Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.

CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.

Palbociclib inhibits the progression of head and neck cancer and improves the Cetuximab response under specific condition in vitro and in vivo.

BACKGROUND: In head and neck squamous cell carcinoma (HNSCC) with human papillomavirus (HPV)-negative, deregulation of cell cycle is partly due to inactivation of p16INK4 and overexpression of cyclin D1. Here we investigated the in vitro and in vivo effects of the CDK4/6 inhibitor Palbociclib alone or combined with EGFR inhibitor Cetuximab in HNSCC. METHODS AND RESULTS: CCK-8, soft agar assay, colony formation assay, wound healing assay and transwell assay, ß-galactosidase assay, western blotting, and cell-derived xenografts were used to investigated the in vitro and in vivo activity of drugs. Cell viability and colony formation decreased in a dose-dependent manner in Tu686, AMC-HN8, and Fadu cells under Palbociclib treatment. Palbociclib remarkably inhibited migration, invasion and the expression MMP-9 in HNSCC cells. Palbociclib also induced senescence. Palbociclib caused the dephosphorylation of RB but increased the cyclin D1 level in a dose-dependent manner. Moreover, combination with Cetuximab could significantly prevent the induction of cyclin D1 and activation of EGFR signals from Palbociclib treatment. Nevertheless, only within the range of certain concentrations, a synergistic inhibitory effect on cell growth was observed when combined with Palbociclib and Cetuximab. Although the synergistic effect in Palbociclib/Cetuximab combined therapy was comparable to that in traditional chemotherapeutic regimens (cisplatin/Cetuximab) in Fadu tumor xenograft, the combination therapy was less active than Cetuximab monotherapy in Tu686 tumor xenograft. CONCLUSION: In HPV-negative HNSCC, CDK4/6 inhibitor shows promising anti-tumor effects, which exhibits a synergistic effect when combined with EGFR inhibitor only in specific drug concentration and mouse model.

Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18.

Mobile genetic elements such as phages and plasmids have evolved anti-CRISPR proteins (Acrs) to suppress CRISPR-Cas adaptive immune systems. Recently, several phage and non-phage derived Acrs including AcrIIA17 and AcrIIA18 have been reported to inhibit Cas9 through modulation of sgRNA. Here, we show that AcrIIA17 and AcrIIA18 inactivate Cas9 through distinct mechanisms. AcrIIA17 inhibits Cas9 activity through interference with Cas9-sgRNA binary complex formation. In contrast, AcrIIA18 induces the truncation of sgRNA in a Cas9-dependent manner, generating a shortened sgRNA incapable of triggering Cas9 activity. The crystal structure of AcrIIA18, combined with mutagenesis studies, reveals a crucial role of the N-terminal ß-hairpin in AcrIIA18 for sgRNA cleavage. The enzymatic inhibition mechanism of AcrIIA18 is different from those of the other reported type II Acrs. Our results add new insights into the mechanistic understanding of CRISPR-Cas9 inhibition by Acrs, and also provide valuable information in the designs of tools for conditional manipulation of CRISPR-Cas9.

Target RNA-guided protease activity in type III-E CRISPR-Cas system.

The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.

Number of positive lymph nodes and lymph node ratio predict recurrence and survival in hypopharyngeal cancer based on SEER database and validation of real-world data.

PURPOSE: This study investigated the impacts of the number of positive lymph nodes (NPLN) and lymph node ratio (LN ratio) for patients with hypopharyngeal squamous cell carcinoma (HPSCC) based on SEER database, which were validated in the real-world data of China. METHODS: A total of 520 patients from SEER database were analyzed. Then 195 patients with pathologically stage III or IV HPSCC in our center were retrospectively studied. RESULTS: In the SEER database, NPLN ≥ 3 was found in 36.9% of patients. Multivariate analysis revealed that LN ratio ≥ 0.138 was significant with poorer overall survival (OS) (hazard ratio [HR] = 1.525, p = 0.001) and cancer-specific survival (CSS) (HR = 1.697, p < 0.001), so was the NPLN ≥ 3 (HR = 1.388, p = 0.013; HR = 1.479, p = 0.008). Patients with NPLN ≥ 3 were found in 103 (52.8%) in our center. Multivariate analysis confirmed a significant association regarding OS (p = 0.005) or CSS (p = 0.003) between patients with LN ratio ≥ 0.138 or not. In addition, disease recurrence rate differed significantly between the patients with NPLN ≥ 3 (27.2%) and NPLN < 3 (14.1%, p = 0.026). Moreover, postoperative chemoradiotherapy (CCRT) was significantly associated with better prognosis in patients with NPLN ≥ 3. CONCLUSION: In the SEER database, NPLN ≥ 3 and LN ratio ≥ 0.138 were independent poor prognostic factors for patients with HPSCC. Whereas identifying worldwide cut-off values for LN ratio is difficult and surgeon-dependent. In our cohort, adjuvant CCRT was beneficial for OS in patients with NPLN ≥ 3.

