Supplementary Material for: L-Carnitine Ameliorates Cancer Cachexia in Mice Partly via the Carnitine Palmitoyltransferase-Associated PPAR-γ Signaling Pathway

<b><i>Background:</i></b>L-Carnitine has been demonstrated to ameliorate cachectic symptoms. In the present study, we sought to investigate the role of the peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling pathway in the ameliorative effects of L-carnitine on cancer cachexia in a colon-26 tumor-bearing mouse model. <b><i>Methods:</i></b> The cachectic mice received L-carnitine (p.o.) or etomoxir (i.p.), or pioglitazone hydrochloride (p.o.) or GW9662 (i.p.). The physiological cachexia parameters, biochemical parameters, and serum cytokines were measured. The expression levels of representative molecules in the PPAR-γ signaling pathway were measured by using quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot analysis. <b><i>Results:</i></b> Oral administration of L-carnitine at 9 mg/kg/day improved the cachexia parameters and biochemical parameters in cancer cachectic mice. The elevated serum concentrations of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) were decreased by L-carnitine. These ameliorative effects of L-carnitine were lessened by the carnitine palmitoyltransferase I (CPT I) inhibitor, etomoxir. The mRNA and protein expression levels of PPAR-α and PPAR-γ were decreased in the livers of cancer cachectic mice and increased after L-carnitine administration, which attenuated the increased mRNA expression levels of sterol-regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS). Similar to pioglitazone, L-carnitine augmented the phosphorylation of PPAR-γ and attenuated the expression levels of phospho-p65 and cyclooxygenase (COX)-2. Additionally, the above-mentioned effects of L-carnitine were reversed by GW9662. <b><i>Conclusion:</i></b>L-Carnitine exerts its ameliorative effects in cancer cachexia in association with the PPAR-γ signaling pathway

Supplementary Material for: Hearing Restoration for Adults with Vestibular Schwannoma in the Only Hearing Ear: Ipsilateral or Contralateral Cochlear Implantation?

<i>Objective:</i> To explore auditory outcomes following cochlear implantation (CI) in patients with vestibular schwannoma (VS) in the only hearing ear. <i>Methods:</i> Three patients, all with a long history of hearing loss on one side and with newly diagnosed VS on the other side, underwent ipsilateral or contralateral CI. Their clinical data were collected retrospectively. Postoperative hearing outcomes were measured during follow-up and compared with the preoperative test results. A thorough search of the English-language literature was performed. <i>Results:</i> Patients 1 and 2 underwent CI in the ipsilateral and contralateral ear, respectively, without tumor removal; patient 3 underwent CI after tumor resection. At the last follow-up, the result of pure-tone audiometry was 25, 45, and 25 dB, respectively. An open-set speech discrimination score was achieved in all 3 patients, with monosyllabic word recognition of 60, 30, and 75%, respectively. Besides the patients included in our study, 28 CI cases with VS in the only hearing ear have been reported up to now. <i>Conclusions:</i> In patients with VS in the only hearing ear, significant hearing deterioration with no obvious tumor growth is a good indication for ipsilateral CI. Long-term deafness in the tumor-free ear is not an absolute contraindication for CI

Supplementary Material for: Identification and Characterization of Long Non-Coding RNAs in Osteogenic Differentiation of Human Adipose-Derived Stem Cells

