Concurrent loss of LKB1 and KEAP1 enhances SHMT-mediated antioxidant defence in KRAS-mutant lung cancer.

Non-small-cell lung cancer (NSCLC) with concurrent mutations in KRAS and the tumour suppressor LKB1 (KL NSCLC) is refractory to most therapies and has one of the worst predicted outcomes. Here we describe a KL-induced metabolic vulnerability associated with serine-glycine-one-carbon (SGOC) metabolism. Using RNA-seq and metabolomics data from human NSCLC, we uncovered that LKB1 loss enhanced SGOC metabolism via serine hydroxymethyltransferase (SHMT). LKB1 loss, in collaboration with KEAP1 loss, activated SHMT through inactivation of the salt-induced kinase (SIK)-NRF2 axis and satisfied the increased demand for one-carbon units necessary for antioxidant defence. Chemical and genetic SHMT suppression increased cellular sensitivity to oxidative stress and cell death. Further, the SHMT inhibitor enhanced the in vivo therapeutic efficacy of paclitaxel (first-line NSCLC therapy inducing oxidative stress) in KEAP1-mutant KL tumours. The data reveal how this highly aggressive molecular subtype of NSCLC fulfills their metabolic requirements and provides insight into therapeutic strategies.

Transcanal endoscopic ear surgery for glomus tympanicum removal: A case series on the necessity of preoperative embolization.

BACKGROUND: Transcanal endoscopic ear surgery (TEES) has become popular in recent years in the treatment of glomus tympanicum tumors (GTT). The most significant risk for TEES is bleeding. In some cases, preoperative vascular embolization is performed to mitigate bleeding during TEES. However, guidelines regarding the necessity and efficacy of preoperative vascular embolization have not been established yet. CASE PRESENTATION: This report aimed to assess the necessity and usefulness of preoperative vascular embolization in TEES for GTT by comparing the surgical findings of TEES without preoperative vascular embolization (Case 1) and TEES with preoperative vascular embolization (Case 2). Compared to Case 1, Case 2 included less bleeding and a more convenient procedure. However, no significant difference was observed. CONCLUSIONS: For GTT confined to the middle ear cavity (Glasscock-Jackson Grade II or less), when performed by a proficient otolaryngologist, TEES alone is sufficient without preoperative vascular embolization.

Prospective Study of Nonsurgical Auricular Correction According to Timing of Treatment.

BACKGROUND AND PURPOSE: Many studies recommend nonsurgical auricular correction during the early postnatal period, when cartilage plasticity is high; however, many patients are not eligible for the procedure. This study compared different timings of nonsurgical auricular correction to investigate benefit after the optimal period for correction. METHODS: In this prospective study, 53 ears from 35 patients with congenital auricular anomaly were assigned to two groups according to age at correction: the "early-group" with correction within 2 weeks of birth and "late-group" with correction 8 weeks after birth. Aesthetic outcomes, caregiver satisfaction, detachment rates and mean device-wearing periods, were compared. RESULTS: Thirty-one ears from 20 patients comprised the early-group, and 18 ears from 12 patients comprised the late-group. Mean time to treatment after birth was 9.09 days in the early-group and 134.7 days in the late-group. In the early-group, detachment occurred in 4/31 ears (12.9%), and in the late-group, detachment occurred in 12/18 ears (66.7%), which was statistically significant (p<0.01). The average period of applying devices was 4.7 ± 1.2 weeks in the early-group and 8.5 ± 4.1 weeks in the late-group, with a significantly longer treatment time in the late-group (p=0.001). The early-group had 87.1% "good" results vs. 55.6% in the late-group, with a statistically significant difference. CONCLUSIONS: The correction period was shorter, detachment rate was lower, and treatment outcome was better in the early-group. However, successful correction was also present in the late-group, showing that the patients who have passed the optimum correction period should proceed after counselling.

Effects of Particulate Matter Exposure on the Eustachian Tube and Middle Ear Mucosa of Rats.

