Modified Unit-Mediated Strand Displacement Reactions for Direct Detection of Single Nucleotide Variants in Active Double-Stranded DNA.

Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function.

BACKGROUND: Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown. METHODS: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs. RESULTS: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αß T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb. CONCLUSION: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

DNA-based molecular classifiers for the profiling of gene expression signatures.

Although gene expression signatures offer tremendous potential in diseases diagnostic and prognostic, but massive gene expression signatures caused challenges for experimental detection and computational analysis in clinical setting. Here, we introduce a universal DNA-based molecular classifier for profiling gene expression signatures and generating immediate diagnostic outcomes. The molecular classifier begins with feature transformation, a modular and programmable strategy was used to capture relative relationships of low-concentration RNAs and convert them to general coding inputs. Then, competitive inhibition of the DNA catalytic reaction enables strict weight assignment for different inputs according to their importance, followed by summation, annihilation and reporting to accurately implement the mathematical model of the classifier. We validated the entire workflow by utilizing miRNA expression levels for the diagnosis of hepatocellular carcinoma (HCC) in clinical samples with an accuracy 85.7%. The results demonstrate the molecular classifier provides a universal solution to explore the correlation between gene expression patterns and disease diagnostics, monitoring, and prognosis, and supports personalized healthcare in primary care.

Targeted plasma proteomic analysis uncovers a high-performance biomarker panel for early diagnosis of gastric cancer.

BACKGROUND: Gastric cancer (GC) is characterized by high morbidity, high mortality and low early diagnosis rate. Early diagnosis plays a crucial role in radically treating GC. The aim of this study was to identify plasma biomarkers for GC and early GC diagnosis. METHODS: We quantified 369 protein levels with plasma samples from discovery cohort (n = 88) and validation cohort (n = 50) via high-throughput proximity extension assay (PEA) utilizing the Olink-Explore-384-Cardiometabolic panel. The multi-protein signatures were derived from LASSO and Ridge regression models. RESULTS: In the discovery cohort, 13 proteins (GDF15, ITIH3, BOC, DPP7, EGFR, AMY2A, CCDC80, CD163, GPNMB, LTBP2, CTSZ, CCL18 and NECTIN2) were identified to distinguish GC (Stage I-IV) and early GC (HGIN-I) groups from control group with AUC of 0.994 and AUC of 0.998, severally. The validation cohort yielded AUC of 0.930 and AUC of 0.818 for GC and early GC, respectively. CONCLUSIONS: This study identified a multi-protein signature with the potential to benefit clinical GC diagnosis, especially for Asian and early GC patients, which may contribute to the development of a less-invasive, convenient, and efficient early screening tool, promoting early diagnosis and treatment of GC and ultimately improving patient survival.

Rapamycin inhibits B16 melanoma cell viability invitro and invivo by inducing autophagy and inhibiting the mTOR/p70­S6k pathway.

Rapamycin is an immunosuppressant that has been shown to prevent tumor growth following organ transplantation. However, its exact mode of antitumor action remains unknown. The present study used the B16-F10 (B16) murine melanoma model to explore the antitumor mechanism of rapamycin, and it was revealed that rapamycin reduced B16 cell viability in vitro and in vivo. In addition, in vitro and in vivo, the results of western blotting showed that rapamycin reduced Bcl2 expression, and enhanced the protein expression levels of cleaved caspase 3 and Bax, indicating that it can induce the apoptosis of B16 melanoma cells. Furthermore, the results of cell cycle analysis and western blotting showed that rapamycin induced B16 cell cycle arrest in the G1 phase, based on the reduction in the protein expression levels of CDK1, cyclin D1 and CDK4, as well as the increase in the percentage of cells in G1 phase. Rapamycin also significantly increased the number of autophagosomes in B16 melanoma cells, as determined by transmission electron microscopy. Furthermore, the results of RT-qPCR and western blotting showed that rapamycin upregulated the protein expression levels of microtubule-associated protein light chain 3 (LC3) and Beclin-1, while downregulating the expression of p62 in vitro and in vivo, thus indicating that rapamycin could trigger cellular autophagy. The present study revealed that rapamycin in combination with chloroquine (CQ) further increased LC3 expression compared with that in the CQ group, suggesting that rapamycin induced an increase in autophagy in B16 cells. Furthermore, the results of western blotting showed that rapamycin blocked the phosphorylation of p70 ribosomal S6 kinase (p70-S6k) and mammalian target of rapamycin (mTOR) proteins in vitro and in vivo, thus suggesting that rapamycin may exert its antitumor effect by inhibiting the phosphorylation of the mTOR/p70-S6k pathway. In conclusion, rapamycin may inhibit tumor growth by inducing cellular G1 phase arrest and apoptosis. In addition, rapamycin may exert its antitumor effects by inducing the autophagy of B16 melanoma cells in vitro and in vivo, and the mTOR/p70-S6k signaling pathway may be involved in this process.