<strong><em>Background/Aims:</em></strong> Long noncoding RNAs (lncRNAs) play important roles in stem cell differentiation. However, their role in osteogenesis of human adipose-derived stem cells (ASCs), a promising cell source for bone regeneration, remains unknown. Here, we investigated the expression profile and potential roles of lncRNAs in osteogenic differentiation of human ASCs. <b><i>Methods:</i></b> Human ASCs were induced to differentiate into osteoblasts <i>in vitro</i>, <i>and</i> the expression profiles of lncRNAs and mRNAs in undifferentiated and osteogenic differentiated ASCs were obtained by microarray. Bioinformatics analyses including subgroup analysis, gene ontology analysis, pathway analysis and co-expression network analysis were performed. The function of lncRNA <i>H19</i> was determined by <i>in vitro</i> knockdown and overexpression. Quantitative reverse transcription polymerase chain reaction was utilized to examine the expression of selected genes. <b><i>Results:</i></b> We identified 1,460 upregulated and 1,112 downregulated lncRNAs in osteogenic differentiated human ASCs as compared with those of undifferentiated cells (Fold change ≥ 2.0, <i>P</i> < 0.05). Among these, 94 antisense lncRNAs, 85 enhancer-like lncRNAs and 160 lincRNAs were further recognized. We used 12 lncRNAs and 157 mRNAs to comprise a coding-non-coding gene expression network. Additionally, silencing of <i>H19</i> caused a significantly increase in expression of osteogenesis-related genes, including <i>ALPL</i> and <i>RUNX2</i>, while a decrease was observed after <i>H19</i> overexpression. <b><i>Conclusion:</i></b> This study revealed for the first time the global expression profile of lncRNAs involved in osteogenic differentiation of human ASCs and provided a foundation for future investigations of lncRNA regulation of human ASC osteogenesis

Supplementary Material for: The Long Intergenic Noncoding RNA 00707 Promotes Lung Adenocarcinoma Cell Proliferation and Migration by Regulating Cdc42

<b><i>Background/Aims:</i></b> Lung cancer (LC) is a serious disease with high morbidity and mortality. Long noncoding RNAs (lncRNAs) have garnered attention because they participate in diverse human disorders, including cancer. Our study examined the long intergenic noncoding RNA 00707 (LINC00707). The effects of LINC00707 on lung adenocarcinoma (LAD) and molecular mechanisms are unclear. This study is aimed to investigate the role of LINC00707 in the malignant processes of LAD. <b><i>Methods:</i></b> Quantitative reverse transcription PCR (qRT-PCR) was used to examine the expression level of LINC00707 in tissues and cell lines. The association of LINC00707 expression and postoperative prognosis was analyzed by the Kaplan-Meier method and log-rank test. Cell proliferation was evaluated <i>in vitro</i> and <i>in vivo</i>. Transwell assays were performed to examine cell migration. Cell cycle and apoptosis was determined by flow -cytometric and western blot analyses. Microarray analysis was conducted to screen for the downstream target gene Cdc42 of LINC00707, which was identified by qRT-PCR, functional analysis, and rescue experiment. <b><i>Results:</i></b> The expression level of LINC00707 was clearly upregulated in LAD tissues compared to that in corresponding normal tissues. Its overexpression was related to advanced TNM stage, larger tumor size, lymphatic metastasis, and poor prognosis. Functional assays revealed that LINC00707 knockdown repressed LAD cell proliferation both <i>in vitro</i> and <i>in vivo</i>. This process may involve the inducing of G1 arrest and apoptosis. Moreover, cell migration was impaired after LINC00707 inhibition. Microarray analysis and rescue assays suggested that Cdc42 is an important target gene involved in the carcinogenesis of LINC00707. <b><i>Conclusions:</i></b> In summary, LINC00707 is a noncoding oncogene that exerts important regulatory functions in LAD, suggesting its potential as a biomarker in the prognosis and treatment of LAD

Supplementary Material for: Chromosome Nomenclature and Cytological Characterization of Sacred Lotus

<p>Sacred lotus is a basal eudicot plant that has been cultivated in Asia for over 7,000 years for its agricultural, ornamental, religious, and medicinal importance. A notable characteristic of lotus is the seed longevity. Extensive endeavors have been devoted to dissect its genome assembly, including the variety China Antique, which germinated from a 1,300-year-old seed. Here, cytogenetic markers representing the 10 largest megascaffolds, which constitute approximately 70% of the lotus genome assembly, were developed. These 10 megascaffolds were then anchored to the corresponding lotus chromosomes by fluorescence in situ hybridization using these cytogenetic markers, and a set of chromosome-specific cytogenetic markers that could unambiguously identify each of the 8 chromosomes was generated. Karyotyping was conducted, and a nomenclature based on chromosomal length was established for the 8 chromosomes of China Antique. Comparative karyotyping revealed relatively conserved chromosomal structures between China Antique and 3 modern cultivars. Interestingly, significant variations in the copy number of 45S rDNA were detected between China Antique and modern cultivars. Our results provide a comprehensive view on the chromosomal structure of sacred lotus and will facilitate further studies and the genome assembly of lotus.</p

