Is clinical target volume necessary for locally advanced non-small cell lung cancer treated with 4D-CT intensity-modulated radiation therapy.

BACKGROUND: A dosimetric evaluation is still lacking in terms of clinical target volume (CTV) omission in stage III patients treated with 4D-CT Intensity-Modulated Radiation Therapy (IMRT). METHODS: 49 stage III NSCLC patients received 4D-CT IMRT were reviewed. Target volumes and organs at risk (OARs) were re-delineated. Four IMRT plans were conducted retrospectively to deliver different prescribed dose (74 Gy-60 Gy), and with or without CTV implementation. Dose and volume histogram (DVH) parameters were collected and compared. RESULTS: In the PTV-g 60 Gy plan (PTV-g refers to the PTV generated from the internal gross tumor volume), only 5 of 49 patients had the isodose ≥ 50 Gy line covering at least 95% of the PTV-c (PTV-c refers to the PTV generated from the internal CTV) volume. When the prescribed dose was elevated to 74 Gy to the PTV-g, 33 of 49 patients could have the isodose ≥ 50 Gy line covering at least 95% of the PTV-c volume. In terms of OARs protection, the SIB-IMRT plan showed the lowest value of V5, V20, and mean dose of lung, had the lowest V55 of esophagus, and the lowest estimated radiation doses to immune cells (EDIC). The V20, V30, and mean dose of heart was lower in the simultaneous integrated boost (SIB) IMRT (SIB-IMRT) plan than that of the PTV-c 60 Gy plan. CONCLUSIONS: CTV omission was not suitable for stage III patients when the prescribed dose to PTV-g was 60 Gy in the era of 4D-CT IMRT. CTV omission plus high dose to PTV-g (74 Gy for example) warranted further exploration. The SIB-IMRT plan had the best protection to normal tissue including lymphocytes, and might be the optimal choice.

Long-term follow-up of protective effects on salivary and swallowing structures and improvement of late xerostomia and dysphagia by level IIb optimisation in clinical target volume of nasopharyngeal carcinoma.

BACKGROUND: This study aimed to assess the long-term effect of level IIb clinical target volume (CTV) optimisation on survival, xerostomia, and dysphagia in patients with nasopharyngeal carcinoma (NPC). METHODS: Clinical data of 415 patients with NPC treated with intensity-modulated radiotherapy between December 2014 and October 2018 were retrospectively analysed. The patients were categorised into modified and comparison groups. Late xerostomia and dysphagia were evaluated using Radiation Therapy Oncology Group/European Organisation for Research and Treatment of Cancer scoring. Survival analysis was performed using the Kaplan-Meier method. Differences in late toxicity and dose parameters between both groups were compared. Prognostic factors for survival and late toxicity were assessed using regression analyses. RESULTS: Patients in the modified group developed late xerostomia and dysphagia less frequently than those in the comparison group did (P < 0.001). The mean dose (Dmean) and V26 of parotid glands; Dmean and V39 of submandibular glands; and Dmean of sublingual glands, oral cavity, larynx, and superior, middle, and lower pharyngeal constrictor muscles were lower in the modified group than those in the comparison group (all P < 0.001). Both groups had no significant differences in overall, local recurrence-free, distant metastasis-free, or progression-free survival. The Dmean of the parotid and sublingual glands was a risk factor for xerostomia. The Dmean of the parotid and sublingual glands and middle pharyngeal constrictor muscle was a risk factor for dysphagia. CONCLUSIONS: Level IIb optimisation in NPC patients who meet certain criteria specially the exclusion of positive retropharyngeal nodes treated with intensity-modulated radiotherapy has the potential to better protect the salivary and swallowing structures, decreasing the development of late radiation-induced xerostomia and dysphagia while maintaining long-term survival.

A deep learning-based 3D Prompt-nnUnet model for automatic segmentation in brachytherapy of postoperative endometrial carcinoma.