A Circular RNA Generated from Nebulin (NEB) Gene Splicing Promotes Skeletal Muscle Myogenesis in Cattle as Detected by a Multi-Omics Approach.

Cattle and the draught force provided by its skeletal muscle have been integral to agro-ecosystems of agricultural civilization for millennia. However, relatively little is known about the cattle muscle functional genomics (including protein coding genes, non-coding RNA, etc.). Circular RNAs (circRNAs), as a new class of non-coding RNAs, can be effectively translated into detectable peptides, which enlightened us on the importance of circRNAs in cattle muscle physiology function. Here, RNA-seq, Ribosome profiling (Ribo-seq), and peptidome data are integrated from cattle skeletal muscle, and detected five encoded peptides from circRNAs. It is further identified and functionally characterize a 907-amino acids muscle-specific peptide that is named circNEB-peptide because derived by the splicing of Nebulin (NEB) gene. This peptide localizes to the nucleus and cytoplasm and directly interacts with SKP1 and TPM1, key factors regulating physiological activities of myoblasts, via ubiquitination and myoblast fusion, respectively. The circNEB-peptide is found to promote myoblasts proliferation and differentiation in vitro, and induce muscle regeneration in vivo. These findings suggest circNEB-peptide is an important regulator of skeletal muscle regeneration and underscore the possibility that more encoding polypeptides derived by RNAs currently annotated as non-coding exist.

CUL4B enhances the malignant phenotype of esophageal squamous cell carcinoma by suppressing TGFBR3 expression.

Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through epigenetic mechanisms. However, the expression and function of CUL4B in esophageal squamous cell carcinoma (ESCC) have not been well illustrated. In this study, we show that upregulation of CUL4B in ESCC cells enhances proliferation, invasion and cisplatin (CDDP)-resistance, while knockdown of CUL4B significantly represses the malignant activities. Mechanistically, we demonstrate that CUL4B promotes proliferation and migration of ESCC cells through inhibiting expression of transforming growth factor beta receptor III (TGFBR3). CRL4B complex binds to the promoter of TGFBR3, and represses its transcription by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes. Taken together, our findings establish a critical role for the CUL4B/TGFBR3 axis in the regulation of ESCC malignancy.

Oxymatrine inhibits TGF­ß1­mediated mitochondrial apoptotic signaling in alveolar epithelial cells via activation of PI3K/AKT signaling.

Although pulmonary fibrosis (PF) causes respiratory failure and death, effective therapies for PF have not been developed. Oxymatrine (OMT), an active ingredient in the Chinese herb Sophora flavescens, exerts antifibrotic effects; however, its effect on PF remains unclear. The present study aimed to determine whether OMT decreases transforming growth factor-ß1 (TGF-ß1)-induced PF in human lung cancer A549 cells by inhibiting apoptosis and targeting the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. To construct a PF cell model, A549 cells were stimulated with TGF-ß1. The experimental groups were as follows: control (untreated cells grown in complete medium), TGF-ß1 (cells treated with 5 ng/ml TGF-ß1), OMT (cells treated with 5 ng/ml TGF-ß1 and 0.25, 0.50, or 1.00 mg/ml OMT), and OMT + LY294002 (cells treated with 5 ng/ml TGF-ß1, 1.0 mg/ml OMT. and 25 µmol/l LY294002). The effects of OMT on cell morphology (via electron microscopy), apoptosis (via Annexin V-PI staining), mitochondrial apoptosis signaling [using JC-1 method to analyze mitochondrial membrane potential (MMP)], and Bcl-2, as well as Bax expression (via western blotting and reverse transcription-quantitative polymerase chain reaction), were analyzed. OMT significantly protected cells against TGF-ß1-induced PF by inhibiting apoptosis. The specific manifestations were cell injury, as evidenced by morphological changes and decreased MMP. Following OMT treatment, the expression of the pro-apoptotic protein Bax increased, whereas that of the anti-apoptotic protein Bcl-2 decreased. The PI3K/AKT-specific inhibitor LY294002 significantly inhibited the ameliorative effects of OMT on TGF-ß1-induced apoptosis. Collectively, OMT attenuated TGF-ß1-mediated mitochondrial apoptosis of alveolar epithelial cells by activating the PI3K/AKT signaling pathway. Therefore, OMT may be a promising drug for PF treatment.