Application of MR images in radiotherapy planning for brain tumor based on deep learning.

PURPOSE: Explore the function and dose calculation accuracy of MRI images in radiotherapy planning through deep learning methods. METHODS: 131 brain tumor patients undergoing radiotherapy with previous MR and CT images were recruited for this study. A new series of MRI from the aligned MR was firstly registered to CT images strictly using MIM software and then resampled. A deep learning method (U-NET) was used to establish a MRI-to-CT conversion model, for which 105 patient images were used as the training set and 26 patient images were used as the tuning set. Data from additional 8 patients were collected as the test set, and the accuracy of the model was evaluated from a dosimetric standpoint. RESULTS: Comparing the synthetic CT images with the original CT images, the difference in dosimetric parameters D98, D95, D2 and Dmean of PTV in 8 patients was less than 0.5%. The gamma passed rates of PTV and whole body volume were: 1%/1 mm: 93.96%±6.75%, 2%/2 mm: 99.87%±0.30%, 3%/3 mm: 100.00%±0.00%; and 1%/1 mm: 99.14%±0.80%, 2%/2 mm: 99.92%±0.08%, 3%/3 mm: 99.99%±0.01%. CONCLUSION: MR images can be used both in delineation and treatment efficacy evaluation and in dose calculation. Using the deep learning way to convert MR image to CT image is a viable method and can be further used in dose calculation.

Nucleic Acid Detection through RNA-Guided Protease Activity in Type III-E CRISPR-Cas Systems.

RNA-guided protease activity was recently discovered in the type III-E CRISPR-Cas systems (Craspase), providing a novel platform for engineering a protein probe instead of the commonly used nucleic acid probe in nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity- and fluorescent protein dual-tagged Csx30 protein substrate, we have established an assay monitoring Csx30 cleavage by target ssRNA-activated Craspase. Four Craspase-based nucleic acid detection systems for genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, and the influenza virus (IFV) were reconstituted with demonstrated specificity. The assay could reliably detect target ssRNAs at concentrations down to 25 pM, which could be further improved approximately 15 000-fold (ca. 2 fM) by incorporating a recombinase polymerase isothermal preamplification step. Importantly, the species-specific substrate cleavage specificity of Craspase enabled multiplexed diagnosis, as demonstrated by the reconstituted composite systems for simultaneous detection of two genes from the same virus (SARS-CoV-2, spike and nsp12) or two types of viruses (SARS-CoV-2 and IFV). The assay could be further expanded by diversifying the fluorescent tags in the substrate and including Craspase systems from various species, thus potentially providing an easily adaptable platform for clinical diagnosis.

Is radical radiotherapy with/without surgery an effective treatment in the lymphoepithelial carcinoma of the salivary gland？.

BACKGROUND: There is limited information of radical radiotherapy (RT) on lymphoepithelial carcinoma of salivary gland (LECSG) regarding to the rarity of the disease. We conducted this retrospective study that evaluated the feasibility and efficacy of radical RT with/without surgery in LECSG. METHODS: We retrospectively reviewed patients that were pathologically diagnosed of LECSG and had definite or suspicious residual disease. The prescribed dose given to P-GTV and/or P-GTV-LN was 66 to 70.4 Gy. The clinical target volume (CTV) involved ipsilateral salivary gland and corresponding lymph node drainage area. RESULTS: A total of 56 patients were included. With a median follow-up of 60 months (range: 8 to 151 months), the 1-, 5-, and 10-year progression-free survival (PFS) rates were 94.6%, 84.7% and 84.7%; locoregional progression-free survival (LRPFS) rates were 98.2%, 87.4% and 87.4%; distance metastasis-free survival (DMFS) rates were 94.6%, 86.7% and 86.7%; and overall survival (OS) rates were 98.2%, 92.4% and 89.0%, respectively. A total of 7 patients without surgery were included. All patients were alive and only one patient experienced failure of distant metastasis four months after RT. The results of univariate analysis showed that compared with N stage, the number of positive lymph nodes (2 positive lymph nodes) was better prognostic predictor especially in PFS. There were no treatment-related deaths and most toxicities of RT were mild. CONCLUSIONS: Radical RT with/without surgery in LECSG for definite or suspicious residual disease is feasibility and efficacy. Most toxicities of RT were mild due to the target volume involved ipsilateral area.