PURPOSE: To create and evaluate a three-dimensional (3D) Prompt-nnUnet module that utilizes the prompts-based model combined with 3D nnUnet for producing the rapid and consistent autosegmentation of high-risk clinical target volume (HR CTV) and organ at risk (OAR) in high-dose-rate brachytherapy (HDR BT) for patients with postoperative endometrial carcinoma (EC). METHODS AND MATERIALS: On two experimental batches, a total of 321 computed tomography (CT) scans were obtained for HR CTV segmentation from 321 patients with EC, and 125 CT scans for OARs segmentation from 125 patients. The numbers of training/validation/test were 257/32/32 and 87/13/25 for HR CTV and OARs respectively. A novel comparison of the deep learning neural network 3D Prompt-nnUnet and 3D nnUnet was applied for HR CTV and OARs segmentation. Three-fold cross validation and several quantitative metrics were employed, including Dice similarity coefficient (DSC), Hausdorff distance (HD), 95th percentile of Hausdorff distance (HD95%), and intersection over union (IoU). RESULTS: The Prompt-nnUnet included two forms of parameters Predict-Prompt (PP) and Label-Prompt (LP), with the LP performing most similarly to the experienced radiation oncologist and outperforming the less experienced ones. During the testing phase, the mean DSC values for the LP were 0.96 ± 0.02, 0.91 ± 0.02, and 0.83 ± 0.07 for HR CTV, rectum and urethra, respectively. The mean HD values (mm) were 2.73 ± 0.95, 8.18 ± 4.84, and 2.11 ± 0.50, respectively. The mean HD95% values (mm) were 1.66 ± 1.11, 3.07 ± 0.94, and 1.35 ± 0.55, respectively. The mean IoUs were 0.92 ± 0.04, 0.84 ± 0.03, and 0.71 ± 0.09, respectively. A delineation time < 2.35 s per structure in the new model was observed, which was available to save clinician time. CONCLUSION: The Prompt-nnUnet architecture, particularly the LP, was highly consistent with ground truth (GT) in HR CTV or OAR autosegmentation, reducing interobserver variability and shortening treatment time.

Clinical research of the value of high-risk CTV setting on intensity-modulated radiotherapy for stage IIB-IVA cervical cancer.

BACKGROUND: This study aims to evaluate the clinical efficacy and side effects of setting up a high-risk clinical target volume (CTV-hr) alongside simultaneous integrated boost intensity-modulated radiotherapy (IMRT-SIB) in patients diagnosed with stage IIB-IVA cervical cancer. METHODS: This study retrospectively analysed patients with stage IIB-IVA cervical cancer who received radical radiotherapy at the Affiliated Hospital of Qingdao University between November 2014 and September 2019. The patients were divided into experimental and control groups based on whether CTV-hr was set. All patients received a combined treatment of radiotherapy and chemotherapy. The dosage for paclitaxel was 135 mg/m2, while for cisplatin it was 75 mg/m2 or for carboplatin it was AUC 4-6, given in a cycle of 21 days. Radiotherapy (RT) included external beam radiation therapy (EBRT) and intracavitary brachytherapy (ICBT). In the control group, positive lymph nodes (GTV-n) were treated at a dose of 58-62 Gy/26-28 fractions(f), while clinical target volumes (CTV) were treated with a dose of 46-48 Gy/26-28f. The experimental group received a simultaneous integrated boost (SIB) to CTV-hr at a dose of 54-56 Gy/26-28f, with the same CTV and GTV-n as the control group. Both groups were combined with brachytherapy with a total dose (EQD2, the equivalent dose in 2 Gy/f) of 80-90 Gy. The study measured objective remission rate (ORR), 3-year progression-free survival (PFS) rate, 3-year overall survival (OS) rate, recurrence rate, and side effects as endpoints. RESULTS: The study enrolled 217 patients, with 119 in the experimental group and 98 in the control group. Results showed that the experimental group had a higher 3-year OS rate (87.4% vs. 71.4%, p = 0.001) and 3-year PFS rate (72.3% vs. 51.0%, p = 0.000) compared to the control group. Additionally, the experimental group had significantly lower rates of overall recurrence (26.1% vs. 50.0%, p = 0.003), in-field recurrence (15.1% vs. 36.7%, p = 0.000), and out-field recurrence(13.4% vs. 35.7%, p = 0.000) compared to the control group. All observed differences were found to be statistically significant. However, the experimental and control groups had no statistically significant difference in ORR and radiological side effects, such as radiation cystitis and enteritis (p > 0.05). CONCLUSIONS: Setting CTV-hr and performing IMRT-SIB on patients with stage IIB-IVA cervical cancer effectively increased the 3-year OS rate, 3-year PFS rate and reduced recurrence rate, with no significant differences in side effects.