Design, Synthesis, Fungicidal and Insecticidal Activities of Novel Diamide Compounds Combining Pyrazolyl and Polyfluoro-Substituted Phenyl into Alanine or 2-Aminobutyric Acid Skeletons.

Thirty novel diamide compounds combining pyrazolyl and polyfluoro-substituted phenyl groups into alanine or 2-aminobutyric acid skeletons were designed and synthesized with pyflubumide as the lead compound to develop potent and environmentally friendly pesticides. The preliminary bioassay results indicated that the new compounds containing the para-hexa/heptafluoroisopropylphenyl moiety exhibit fungicidal, insecticidal, and acaricidal activities. This is the first time that the para-hexa/heptafluoroisopropylphenyl group is a key fragment of the fungicidal activity of new N-phenyl amide compounds. Most of the target compounds exhibited moderate to good insecticidal activity against Aphis craccivora at a concentration of 400 µg/mL, and some showed moderate activity at a concentration of 200 µg/mL; in particular, compounds I-4, II-a-10, and III-26 displayed higher than 78% lethal rates at 200 µg/mL. Compound II-a-14 exhibited a 61.1% inhibition at 200 µg/mL for Tetranychus cinnabarinus. In addition, some of the target compounds exhibited good insecticidal activities against Plutella xylostella at a concentration of 200 µg/mL; the mortalities of compounds I-1, and II-a-15 were 76.7% and 70.0%, respectively. Preliminary analysis of the structure-activity relationship (SAR) indicated that the insecticidal and acaricidal activities varied significantly depending on the type of substituent and substitution pattern. The fungicidal activity results showed that compounds I-1, II-a-10, II-a-17, and III-26 exhibited good antifungal effects. Enzymatic activity experiments and in vivo efficacy of compound II-a-10 were conducted and discussed.

Comprehensive Analysis of PANoptosis-Related Gene Signature of Ulcerative Colitis.

Accumulating evidence shows that the abnormal increase in the mortality of intestinal epithelial cells (IECs) caused by apoptosis, pyroptosis, and necroptosis is closely related to the function of mucous membrane immunity and barrier function in patients with ulcerative colitis (UC). As a procedural death path that integrates the above-mentioned many deaths, the role of PANoptosis in UC has not been clarified. This study aims to explore the characterization of PANoptosis patterns and determine the potential biomarkers and therapeutic targets. We constructed a PANoptosis gene set and revealed significant activation of PANoptosis in UC patients based on multiple transcriptome profiles of intestinal mucosal biopsies from the GEO database. Comprehensive bioinformatics analysis revealed five key genes (ZBP1, AIM2, CASP1/8, IRF1) of PANoptosome with good diagnostic value and were highly correlated with an increase in pro-inflammatory immune cells and factors. In addition, we established a reliable ceRNA regulatory network of PANoptosis and predicted three potential small-molecule drugs sharing calcium channel blockers that were identified, among which flunarizine exhibited the highest correlation with a high binding affinity to the targets. Finally, we used the DSS-induced colitis model to validate our findings. This study identifies key genes of PANoptosis associated with UC development and hypothesizes that IRF1 as a TF promotes PANoptosome multicomponent expression, activates PANoptosis, and then induces IECs excessive death.

Liquid-Liquid Phase Separation in Cardiovascular Diseases.

Liquid-liquid phase separation (LLPS) is a biochemical process in cells that can drive proteins, RNA, and other molecules to concentrate into droplets. These droplets do not have a lipid membrane but rather exist as distinct organelles relative to the surrounding environment, and act as biochemical reaction chambers. In recent years, significant progress has been made in the study of LLPS, especially in the neurodegenerative disease, cancer, and virology fields, but little is known about LLPS in cardiovascular disease (CVD). In this review, we discuss the current understanding of the mechanism and biological functions of LLPS, particularly its roles in regulating CVD.