Supplementary Material for: Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

<p><b><i>Aims:</i></b> This work investigated the relative strengths of different <i>bla</i>_SHV promoter-associated sequences and their regulation function in <i>bla</i>_SHV expression and β-lactam resistance. <b><i>Methods:</i></b> Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), <i>tac</i> promoter, and combined fragments of promoter and <i>bla</i>_SHV were separately constructed and transformed into <i>Escherichia coli</i> DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of <i>bla</i>_SHV were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. <b><i>Results:</i></b> The relative strengths were ranked as P-<i>tac</i> > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of <i>gfp</i> and <i>bla</i>_SHV mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain <i>bla</i>_SHV groups. In relation to the different promoter-associated sequences,<i> bla</i>_SHV-ESBLs displayed significantly different change modes of β-lactam resistance compared with <i>bla</i>_{SHV-non-ESBLs}. <b><i>Conclusion:</i></b> The mRNA expression and β-lactam resistance of the <i>bla</i>_SHV showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.</p

Supplementary Material for: Long Noncoding RNA Linc00152 Functions as a Tumor Propellant in Pan-Cancer

<b><i>Background/Aims:</i></b> The oncogenic role of linc00152 in pan-cancer is unclear. <b><i>Methods:</i></b> In this study, RNA-Seq of 33 breast specimens was performed, and the expression of linc00152 was validated by qPCR using 50 paired breast cancer tissues and adjacent normal tissues. This result combined with the expression of linc00152 in pan-cancer was revalidated by Gene Expression Omnibus and The Cancer Genome Atlas data. Next, the oncogenic roles of linc00152 in view of prognosis, chemoresistance, genomic and epigenetic regulation, including DNA methylation and histone modification, potential biological function enrichment, and basic molecular function in pan-cancer, were also evaluated <i>in vitro</i> and <i>in vivo</i>. <b><i>Results:</i></b> Linc00152 is upregulated in pan-cancer, especially in progressive cancer, and the high expression of linc00152 may lead to a worse prognosis and chemoresistance in pan-cancer patients. Amplification, DNA hypomethylation, promoter-like lncRNA characteristics and super-enhancer regulation are the drivers that lead to the upregulation of linc00152 in pan-cancer. Meanwhile, linc00152 was involved in cancer-related pathways, infection and immune response-associated pathways by enriched analysis using TCGA data. Finally, linc00152 was confirmed to promote the proliferation, migration and invasion in MDA-MB-231, SGC-7901 and 786-O. Moreover, RIP and RNA pull-down assays indicated that linc00152 can bind to EZH2 directly. <b><i>Conclusion:</i></b> All of the results indicated that linc00152 acted as an oncogenic propellant from various perspectives, and it may be an effective therapy target in pan-cancer

Supplementary Material for: Clinical features, diagnosis, and treatment of congenital and neonatal tuberculosis: A retrospective study

