BACKGROUND: Continuous interscalene brachial plexus block (CISB) is well known to reduce postoperative pain and to improve patient satisfaction. However, the effect of CISB on the quality of postoperative recovery is unknown. We Compared the quality of recovery from arthroscopic rotator cuff repair in patients who received CISB or single interscalene brachial plexus block (SISB). METHODS: This prospective non-randomized controlled trial with propensity score matching enrolled 134 patients undergoing arthroscopic surgery for rotator cuff repair. Each patient received an interscalene block before surgery. One group had a catheter insertion 30 min after the end of surgery and started patient-controlled regional analgesia (PCRA, n = 49). The other group received intravenous patient-controlled analgesia (IV-PCA, n = 85). The primary outcome was the quality of recovery (QoR-40) score. Also, postoperative analgesia, sleep quality, and postoperative complications were evaluated. RESULTS: The two groups had similar QoR-40 score on postoperative day-1 (POD1), but the PCRA group had a significantly greater QoR-40 score on POD2 (156.0, IQR: 143.0, 169.0 vs. 171.0, IQR: 159.0, 178.0; p < 0.001). The IV-PCA group received more analgesics during the 2 days after surgery, especially during night-time, and had a higher prevalence of sleep disturbances. The time to first additional analgesics request was significantly longer in PCRA group (14 hours, 95% CI: 13-16 vs. 44 hours, 95% CI: 28-not applicable). The incidence of postoperative nausea and vomiting significantly lower in the PCRA group (16.3% vs 46.9%, p = 0.002). CONCLUSION: CISB showed a higher quality of recovery score than SISB with IV-PCA in arthroscopic rotator cuff repair, probably related to the effective analgesia, improved sleep quality, and reduced opioid-related complications.

Effects of Sevoflurane on Lewis Lung Carcinoma Cell Proliferation In Vivo and In Vitro.

Background and objectives: There are several studies that sevoflurane could enhance proliferation of cancer cells, while others suggest no effect on clinical outcome. We conducted in vivo and in vitro experiments to investigate the effects of sevoflurane, a volatile anesthetic, on proliferation and outcomes of Lewis lung carcinoma (LLC) cells. Materials and Methods: A total of 37 mice were injected with LLC cells to compare the tumor size and survival of the sevoflurane exposed group (sevo group) and control group. The sevo group was exposed to 2% sevoflurane and 4 L/min of oxygen for 1 h per day 3 times per week, and the control group was exposed only to 4 L/min of oxygen. In vitro study, 12 plates incubated with LCC cells. 6 plates were exposed to 2% sevoflurane for 1 hr/day for 3 days and 6 plates were not exposed, and cell proliferation was compared after 3 days. Results: There were no significant differences in survival or tumor size between mice exposed to sevoflurane and control mice (survival: 29.06 ± 4.45 vs. 28.76 ± 3.75, p = 0.836; tumor size: 0.75 (0.41-1.02) vs. 0.49 (0.11-0.79), p = 0.153). However, in vitro study, the proliferation of LLC cells exposed to sevoflurane increased by 9.2% compared to the control group (p = 0.018). Conclusions: Sevoflurane (2 vol%) exposure could promote proliferation of LLC cells in vitro environment, but may not affect proliferation of LLC cells in vivo environment. These results suggest that in vitro studies on the effects of anesthetics on cancer may differ from those of in vivo or clinical studies.

Interval-dependent neurotoxicity after multiple ketamine injections in late postnatal mice.