Outcomes of surgical repair of anomalous origin of the left coronary artery from the pulmonary artery in infants and children.

OBJECTIVES: Anomalous origin of the left coronary artery from the pulmonary artery is associated with high mortality if not timely surgery. We reviewed our experience with anomalous origin of the left coronary artery from the pulmonary artery to assess the preoperative variables predictive of outcome and post-operative recovery of left ventricular function. METHODS: A retrospective review was conducted and collected data from patients who underwent anomalous origin of the left coronary artery from the pulmonary artery repair at our institute from April 2005 to December 2019. Left ventricular function was assessed by ejection fraction and the left ventricular end-diastolic dimension index. The outcomes of reimplantation repair were analysed. RESULTS: A total of 30 consecutive patients underwent anomalous origin of the left coronary artery from the pulmonary artery repair, with a median age of 14.7 months (range, 1.5-59.6 months), including 14 females (46.67%). Surgery was performed with direct coronary reimplantation in 12 patients (40%) and the coronary lengthening technique in 18 (60%). Twelve patients had concomitant mitral annuloplasty. There were two in-hospital deaths (6.67%), no patients required mechanical support, and no late deaths occurred. Follow-up echocardiograms demonstrated significant improvement between the post-operative time point and the last follow-up in ejection fraction (49.43%±19.92% vs 60.21%±8.27%, p < 0.01) and in moderate or more severe mitral regurgitation (19/30 vs 5/28, p < 0.01). The left ventricular end-diastolic dimension index decreased from 101.91 ± 23.07 to 65.06 ± 12.82 (p < 0.01). CONCLUSIONS: Surgical repair of anomalous origin of the left coronary artery from the pulmonary artery has good mid-term results with low mortality and reintervention rates. The coronary lengthening technique has good operability and leads to excellent cardiac recovery. The decision to concomitantly correct mitral regurgitation should be flexible and be based on the pathological changes of the mitral valve and the degree of mitral regurgitation.

Novel Cyclic Endomorphin Analogues with Multiple Modifications and Oligoarginine Vector Exhibit Potent Antinociception with Reduced Opioid-like Side Effects.

Endomorphins (EMs) are potent pharmaceuticals for the treatment of pain. Herein, we investigated several novel EM analogues with multiple modifications and oligoarginine conjugation. Our results showed that analogues 1-6 behaved as potent µ-opioid agonists and enhanced stability and lipophilicity. Analogues 5 and 6 administered centrally and peripherally induced significant and prolonged antinociceptive effects in acute pain. Both analogues also produced long-acting antiallodynic effects against neuropathic and inflammatory pain. Furthermore, they showed a reduced acute antinociceptive tolerance. Analogue 6 decreased the extent of chronic antinociceptive tolerance, and analogue 5 exhibited no tolerance at the supraspinal level. Particularly, they displayed nontolerance-forming antinociception at the peripheral level. In addition, analogues 5 and 6 exhibited reduced or no opioid-like side effects on gastrointestinal transit, conditioned place preference (CPP), and motor impairment. The present investigation established that multiple modifications and oligoarginine-vector conjugation of EMs would be helpful in developing novel analgesics with fewer side effects.

Screening and identification of female-specific DNA sequences in octaploid sturgeon using comparative genomics with high-throughput sequencing.

In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.

Cardiac lipoblastoma of the pulmonary valve: A rare tumor in an unusual location.

BACKGROUD: Lipoblastoma is a rare, benign, fatty tissue tumour tumor that occurs in infancy and early childhood. Intrathoracic and mediastinal involvement of this tumour is rare, and an intracardiac location is even rarer. AIMS: To report the first case of a lipoblastoma in the pulmonary valve. MATERIALS: In this study, we report the case of a 15-month-old girl who presented with a tumour in the pulmonary valve. RESULTS: The tumour was completely resected macroscopically. Histological examination of the lesion confirmed the diagnosis of lipoblastoma. DISCUSSION: Most primary cardiac tumours are benign in the sense they are not invasive. However, benign tumours maintain the potential for serious illness related to significant haemodynamic compromise or life-threatening dysrhythmias. Due to the association of lipoblastoma with other heart malformations, preoperative diagnosis remains challenging. CONCLUSION: Tumours on the heart valve are more likely to cause haemodynamic disturbances. Complete resection yields an excellent prognosis.