CTV Based Sensor for the Detection of Ni2+ Ions With Real Sample Analysis Based on Mechanism of Fluorescence Along with Computational Insights.

Identification and detection of harmful contaminants such as nickel and other materials from soil and water is critical necessity at the present moment. So with this motive to detect and identify harmful pollutants, a novel cyclotriveratrylene based derivative was prepared for the detection and binding of harmful pollutants which had the properties of fluorescence. The newly derivative of Cyclotriveratrylene was found to be highly sensitive and selective towards Ni2+ ions. The complexation behaviour of this newly synthesised molecule was studied in presence of transition elements. Also computational methods such as docking, molecular modelling and DFT were used to study the molecular orbitals and energies of CTG-NBEP. The detection of Ni2+ from water samples were also carried out successfully.

Clinical evaluation of deep learning-based clinical target volume three-channel auto-segmentation algorithm for adaptive radiotherapy in cervical cancer.

OBJECTIVES: Accurate contouring of the clinical target volume (CTV) is a key element of radiotherapy in cervical cancer. We validated a novel deep learning (DL)-based auto-segmentation algorithm for CTVs in cervical cancer called the three-channel adaptive auto-segmentation network (TCAS). METHODS: A total of 107 cases were collected and contoured by senior radiation oncologists (ROs). Each case consisted of the following: (1) contrast-enhanced CT scan for positioning, (2) the related CTV, (3) multiple plain CT scans during treatment and (4) the related CTV. After registration between (1) and (3) for the same patient, the aligned image and CTV were generated. Method 1 is rigid registration, method 2 is deformable registration, and the aligned CTV is seen as the result. Method 3 is rigid registration and TCAS, method 4 is deformable registration and TCAS, and the result is generated by a DL-based method. RESULTS: From the 107 cases, 15 pairs were selected as the test set. The dice similarity coefficient (DSC) of method 1 was 0.8155 ± 0.0368; the DSC of method 2 was 0.8277 ± 0.0315; the DSCs of method 3 and 4 were 0.8914 ± 0.0294 and 0.8921 ± 0.0231, respectively. The mean surface distance and Hausdorff distance of methods 3 and 4 were markedly better than those of method 1 and 2. CONCLUSIONS: The TCAS achieved comparable accuracy to the manual delineation performed by senior ROs and was significantly better than direct registration.

Three-dimensional deep neural network for automatic delineation of cervical cancer in planning computed tomography images.

PURPOSE: Radiation therapy is an essential treatment modality for cervical cancer, while accurate and efficient segmentation methods are needed to improve the workflow. In this study, a three-dimensional V-net model is proposed to automatically segment clinical target volume (CTV) and organs at risk (OARs), and to provide prospective guidance for low lose area. MATERIAL AND METHODS: A total of 130 CT datasets were included. Ninety cases were randomly selected as the training data, with 10 cases used as the validation data, and the remaining 30 cases as testing data. The V-net model was implemented with Tensorflow package to segment the CTV and OARs, as well as regions of 5 Gy, 10 Gy, 15 Gy, and 20 Gy isodose lines covered. The auto-segmentation by V-net was compared to auto-segmentation by U-net. Four representative parameters were calculated to evaluate the accuracy of the delineation, including Dice similarity coefficients (DSC), Jaccard index (JI), average surface distance (ASD), and Hausdorff distance (HD). RESULTS: The V-net and U-net achieved the average DSC value for CTV of 0.85 and 0.83, average JI values of 0.77 and 0.75, average ASD values of 2.58 and 2.26, average HD of 11.2 and 10.08, respectively. As for the OARs, the performance of the V-net model in the colon was significantly better than the U-net model (p = 0.046), and the performance in the kidney, bladder, femoral head, and pelvic bones were comparable to the U-net model. For prediction of low-dose areas, the average DSC of the patients' 5 Gy dose area in the test set were 0.88 and 0.83, for V-net and U-net, respectively. CONCLUSIONS: It is feasible to use the V-Net model to automatically segment cervical cancer CTV and OARs to achieve a more efficient radiotherapy workflow. In the delineation of most target areas and OARs, the performance of V-net is better than U-net. It also offers advantages with its feature of predicting the low-dose area prospectively before radiation therapy (RT).