PURPOSE: Measuring the neurotoxic effects of multiple anesthetic exposures during neurodevelopment is complex due to the numerous factors that can affect the outcome. While we recently discovered that the interval between multiple sevoflurane exposures can affect the level of neurotoxicity, the significance of interval for other anesthetic agents is unknown. Thus, we evaluated the significance of dosing interval in the neurotoxic effects of multiple ketamine injections in postnatal day (PND) 17 mice. METHODS: PND17 mice of both sexes were intraperitoneally injected with ketamine (35 mg/kg) three times at short (2 h) or long (24 h) intervals. Changes in synaptic transmission were measured in hippocampal pyramidal neurons 5 days after the last injection, and behavioral changes were assessed at the age of 8 weeks. Values are presented as mean ± SD. RESULTS: Whereas short-interval ketamine injections enhanced excitatory synaptic transmission, as evidenced by an increased frequency of miniature excitatory postsynaptic currents (mEPSCs; ketamine, 0.09 ± 0.07 Hz; control, 0.06 ± 0.03 Hz), long-interval ketamine injections did not; instead, they decreased the amplitude of miniature inhibitory postsynaptic currents (mIPSCs; ketamine, 47.72 ± 6.90 pA; control, 51.21 ± 7.65 pA,). However, only long-interval ketamine injections induced long-term changes in anxiety behavioral in the open-field test (decrease in center duration; ketamine, 400.1 ± 162.8 s; control, 613.3 ± 312.7 s). CONCLUSIONS: Multiple ketamine injections induce interval-dependent, long-lasting synaptic changes and behavioral impairments. Future studies should carefully consider the dosing interval as a significant factor when studying the neurotoxic effects of multiple anesthetic exposures.

Identifying the ideal tracheostomy site based on patient characteristics during percutaneous dilatational tracheostomy without bronchoscopy.

BACKGROUND: We previously reported that percutaneous dilatational tracheostomy (PDT) can be safely performed 2 cm below the cricothyroid membrane without the aid of a bronchoscope. Although our simplified method is convenient and does not require sophisticated equipment, the precise location for tracheostomy cannot be confirmed. Because it is recommended that tracheostomy be performed at the second tracheal ring, we assessed whether patient characteristics could predict the distance between the cricothyroid membrane and the second tracheal ring. METHODS: Data from 490 patients who underwent three-dimensional neck computed tomography from January 2012 to December 2015 were analyzed, and the linear distance from the upper part of the cricoid cartilage (CC) to the lower part of the second tracheal ring (2TR) was measured in the sagittal plane. RESULTS: The mean CC-to-2TR distance was 25.26 mm (95% CI 25.02-25.48 mm). Linear regression analysis showed that the predicted CC-to-2TR distance could be calculated as -5.73 + 0.2 × height (cm) + 1.22 × sex (male: 1, female: 0) + 0.01 × age (yr) -0.03 × weight (kg) (adj. R2 = 0.55). CONCLUSIONS: These results suggest that height and sex should be considered when performing PDT without bronchoscope guidance.

Anesthesia affects excitatory/inhibitory synapses during the critical synaptogenic period in the hippocampus of young mice: Importance of sex as a biological variable.

BACKGROUND: Sex plays an important yet often underexplored role in neurodevelopment and neurotoxicity. While several studies report the importance of sex regarding anesthesia-induced neurotoxicity in neonatal mice, only few have focused on the late postnatal period. Here, to further understand the importance of sex regarding the neurobiological changes after early anesthesia during the critical synaptogenic period, we exposed postnatal day 16, 17 (PND 16, 17) mice to sevoflurane in pediatric patients and performed detailed evaluations in the hippocampus. METHODS: PND 16, 17 mice received a single exposure of oxygen with or without sevoflurane (2.5%) for 2 h. Changes of the hippocampus were analyzed in male and female mice 6 h after exposure: excitatory/inhibitory synaptic transmission, protein/mRNA expression levels of excitatory/inhibitory synaptic molecules (GluR1, GluR2, PSD95, gephyrin, GAD65), and number of excitatory synapses. RESULTS: Sevoflurane exposure increased the frequency of miniature excitatory postsynaptic currents specifically in male mice (control: 0.07 ± 0.04 [Hz]; sevoflurane: 14.72 ± 0.08 [Hz]), while miniature inhibitory postsynaptic currents were affected specifically in female mice. The protein/mRNA expression levels of excitatory synaptic molecules were also increased specifically in male mice. Unexpectedly, protein/mRNA expression levels of inhibitory synaptic molecules were increased in both sexes, and there was no male-specific increase of excitatory synapse number. CONCLUSIONS: Exposure of mice to sevoflurane during the critical, late postnatal period induces sex-dependent changes in the hippocampus. Although often disregarded, our results confirm the importance of sex as a biological variable when studying the changes triggered by early anesthesia.

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer.

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.