Structurally diverse polyprenylated acylphloroglucinols from Hypericum uralum Buch.-Ham. ex D. Don.

Uralins A - D, four undescribed polycyclic polyprenylated acylphloroglucinols (PPAPs) featuring an unprecedented fused hexacyclic architecture, a unique monocyclic tetra-seco-tetranor-b-PPAP, an oxidative b-PPAP and a rare norspiroindane-type m-PPAP, respectively, were isolated from the aerial parts of Hypericum uralum, along with ten known PPAPs. Their structures and absolute configurations were elucidated by extensive spectroscopic techniques (MS, NMR, [α]D, CD), conceivable biogenetic pathways and time-dependent density functional theory-based electronic circular dichroism (TDDFT-ECD) calculations. Biological assays showed three b-PPAPs had moderate antioxidative damage activities, while spiroindanes exhibited moderate cytotoxic effects.

A Novel XGBoost Method to Identify Cancer Tissue-of-Origin Based on Copy Number Variations.

The discovery of cancer of unknown primary (CUP) is of great significance in designing more effective treatments and improving the diagnostic efficiency in cancer patients. In the study, we develop an appropriate machine learning model for tracing the tissue of origin of CUP with high accuracy after feature engineering and model evaluation. Based on a copy number variation data consisting of 4,566 training cases and 1,262 independent validation cases, an XGBoost classifier is applied to 10 types of cancer. Extremely randomized tree (Extra tree) is used for dimension reduction so that fewer variables replace the original high-dimensional variables. Features with top 300 weights are selected and principal component analysis is applied to eliminate noise. We find that XGBoost classifier achieves the highest overall accuracy of 0.8913 in the 10-fold cross-validation for training samples and 0.7421 on independent validation datasets for predicting tumor tissue of origin. Furthermore, by contrasting various performance indices, such as precision and recall rate, the experimental results show that XGBoost classifier significantly improves the classification performance of various tumors with less prediction error, as compared to other classifiers, such as K-nearest neighbors (KNN), Bayes, support vector machine (SVM), and Adaboost. Our method can infer tissue of origin for the 10 cancer types with acceptable accuracy in both cross-validation and independent validation data. It may be used as an auxiliary diagnostic method to determine the actual clinicopathological status of specific cancer.

Risk-adapted stereotactic body radiation therapy for central and ultra-central early-stage inoperable non-small cell lung cancer.

To determine the therapeutic efficacy and safety of risk-adapted stereotactic body radiation therapy (SBRT) schedules for patients with early-stage central and ultra-central inoperable non-small cell lung cancer. From 2006 to 2015, 80 inoperable T1-2N0M0 NSCLC patients were treated with two median dose levels: 60 Gy in six fractions (range, 48-60 Gy in 4-8 fractions) prescribed to the 74% isodose line (range, 58%-79%) for central lesions (ie within 2 cm of, but not abutting, the proximal bronchial tree; n = 43), and 56 Gy in seven fractions (range, 48-60 Gy in 5-10 fractions) prescribed to the 74% isodose line (range, 60%-80%) for ultra-central lesions (ie abutting the proximal bronchial tree; n = 37) on consecutive days. Primary endpoint was overall survival (OS); secondary endpoints included progression-free survival (PFS), tumor local control rate (LC), and toxicity. Median OS and PFS were 64.47 and 32.10 months (respectively) for ultra-central patients, and not reached for central patients. Median time to local failure, regional failure, and any distant failures for central versus ultra-central lesions were: 27.37 versus 26.07 months, 20.90 versus 12.53 months, and 20.85 versus 15.53 months, respectively, all P < .05. Multivariate analyses showed that tumor categorization (ultra-central) and planning target volume ≥52.76 mL were poor prognostic factors of OS, PFS, and LC, respectively (all P < .05). There was one grade 5 toxicity; all other toxicities were grade 1-2. Our results showed that ultra-central tumors have a poor OS, PFS, and LC compared with central patients because of the use of risk-adapted SBRT schedules that allow for equal and favorable toxicity profiles.