Deep learning-based auto-segmentation of clinical target volumes for radiotherapy treatment of cervical cancer.

OBJECTIVES: Because radiotherapy is indispensible for treating cervical cancer, it is critical to accurately and efficiently delineate the radiation targets. We evaluated a deep learning (DL)-based auto-segmentation algorithm for automatic contouring of clinical target volumes (CTVs) in cervical cancers. METHODS: Computed tomography (CT) datasets from 535 cervical cancers treated with definitive or postoperative radiotherapy were collected. A DL tool based on VB-Net was developed to delineate CTVs of the pelvic lymph drainage area (dCTV1) and parametrial area (dCTV2) in the definitive radiotherapy group. The training/validation/test number is 157/20/23. CTV of the pelvic lymph drainage area (pCTV1) was delineated in the postoperative radiotherapy group. The training/validation/test number is 272/30/33. Dice similarity coefficient (DSC), mean surface distance (MSD), and Hausdorff distance (HD) were used to evaluate the contouring accuracy. Contouring times were recorded for efficiency comparison. RESULTS: The mean DSC, MSD, and HD values for our DL-based tool were 0.88/1.32 mm/21.60 mm for dCTV1, 0.70/2.42 mm/22.44 mm for dCTV2, and 0.86/1.15 mm/20.78 mm for pCTV1. Only minor modifications were needed for 63.5% of auto-segmentations to meet the clinical requirements. The contouring accuracy of the DL-based tool was comparable to that of senior radiation oncologists and was superior to that of junior/intermediate radiation oncologists. Additionally, DL assistance improved the performance of junior radiation oncologists for dCTV2 and pCTV1 contouring (mean DSC increases: 0.20 for dCTV2, 0.03 for pCTV1; mean contouring time decrease: 9.8 min for dCTV2, 28.9 min for pCTV1). CONCLUSIONS: DL-based auto-segmentation improves CTV contouring accuracy, reduces contouring time, and improves clinical efficiency for treating cervical cancer.

Connecting the Dots: Translating the Vaginal Microbiome Into a Drug.

A Lactobacillus-dominated vaginal microbiota (VMB) has been associated with health and considered an important host defense mechanism against urogenital infections. Conversely, depletion of lactobacilli and increased microbial diversity, amplifies the risk of adverse gynecologic and obstetric outcomes. A common clinical condition that exemplifies dysbiosis is bacterial vaginosis (BV). BV is currently treated with antibiotics, but frequently recurs, due in part to persistent dysbiosis and failure of lactobacilli to repopulate the vagina. New treatment options are needed to address BV. The VMB is relatively simple and optimally dominated by one or several species of Lactobacillus. Lactobacillus crispatus is strongly associated with vaginal health and depleted in dysbiosis. Replenishing the dysbiotic VMB with protective L. crispatus CTV-05 is a promising approach to prevent recurrent infections and improve women's health. Here we discuss confirmation of this approach with the microbiome-based biologic drug, LACTIN-V (L. crispatus CTV-05), focusing on prevention of BV recurrence.

Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids.

BACKGROUND: High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. METHODS: Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. RESULTS: The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. CONCLUSIONS: This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

Tailoring PTV expansion to improve the dosimetry of post modified radical mastectomy intensity-modulated radiotherapy for left-sided breast cancer patients by using 4D CT combined with cone beam CT.