OBJECTIVES: Particulate matter (PM) is a risk factor for various diseases. Recent studies have established an association between otitis media (OM) and PM exposure. To confirm this relationship, we developed a novel exposure model designed to control the concentration of PM, and we observed the effects of PM exposure on the Eustachian tube (ET) and middle ear mucosa of rats. METHODS: Forty healthy, 10-week-old, male Sprague-Dawley rats were divided into 3-day, 7-day, 14-day exposure, and control groups (each, n=10). The rats were exposed to incense smoke as the PM source for 3 hours per day. After exposure, bilateral ETs and mastoid bullae were harvested, and histopathological findings were compared using microscopy and transmission electron microscopy (TEM). The expression levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor (VEGF) in the middle ear mucosa of each group were compared using real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: In the ET mucosa of the exposure group, the goblet cell count significantly increased after PM exposure (P=0.032). In the middle ear mucosa, subepithelial space thickening, increased angio-capillary tissue, and inflammatory cell infiltration were observed. Moreover, the thickness of the middle ear mucosa in the exposure groups increased compared to the control group (P<0.01). The TEM findings showed PM particles on the surface of the ET and middle ear mucosa, and RT-PCR revealed that messenger RNA (mRNA) expression of IL-1ß significantly increased in the 3-day and 7-day exposure groups compared to the control group (P=0.035). VEGF expression significantly increased in the 7-day exposure group compared to the control and 3-day exposure groups (P<0.01). CONCLUSION: The ET and middle ear mucosa of rats showed histopathologic changes after acute exposure to PM that directly reached the ET and middle ear mucosa. Therefore, acute exposure to PM may play a role in the development of OM.

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Hearing recovery prediction and prognostic factors of idiopathic sudden sensorineural hearing loss: a retrospective analysis with a deep neural network model

Abstract Objective Idiopathic Sudden Sensorineural Hearing Loss (ISSHL) is an otologic emergency, and an early prediction of prognosis may facilitate proper treatment. Therefore, we investigated the prognostic factors for predicting the recovery in patients with ISSHL treated with combined treatment method using machine learning models. Methods We retrospectively reviewed the medical records of 298 patients with ISSHL at a tertiary medical institution between January 2015 and September 2020. Fifty-two variables were analyzed to predict hearing recovery. Recovery was defined using Siegel's criteria, and the patients were categorized into recovery and non-recovery groups. Recovery was predicted by various machine learning models. In addition, the prognostic factors were analyzed using the difference in the loss function. Results There were significant differences in variables including age, hypertension, previous hearing loss, ear fullness, duration of hospital admission, initial hearing level of the affected and unaffected ears, and post-treatment hearing level between recovery and non-recovery groups. The deep neural network model showed the highest predictive performance (accuracy, 88.81%; area under the receiver operating characteristic curve, 0.9448). In addition, initial hearing level of affected and non-affected ear, post-treatment (2-weeks) hearing level of affected ear were significant factors for predicting the prognosis. Conclusion The deep neural network model showed the highest predictive performance for recovery in patients with ISSHL. Some factors with prognostic value were identified. Further studies using a larger patient population are warranted. Level of evidence: Level 4.

Treatment results for congenital cholesteatoma using transcanal endoscopic ear surgery.

PURPOSE: Transcanal endoscopic ear surgery for congenital cholesteatoma provides a smaller incision and better visibility than conventional surgical microscope ear surgery. We aimed to evaluate the treatment results of transcanal endoscopic ear surgery in ameliorating the recurrence and post-operative complications of pediatric congenital cholesteatoma. MATERIALS AND METHODS: A retrospective chart review was conducted on patients with congenital cholesteatoma who underwent transcanal endoscopic ear surgery at a tertiary referral medical center from January 2012 to December 2020. We categorized the patients into two groups according to the presence of remnant cholesteatoma and compared their characteristics. RESULTS: This study included 46 patients (32 males and 14 females; 46 ears). The mean age was 3.0 ± 2.6 (1-17) years. Congenital cholesteatoma was predominantly located in the anterior-superior quadrant (63.0 %), and ossicular involvement was confirmed in six cases. The mean cholesteatoma size identified by temporal bone computed tomography was 3.9 ± 2.0 (1.2-13) mm. Seven cases had remnant cholesteatoma lesions (15.2 %); four improved after revision surgery, and three were followed up without immediate further intervention. Postoperative complications (e.g., tympanic membrane perforation, retraction) were observed in 10 cases. Comparative evaluations of various characteristics after categorizing the participants into residual and non-residual lesion groups revealed no significant differences. CONCLUSIONS: Consequently, transcanal endoscopic ear surgery can be considered an effective and safe operation for treating congenital cholesteatoma in the pediatric population with no serious side effects. Further large-scale research with hearing test results should be conducted meticulously as a follow-up to this study.

A new role for ammonia in tumorigenesis?

Tumor-supporting roles of ammonia have gained greater appreciation in recent years and normally focus on ammonia's role as a nitrogen source. Recently in Nature Metabolism,Cheng et al. (2022) demonstrate a novel, non-nitrogen, metabolism-related role of ammonia as a key activator for lipogenesis by facilitating SREBP-1 activation.

Effects of cigarette smoke on Haemophilus influenzae-induced otitis media in a rat model.