Prognostic value of post-radiotherapy neutrophil-to-lymphocyte ratio in locally advanced nasopharyngeal carcinoma.

PURPOSE: To explore the temporal profile of the peripheral neutrophil-to-lymphocyte ratio (NLR) in patients with locally advanced nasopharyngeal carcinoma (LANPC) and the potential prognostic value of its dynamic changes. METHODS: Complete blood count of 112 patients from a previous phase II study were retrospectively collected at the timepoints of the initiation of induction chemotherapy (pre-IC), within 1 week before radiotherapy started (pre-RT), and within 1 week after radiotherapy finished (post-RT). Data of 103 patients were fully recorded and Cox regression analysis was used to analyze the correlations of potential risk factors with 5­year overall survival (OS). The performance of the prognostic factor was validated in another independent cohort of 103 matched (by T and N stage) patients selected from 236 consecutive NPC patients treated with IC and concurrent chemoradiation. RESULTS: Multivariate analysis (MVA) identified patient age >50 years old (hazard ratio [HR] = 3.4, p = 0.02), weight loss during RT >7.5% (HR = 3.2, p = 0.03), and post-RT peripheral NLR >7.05 (vs. NLR ≤7.05, HR = 2.5, p = 0.04, 5­year OS 71.4% vs. 87.8%) as unfavorable prognostic factors for OS. There was also a non-significant trend in the MVA that patients with post-RT peripheral NLR >7.05 showed worse progression-free survival (PFS; HR = 1.9, p = 0.06, 5­year PFS 64.1% vs. 81.8%). Post-RT NLR had a good prognostic performance in the validation cohort (concordance index = 0.73, standard error 0.10; p = 0.02, Wilcoxon test). CONCLUSION: Post-RT NLR is an independent prognostic factor for OS in LANPC patients. The dynamic change of the routinely tested inflammatory variable could help selection of appropriate treatment options and follow-up strategies.

[Automatic segmentation of head and neck organs at risk based on three-dimensional U-NET deep convolutional neural network].

The segmentation of organs at risk is an important part of radiotherapy. The current method of manual segmentation depends on the knowledge and experience of physicians, which is very time-consuming and difficult to ensure the accuracy, consistency and repeatability. Therefore, a deep convolutional neural network (DCNN) is proposed for the automatic and accurate segmentation of head and neck organs at risk. The data of 496 patients with nasopharyngeal carcinoma were reviewed. Among them, 376 cases were randomly selected for training set, 60 cases for validation set and 60 cases for test set. Using the three-dimensional (3D) U-NET DCNN, combined with two loss functions of Dice Loss and Generalized Dice Loss, the automatic segmentation neural network model for the head and neck organs at risk was trained. The evaluation parameters are Dice similarity coefficient and Jaccard distance. The average Dice Similarity coefficient of the 19 organs at risk was 0.91, and the Jaccard distance was 0.15. The results demonstrate that 3D U-NET DCNN combined with Dice Loss function can be better applied to automatic segmentation of head and neck organs at risk.

IMRT combined with S-1 concurrent chemoradiotherapy in locally advanced nasopharyngeal carcinoma: a prospective phase II study.