Introduction: Limited studies have explored the clinical features, treatment and prognosis of neonatal tuberculosis (TB). Here, we attempted to delineate the clinical characteristics of neonatal TB, which may help clinicians further understand this disease. Methods: A retrospective analysis of neonates diagnosed with congenital and/or neonatal TB disease from January 2010 to December 2020 was performed. Information on the demographic and epidemiological features, clinical symptoms, laboratory and imaging examinations, therapeutic regimens, and outcomes was collected. Kaplan–Meier analysis was used to present the time to disease onset, time to diagnosis, etc.. Results: Forty-eight cases of neonatal TB were classified into congenital (n=33) and postnatal (n=15) . The median time to disease onset in postnatal group was significantly longer than that in congenital group. Positive results for gastric fluid acid-fast bacilli, TB culture, Xpert MTB/RIF, IGRA and TST were detected in 26/48 (54.2%), 14/34 (41.2%), 11/18 (61.1%), 19/29 (65.5%), and 8/24 (33.3%) patients, respectively. For lymphadenopathy, CT scans showed a higher detection rate than did X-ray (80.0% vs 0). Of the 48 infants, 44/48 (91.7%) received anti-TB therapy, and 33/44 (75%) were clinically improved or cured after 22.1 months (IQR 12.4–27.7) of follow-up. Drug-induced liver injury occurred in 14/44 (31.8%) patients. Discussion/Conclusion: IGRA and Xpert MTB/RIF showed good positive rate in neonatal TB infection/disease. In cases where TB is presumed but etiological evidence is lacking, low dose CT could be considered. Prompt treatment under careful surveillance is important for preventing mortality and avoiding severe adverse effects

Supplementary Material for: Predictive Value of Four-Dimensional Strain Echocardiography for Adverse Cardiovascular Outcomes in ST-Elevation Myocardial Infarction Patients Treated with Primary Percutaneous Coronary Intervention

<b><i>Objectives:</i></b> To investigate the predictive value of four-dimensional (4D) strain echocardiography for major adverse cardiovascular events (MACE) in ST-elevation acute myocardial infarction (STEMI) patients. <b><i>Methods:</i></b> Consecutive STEMI patients who underwent successful primary coronary interven tion (PCI) were enrolled and followed, with 2D and 4D strain echocardiography performed within 1 week after PCI. <b><i>Results:</i></b> Twenty-six first MACE were recorded in 81 patients who finished a ∼3.0 year follow-up. Compared with those without MACE, subjects with MACE were more likely to have anterior MI (73.08 vs. 38.18%, <i>p</i> = 0.003), significantly decreased 2D left ventricular ejection fraction (2DLVEF) and 4DLVEF (all <i>p</i> < 0.05), as well as an overtly compromised 4D strain parameters. The prediction models incorporating infarct location with either 2DLVEF or 4D strain parameters were then developed. Model comparisons revealed that the global area strain (GAS)-based model had the highest discriminative capacity (c statistics = 0.774) and was well calibrated for MACE. Additionally, the clinical utility of the GAS-based prediction model was verified by decision curve analysis showing a consistent positive and larger net benefit compared to the 2DLVEF-based model. <b><i>Conclusions:</i></b> Our data support a superiority of 4D strain echocardiography over conventional 2D echocardiography, especially GAS, for risk stratification in STEMI patients after successful primary PCI

Supplementary Material for: The Wound Healing of Autologous Regenerative Factor on Recurrent Benign Airway Stenosis: A Canine Experimental and Pilot Study

Introduction: Benign airway stenosis (BAS) is a severe pathologic condition. Complex stenosis has a high recurrence rate and requires repeated bronchoscopic interventions for achieving optimal control, leading to recurrent BAS (RBAS) due to intraluminal granulation. Methods: This study explored the potential of autologous regenerative factor (ARF) for treating RBAS using a post-intubation tracheal stenosis canine model. Bronchoscopic follow-ups were conducted, and RNA-seq analysis of airway tissue was performed. A clinical study was also initiated involving 17 patients with recurrent airway stenosis. Results: In the animal model, ARF demonstrated significant effectiveness in preventing further collapse of the injured airway, maintaining airway patency and promoting tissue regeneration. RNA-seq results showed differential gene expression, signifying alterations in cellular components and signaling pathways. The clinical study found that ARF treatment was well-tolerated by patients with no severe adverse events requiring hospitalization. ARF treatment yielded a high response rate, especially for post-intubation tracheal stenosis and idiopathic tracheal stenosis patients. Conclusion: The study concludes that ARF presents a promising, effective, and less-invasive method for treating RBAS. ARF has shown potential in prolonging the intermittent period and reducing treatment failure in patients with recurrent tracheal stenosis by facilitating tracheal mucosal wound repair and ameliorating tracheal fibrosis. This novel approach could significantly impact future clinical applications