Exposure to cigarette smoke (CS) is a factor that could delay or worsen the recovery of otitis media (OM) by causing inflammatory swelling of the Eustachian tube (ET). However, despite the suggested relationship, little is known about the association between OM and CS. Therefore, we aimed to evaluate the effects of CS on the development, progression, and recovery of OM, as well as the histological and molecular changes caused by CS exposure, by using a rat model of OM infected with non-typeable Haemophilus influenzae (NTHi). Eighty Sprague-Dawley rats with normal middle ears (MEs) were divided into four groups (n = 20 rats/group): control, CS, OM, and CS + OM. The CS and CS + OM groups were exposed to CS for 2 weeks. The inflammatory reaction to NTHi was more intense and lasted longer in the CS + OM group than in the other groups. Goblet cell proliferation and mucus secretion in the ET were more significant in the CS and CS + OM groups than in the other groups. These findings suggested that because CS directly affects the ET and ME mucosa, bacterial OM can become more severe and may resolve more slowly in the presence of CS exposure rather than in its absence.

Heterogeneous nuclear ribonucleoprotein A2/B1 regulates the ERK and p53/HDM2 signaling pathways to promote the survival, proliferation and migration of non­small cell lung cancer cells.

Lung cancer is the most frequent cause of cancer­associated mortality worldwide. Upregulation of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) has been reported in non­small cell lung cancer (NSCLC) cells, but its contribution to NSCLC remains poorly understood. hnRNPA2/B1 is involved in carcinogenesis by interacting with a number of proteins; however, little is known about its interaction with p53. The results of the present study revealed that hnRNPA2/B1 expression levels were upregulated in NSCLC cells under tumorsphere culture conditions and cisplatin treatment compared with those in cells under the adherent condition and dimethyl sulfoxide treatment, respectively, suggesting that hnRNPA2/B1 expression is induced under stress conditions. hnRNPA2/B1 knockdown decreased the number and size of NSCLC cell colonies in a clonogenic survival assay and led to a decreased migratory potential of NSCLC cells, suggesting that hnRNPA2/B1 may promote the survival, proliferation and migration of NSCLC cells. hnRNPA2/B1 knockdown induced G0/G1 phase arrest in NSCLC cells through cyclin E degradation and phosphorylation of cyclin­dependent kinase 2. In addition, hnRNPA2/B1 knockdown inhibited extracellular signal­regulated kinase (ERK)1/2 phosphorylation, suggesting that hnRNPA2/B1 may promote the G1/S phase transition in NSCLC cells through ERK signaling. hnRNPA2/B1 knockdown resulted in increased expression levels of p21 and p27 in NSCLC cells, as well as p53 induction and phosphorylation. Additionally, hnRNPA2/B1 knockdown inhibited human double minute 2 protein (HDM2) stability and phosphorylation, whereas overexpression of hnRNPA2 induced the opposite effects. These results suggested that hnRNPA2/B1 may promote the survival, proliferation and migration of NSCLC cells through preventing the activation of p53, which is induced by ERK­mediated HDM2 activation. The results of the present study also indicated that the components of the hnRNPA2/B1/ERK/p53/HDM2 signaling pathway may be novel potential molecular targets for the treatment of patients with NSCLC.

A longitudinal study of changes of congenital auricular deformity regarding self-correction.

This study aimed to investigate the factors associated with congenital auricular deformities and evaluate the long-term frequency of their self-correction. Ninety newborns were enrolled in the study, and data were collected within 2 weeks after birth and at 1 year. The shape of the auricle was classified into seven categories using a digital image. At 2 weeks after birth, several birth-related factors were evaluated in the auricular deformity and normal groups. At 1 year after birth, the images of auricles were compared with the images at birth, and the changes in the auricle shape were investigated. Congenital auricular deformities were observed in 139 out of 180 ears, and the major type noted was helix rim deformity (47 ears), followed by normal ears (41 ears), and cup ears (33 ears). Male sex was found to have a statistically significant association with the occurrence of auricular deformity. In the longitudinal study, among 43 neonates (86 ears) followed-up 12 months later, the self-correction rate was approximately 50%. The normal auricle and prominent ear increased, helix rim deformity and cup ear decreased significantly. The prognosis of deformity varied depending on the type of deformity. Considering the low self-correction rate in the prominent and cup ears, newborns with these deformities might be recommended to undergo management such as auricle molding technique, as required.

Mercury Chloride but Not Lead Acetate Causes Apoptotic Cell Death in Human Lung Fibroblast MRC5 Cells via Regulation of Cell Cycle Progression.