Purpose The current standard treatment for locally advanced nasopharyngeal carcinoma (LANPC) is intensity-modulated radiation therapy (IMRT) plus cisplatin concurrent chemoradiotherapy (CCRT). However, this regimen has well-known hematological and gastrointestinal toxicities. Many studies have reported that S-1 was effective in the treatment of multiple solid cancers with mild toxicities. However, knowledge regarding IMRT plus S-1 CCRT in LANPC is lacking. Therefore, we conducted this prospective phase II trial to evaluate the efficacy and safety of this regimen in LANPC. Patients and Methods Eligible patients with histologically confirmed LANPC were enrolled in this study. IMRT was given in 30-32 fractions five times per week. Concurrently, S-1 was administrated twice per day orally based on the body surface area (BSA < 1.25 m2, 30 mg; BSA: 1.25-1.5 m2, 40 mg; BSA > 1.5 m2, 50 mg). The primary endpoints were progression-free survival (PFS) and adverse events. Results From August 1, 2013, to December 15, 2017, 131 patients were enrolled in this study. The distribution of disease stages among the patients was as follows: 21 patients were in stage II (16.0%), 42 patients were in stage III (32.0%), and 68 patients were in stage IV (52.0%). After CCRT, the 3-year PFS, overall survival (OS), local recurrence-free survival (LRFS) and distant metastasis-free survival (DMFS) rates were 87.4%, 95.7%, 94.7%, and 91.5%, respectively. The severity of most toxicities was mild. Approximately two-thirds of patients had no hematological toxicity. Grade 2 hematological toxicities included leukopenia (11.5%), anemia (1.5%), and thrombocytopenia (0.8%). Grade 3 hematological toxicities were rarely observed. Conclusion The results demonstrated that IMRT plus S-1 CCRT was effective with mild toxicity for patients with LANPC.

Subgroups of parotid gland infiltrating ductal carcinoma benefit from postoperative radiotherapy: a population-based study.

AIM: To analyze the prognostic factors and the impact of postoperative radiotherapy (PORT) in parotid gland infiltrating ductal carcinoma (IDC). MATERIALS & METHODS: 252 patients diagnosed with parotid gland IDC were identified from the SEER database. Kaplan-Meier and Cox regression analysis was performed to evaluate the prognostic factors. Propensity score matching was applied then. RESULTS: Multivariate analysis showed old age and chemotherapy were independent risk factors in parotid gland IDC. Subgroup analysis demonstrated that overall survival (OS) rate of the PORT group was significantly superior to that of the no radiotherapy group in the T3-4 subgroup (p = 0.049), N1 subgroup (p = 0.019) and Tumor, Node, Metastasis (TNM) III subgroup (p = 0.025). CONCLUSION: PORT improved survival of parotid gland IDC patients within T3-4, N1 and TNM III subgroups.

Prognostic values of hematological biomarkers in nasopharyngeal carcinoma patients treated with intensity-modulated radiotherapy.

PURPOSE: In this study, we evaluated the prognostic values of hematological biomarkers in primary nasopharyngeal carcinoma (NPC) patients receiving definitive intensity-modulated radiotherapy (IMRT). METHODS: There were 427 NPC patients enrolled between January 2010 and March 2013 at Fudan University Shanghai Cancer Center. Pre-treatment absolute neutrophil count (ANC), platelet count (APC), lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were collected as prognostic biomarkers. The Kaplan-Meier method and log-rank test were utilized to calculate progression-free survival (PFS) and overall survival (OS). The Cox proportional hazard models were applied to assess variables. RESULTS: ANC, APC and ALC were declined, while NLR and PLR were elevated significantly after therapy (P < 0.001 each). On multivariate analysis, pre-treatment NLR ≥ 2.32 was associated with shortened OS (P = 0.048) and PFS (P = 0.008), whereas PLR ≥ 123.0 was related with inferior OS (P = 0.032), yet it was not correlated with PFS (P = 0.161). CONCLUSIONS: High pre-treatment NLR and PLR indicated poor survival in NPC patients treated with IMRT-based therapy. As easily accessible and economically feasible biomarkers, NLR and PLR can be applied into clinical practice, in combination with current TNM staging, to design a more personalized treatment in these patients.

EZH2 suppresses the nucleotide excision repair in nasopharyngeal carcinoma by silencing XPA gene.

The enhancer of zeste homolog 2 (EZH2) is involved in a number of fundamental pathological processes of cancer. However, its role in DNA repair pathway is still unclear. Here, we have identified XPA as a novel target gene of EZH2 via a DNA repair pathway PCR array. XPA plays a pivot role in nucleotide excision repair (NER). The expression of XPA was significantly increased by EZH2 specific inhibitor GSK126 or lentiviral shEZH2 in nasopharyngeal carcinoma (NPC) CNE and 8F cell lines. Chromatin immunoprecipitation assay demonstrated that EZH2 catalyzes H3K27 trimethylation at the XPA promoters. Furthermore, we validated the negative correlation of EZH2 and XPA in a NPC tissue microarray by immunohistochemistry staining. We also found that high expression of EZH2 was positively correlated with advanced T, N, and AJCC stage of NPC; and low expression of XPA was positively correlated with advanced T and N stage. In NPC cell lines, increased XPA expression by EZH2 inhibition resulted in a more rapid removal of UVC induced 6-4PP- and CPD-DNA adducts, as well as enhanced efficiency of DNA repair after UVC irradiation as detected by the Comet assay and immunofluorescence staining of γH2Ax. Consistently, increased cell clonogenic survival, decreased apoptosis, and necrosis after UVC irradiation, and increased resistance to DNA damaging agent cisplatin was also observed in EZH2 inhibited cells. These results illustrate that EZH2 may promote carcinogenesis and cancer development of NPC by transcriptional repression of XPA gene and inactivation of NER pathway. © 2016 Wiley Periodicals, Inc.

Dose Evaluation of Fractionated Schema and Distance From Tumor to Spinal Cord for Spinal SBRT with Simultaneous Integrated Boost: A Preliminary Study.

BACKGROUND This study investigated and quantified the dosimetric impact of the distance from the tumor to the spinal cord and fractionation schemes for patients who received stereotactic body radiation therapy (SBRT) and hypofractionated simultaneous integrated boost (HF-SIB). MATERIAL AND METHODS Six modified planning target volumes (PTVs) for 5 patients with spinal metastases were created by artificial uniform extension in the region of PTV adjacent spinal cord with a specified minimum tumor to cord distance (0-5 mm). The prescription dose (biologic equivalent dose, BED) was 70 Gy in different fractionation schemes (1, 3, 5, and 10 fractions). For PTV V100, Dmin, D98, D95, and D1, spinal cord dose, conformity index (CI), V30 were measured and compared. RESULTS PTV-to-cord distance influenced PTV V100, Dmin, D98, and D95, and fractionation schemes influenced Dmin and D98, with a significant difference. Distances of ≥2 mm, ≥1 mm, ≥1 mm, and ≥0 mm from PTV to spinal cord meet dose requirements in 1, 3, 5, and 10 fractionations, respectively. Spinal cord dose, CI, and V30 were not impacted by PTV-to-cord distance and fractionation schemes. CONCLUSIONS Target volume coverage, Dmin, D98, and D95 were directly correlated with distance from the spinal cord for spine SBRT and HF-SIB. Based on our study, ≥2 mm, ≥1 mm, ≥1 mm, and ≥0 mm distance from PTV to spinal cord meets dose requirements in 1, 3, 5 and 10 fractionations, respectively.

[Evaluation of the EDOSE for IMRT Delivery Quality Assurance].

Objective: Evaluation of intensity-modulated radiation therapy(IMRT) delivery quality assurance(DQA) using EDOSE software based on accelerator electronic portal imaging device (EPID). Methods: Analysed the dose response uniformity correction of EPID imaging plate in different fields and the gamma pass rates about the fields; And compared the calculated parameters of the EDOSE and the Pinnacle planning station. Results: The uniformities on the homogeneous region of thefi elds are good after who corrected by EDOSE. The gamma(γ, 3mm/3%) pass rates are in the ideal range. The volume of the organs at risk(OAR) have no statistical signifi cance except the right lung; A part of the parameters have statistical signifi cance. Conclusions: The results of the calculation and analysis of EDOSE has a high agreement with the planning system.

[Results of Five-year Quality Control Program and the Change of QA Mode for Helical Tomotherapy Machine].

Helical tomotherapy (HT), as a new IMRT technology, utilizes a fan beam of radiation for treatment. It combines the main characteristics of a modern CT scanner and a linear accelerator to achieve the function of helical tomotherapy. Due to the complexity of the system with a highly integrated and automated features, monitoring its operation, continuing to improve the stability and reliability, and simplifying its quality control procedures has become an important part of quality assurance (QA) for HT. Based on the results of afi ve-year quality control (QC) program, and the initial application of new QA equipment, this study wil summarize the standardization mode of its QA and explore the changes of QA mode.

[Evaluation of Cone Beam CT Image Quality and Analysis of Its Long-term Stability on HU Values under Different Conditions].