Heavy metals are important for various biological systems, but, in excess, they pose a serious risk to human health. Heavy metals are commonly used in consumer and industrial products. Despite the increasing evidence on the adverse effects of heavy metals, the detailed mechanisms underlying their action on lung cancer progression are still poorly understood. In the present study, we investigated whether heavy metals (mercury chloride and lead acetate) affect cell viability, cell cycle, and apoptotic cell death in human lung fibroblast MRC5 cells. The results showed that mercury chloride arrested the sub-G1 and G2/M phases by inducing cyclin B1 expression. In addition, the exposure to mercury chloride increased apoptosis through the activation of caspase-3. However, lead had no cytotoxic effects on human lung fibroblast MRC5 cells at low concentration. These findings demonstrated that mercury chloride affects the cytotoxicity of MRC5 cells by increasing cell cycle progression and apoptotic cell death.

Bisphenol A Exposure Changes the Transcriptomic and Proteomic Dynamics of Human Retinoblastoma Y79 Cells.

Bisphenol A (BPA) is a xenoestrogen chemical commonly used to manufacture polycarbonate plastics and epoxy resin and might affect various human organs. However, the cellular effects of BPA on the eyes have not been widely investigated. This study aimed to investigate the cellular cytotoxicity by BPA exposure on human retinoblastoma cells. BPA did not show cytotoxic effects, such as apoptosis, alterations to cell viability and cell cycle regulation. Comparative analysis of the transcriptome and proteome profiles were investigated after long-term exposure of Y79 cells to low doses of BPA. Transcriptome analysis using RNA-seq revealed that mRNA expression of the post-transcriptional regulation-associated gene sets was significantly upregulated in the BPA-treated group. Cell cycle regulation-associated gene sets were significantly downregulated by exposure to BPA. Interestingly, RNA-seq analysis at the transcript level indicated that alternative splicing events, particularly retained introns, were noticeably altered by low-dose BPA treatment. Additionally, proteome profiling using MALDI-TOF-MS identified a total of nine differentially expressed proteins. These results suggest that alternative splicing events and altered gene/protein expression patterns are critical phenomena affected by long-term low-dose BPA exposure. This represents a novel marker for the detection of various diseases associated with environmental pollutants such as BPA.

Transcriptome Analysis Reveals HgCl2 Induces Apoptotic Cell Death in Human Lung Carcinoma H1299 Cells through Caspase-3-Independent Pathway.

Mercury is one of the detrimental toxicants that can be found in the environment and exists naturally in different forms; inorganic and organic. Human exposure to inorganic mercury, such as mercury chloride, occurs through air pollution, absorption of food or water, and personal care products. This study aimed to investigate the effect of HgCl2 on cell viability, cell cycle, apoptotic pathway, and alters of the transcriptome profiles in human non-small cell lung cancer cells, H1299. Our data show that HgCl2 treatment causes inhibition of cell growth via cell cycle arrest at G0/G1- and S-phase. In addition, HgCl2 induces apoptotic cell death through the caspase-3-independent pathway. Comprehensive transcriptome analysis using RNA-seq indicated that cellular nitrogen compound metabolic process, cellular metabolism, and translation for biological processes-related gene sets were significantly up- and downregulated by HgCl2 treatment. Interestingly, comparative gene expression patterns by RNA-seq indicated that mitochondrial ribosomal proteins were markedly altered by low-dose of HgCl2 treatment. Altogether, these data show that HgCl2 induces apoptotic cell death through the dysfunction of mitochondria.

Ablation of SMUG1 Reduces Cell Viability and Increases UVC-Mediated Apoptosis in Hepatocarcinoma HepG2 Cells.

Uracil is an unavoidable aberrant base in DNA sequences, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires multiple biochemical steps with conformational changes of DNA that inhibit DNA replication and interfere with transcription. However, the relevance of uracil in DNA for cellular physiology and transcriptional regulation is not fully understood. We investigated the functional roles of SMUG1 using knock-down (KD) and knock-out (KO) models. The proliferation ratio of SMUG1 KD and KO cells was decreased compared to WT control cells, and the cell cycle was arrested in the G2/M phases before the transition to mitosis. The apoptotic cell death was increased in KD and KO cell lines through the increase of BAX and active caspase 3 expression. Phospho-gamma-H2AX expression, which reflected accumulated DNA damage, was also increased in KO cells. Moreover, the apoptotic cells by DNA damage accumulation were markedly increased in SMUG1 KD and KO cells after ultraviolet C irradiation. Transcriptomic analysis using RNA-seq revealed that SMUG1 was involved in gene sets expression including cell cycle transition and chromatin silencing. Together, the results implicate SMUG1 as a critical factor in cell cycle and transcriptional regulation.