Objective: To evaluate the cone beam CT (CBCT) image quality and its long-term stability for HUs under different scanning conditions. Methods: With the OBI system, Catphan 504 phantom using the different modes was scanned through one year. Analysis of variance (ANOVA) was used to evaluate the CBCT images. Results: The results of contrast resolution under different scanning conditions met the requirements of acceptance test procedure. Spatial contrast resolution(≥5 lp/cm) of half-fan scanning conditions were inferior to those (≥7 lp/cm) of ful-fan mode. The accuracies of spatial linear distance and angle were within±1%. HUs in air density plug were constant, but HUs of other plugs with different conditions had the signifi cant difference (F=660.84,P=0). Under different conditions in a year, the mean HUs showed a good agreement. Conclusions: HUs of CBCT image under different scanning conditions exist a little difference, but there is a good consistency for the long term stability analysis. It is helpful for CBCT images directly used for dose re-calculation in adaptive radiation therapy (ART).

Insights into the modulation of bacterial NADase activity by phage proteins.

The Silent Information Regulator 2 (SIR2) protein is widely implicated in antiviral response by depleting the cellular metabolite NAD+. The defense-associated sirtuin 2 (DSR2) effector, a SIR2 domain-containing protein, protects bacteria from phage infection by depleting NAD+, while an anti-DSR2 protein (DSR anti-defense 1, DSAD1) is employed by some phages to evade this host defense. The NADase activity of DSR2 is unleashed by recognizing the phage tail tube protein (TTP). However, the activation and inhibition mechanisms of DSR2 are unclear. Here, we determine the cryo-EM structures of DSR2 in multiple states. DSR2 is arranged as a dimer of dimers, which is facilitated by the tetramerization of SIR2 domains. Moreover, the DSR2 assembly is essential for activating the NADase function. The activator TTP binding would trigger the opening of the catalytic pocket and the decoupling of the N-terminal SIR2 domain from the C-terminal domain (CTD) of DSR2. Importantly, we further show that the activation mechanism is conserved among other SIR2-dependent anti-phage systems. Interestingly, the inhibitor DSAD1 mimics TTP to trap DSR2, thus occupying the TTP-binding pocket and inhibiting the NADase function. Together, our results provide molecular insights into the regulatory mechanism of SIR2-dependent NAD+ depletion in antiviral immunity.

International Recommendations on Postoperative Management for Potentially Resectable Locally Recurrent Nasopharyngeal Carcinoma.

PURPOSE: Locally recurrent nasopharyngeal carcinoma (NPC) presents substantial challenges in clinical management. While postoperative re-irradiation (re-RT) has been acknowledged as a potential treatment option, standardized guidelines and consensus regarding the use of re-RT in this context are lacking. This article provides a comprehensive review and summary of international recommendations on postoperative management for potentially resectable locally recurrent NPC, with a special focus on postoperative re-RT. METHODS AND MATERIALS: A thorough search was conducted to identify relevant studies on postoperative re-RT for locally recurrent NPC. Controversial issues, including resectability criteria, margin assessment, indications for postoperative re-RT, and the optimal dose and method of re-RT, were addressed through a Delphi consensus process. RESULTS: The consensus recommendations emphasize the need for a clearer and broader definition of resectability, highlighting the importance of achieving clear surgical margins, preferably through an en bloc approach with frozen section margin assessment. Furthermore, these guidelines suggest considering re-RT for patients with positive or close margins. Optimal postoperative re-RT doses typically range around 60Gy, and hyperfractionation has shown promise in reducing toxicity. CONCLUSION: These guidelines aim to assist clinicians in making evidence-based decisions and improving patient outcomes in the management of potentially resectable locally recurrent NPC. By addressing key areas of controversy and providing recommendations on resectability, margin assessment, and re-RT parameters, these guidelines serve as a valuable resource for the clinical experts involved in the treatment of locally recurrent NPC. SUMMARY: This article provides international recommendations on postoperative management for potentially resectable locally recurrent nasopharyngeal carcinoma (NPC), with a special focus on postoperative re-irradiation (re-RT). The consensus guidelines highlight the importance of achieving clear surgical margins, suggest considering re-RT for patients with positive or close margins, recommend an optimal re-RT dose of around 60Gy, and propose the use of hyperfractionation to reduce toxicity. The aim is to improve patient outcomes in the management of resectable locally recurrent NPC.