Comparative study of endoscopic and microscopic tympanoplasty performed by a single experienced surgeon.

PURPOSE: The use of endoscopes in otologic procedures has been increasing worldwide. This study aimed to compare the efficacy of microscopic tympanoplasty (MT) and endoscopic tympanoplasty (ET) for tympanic membrane and middle ear surgery. MATERIALS AND METHODS: We retrospectively analyzed 81 patients who underwent MT (n = 44) and ET (n = 37) for chronic otitis media with tympanic membrane perforation performed by a single surgeon between January 2013 and September 2019. The hearing outcomes, graft success rate, complications, operation time and hospital stay, and cost-effectiveness were recorded and compared between groups. Hearing outcomes were determined by pure tone audiometry. Cost-effectiveness was determined by the operation cost and total cost. RESULTS: There was no significant difference between the MT and ET groups regarding demographic characteristics, with the exception of the male:female ratio. There was no significant difference in the pre- and postoperative air conduction, bone conduction thresholds, and air-bone gap values between the two groups, but a significant audiologic improvement was observed in both groups (p < 0.05). In terms of recurrence of tympanic membrane perforation, postoperative otorrhea, and discomfort symptoms, there was no significant difference between groups (p > 0.05). The operation time and hospital stay were shorter in the ET group than in the MT group (p < 0.05). There were no significant differences in operation cost between the two groups (p > 0.05), but the total cost was significantly lower in the ET group than the MT group (p < 0.05). CONCLUSION: ET is as safe and medically efficacious as conventional MT, shortens the operation time and hospital stay, and is cost-effective.

The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1 mutant lung cancer.

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumour suppressor STK11 (also known as LKB1) encoding the kinase LKB1 result in aggressive tumours prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analysed tumour metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KRAS/LKB1 co-mutant tumours. KRAS/LKB1 co-mutant cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2). GFPT2 inhibition selectively reduced KRAS/LKB1 co-mutant tumour cell growth in culture, xenografts and genetically modified mice. Our results define a new metabolic vulnerability in KRAS/LKB1 co-mutant tumours and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

Evaluation of the Biodegradation Efficiency of Four Various Types of Plastics by Pseudomonas aeruginosa Isolated from the Gut Extract of Superworms.

Plastic waste worldwide is becoming a serious pollution problem for the planet. Various physical and chemical methods have been tested in attempts to remove plastic dumps. However, these have usually resulted in secondary pollution issues. Recently, the biodegradation of plastic by fungal and bacterial strains has been spotlighted as a promising solution to remove plastic wastes without generating secondary pollution. We have previously reported that a Pseudomonas aeruginosa strain isolated from the gut of a superworm is capable of biodegrading polystyrene (PS) and polyphenylene sulfide (PPS). Herein, we demonstrate the extraordinary biodegradative power of P. aeruginosa in efficiently depolymerizing four different types of plastics: PS, PPS, polyethylene (PE) and polypropylene (PP). We further compared biodegradation rates for these four plastic types and found that PE was biodegraded fastest, whereas the biodegradation of PP was the slowest. Moreover, the growth rates of P. aeruginosa were not always proportional to biodegradation rates, suggesting that the rate of bacterial growth could be influenced by the composition and properties of intermediate molecules produced during plastic biodegradation, and these may supply useful cellular precursors and energy. In conclusion, an initial screening system to select the most suitable bacterial strain to biodegrade certain types of plastic is particularly important and may be necessary to solve plastic waste problems both presently and in the future.

Oncology Therapeutics Targeting the Metabolism of Amino Acids.

Amino acid metabolism promotes cancer cell proliferation and survival by supporting building block synthesis, producing reducing agents to mitigate oxidative stress, and generating immunosuppressive metabolites for immune evasion. Malignant cells rewire amino acid metabolism to maximize their access to nutrients. Amino acid transporter expression is upregulated to acquire amino acids from the extracellular environment. Under nutrient depleted conditions, macropinocytosis can be activated where proteins from the extracellular environment are engulfed and degraded into the constituent amino acids. The demand for non-essential amino acids (NEAAs) can be met through de novo synthesis pathways. Cancer cells can alter various signaling pathways to boost amino acid usage for the generation of nucleotides, reactive oxygen species (ROS) scavenging molecules, and oncometabolites. The importance of amino acid metabolism in cancer proliferation makes it a potential target for therapeutic intervention, including via small molecules and antibodies. In this review, we will delineate the targets related to amino acid metabolism and promising therapeutic approaches.