PURPOSE: Our study aimed to improve the dosimetry of post modified radical mastectomy intensity-modulated radiotherapy (PMRM-IMRT) for left-sided breast cancer patients by tailoring and minimizing PTV expansion three-dimensionally utilizing 4D CT combined with on-board cone beam CT (CBCT). METHODS: We enrolled a total of 10 consecutive left-sided breast cancer patients to undergo PMRM-IMRT. We measured the intra-fractional CTV displacement attributed to respiratory movement by defining 9 points on the left chest wall and quantifying their displacement by using the 4D CT, and measured the inter-fractional CTV displacement resulting from the integrated effect of respiratory movement, thoracic deformation and set up errors by using CBCT. We created 3 different PMRM-IMRT plans for each of the patients using PTVt (tailored PTV expansion three-dimensionally), PTV0.5 and PTV0.7 (isotropic 0.5- cm and isotropic 0.7- cm expanding margin of CTV), respectively. We performed paired samples t test to establish a hierarchy in terms of plan quality and dosimetric benefits. P < 0.05 was considered statistically significant. RESULTS: The inter-fractional CTV displacement (2.6 ± 2.2 mm vertically, 2.8 ± 2.3 mm longitudinally, and 1.7 ± 1.2 mm laterally) measured by CBCT was much larger than the intra-fractional one (0.5 ± 0.5 mm vertically, 0.5 ± 1.0 mm longitudinally, and 0.3 ± 0.3 mm laterally, respectively) measured by 4D CT. Intensity-modulated radiotherapy with tailored PTV expansion based on inter-fractional CTV displacement had dosimetrical advantages over those with PTV0.5 or those with PTV0.7 owing to its perfect PTV dose coverage and better OARs sparing(especially of heart and left lung). CONCLUSION: The CTV displacement in PMRM-IMRT predominantly arises from inter-fraction rather than from intra-fraction during natural respiration and differs in 3 coordinate axes either inter-fractionally or intra-fractionally. Tailoring and minimizing PTV expansion three-dimensionally significantly improves the dosimetry of PMRM-IMRT for left-sided breast cancer patients.

The Venous Occlusion Image Score: A Novel Quantitative Scoring Instrument for Cerebral Venous Sinus Thrombosis.

PURPOSE: This study aims to test the validity of a new quantitative scoring instrument-the Venous Occlusion Image Score (VOIS), and assess the diagnostic and prognostic value of VOIS for cerebral venous sinus thrombosis (CVST). METHODS: The VOIS divided the major cerebral venous sinuses and internal jugular veins into nine parts of interest. CT venography and DSA source images and reconstruction were extracted from the database, then interpreted and scored independently according to VOIS by a panel of three reviewers. Inter-observer and intra-observer reliability were determined using the intraclass correlation coefficient (ICC) and the kappa coefficient (κ). The primary outcome was the 3-month functional outcome and evaluated by modified Rankin Scale (mRS). The sensitivity and specificity of VOIS for the primary outcomes were computed. Logistic regression was applied to evaluate the association between the score on VOIS and the primary outcomes. RESULTS: Fifty-six patients with CVST were included in the study. For 16 patients underwent cerebral CTV and DSA, excellent interobserver agreement was observed for DSA (ICC=0.90, 95%CI = 0.87 - 0.92, P < 0.001), and CTV (ICC = 0.92, 95%CI = 0.84 - 0.93, P < 0.001). The κ coefficient of agreement for the two radiology measures was 0.88 (95%CI = 0.79-0.92), indicating good inter-method agreement. For 56 patients followed up by CTV, baseline VOIS value correlated inversely with the severity of stroke on the National Institutes of Health Stroke Scale (r = -0·53, P < 0·001), and modified Rankin Scale (r = -0·59, P < 0·001). Baseline CTV-VOIS value predicted functional outcome (P < 0·05). CONCLUSION: VOIS may serve as a convenient and reliable method in the treatment guidance and outcome prediction of patients with CVST.

Dosimetric and clinical outcomes of CT based HR-CTV delineation for HDR intracavitary brachytherapy in carcinoma cervix - a retrospective study.

BACKGROUND: Brachytherapy for carcinoma cervix has moved from Point A based planning to optimization of dose based on HR-CTV. Guidelines have been published by GEC ESTRO on HR-CTV delineation based on clinical gynecological examination and MR sequences. These have given significant clinical results in terms of local control. However, many centers around the country and worldwide still use CT based planning, which restricts HR-CTV delineation, as disease and cervix can rarely be differentiated on a planning CT. Various studies have been done to develop CT based contouring guidelines from the available data, but enough evidence is not available on the clinical outcome when treatment is optimized to HR-CTV contoured on CT images. The purpose of this study is to find out the relation between local control and dosimetry of HR-CTV as delineated on CT images. MATERIALS AND METHODS: Patients of locally advanced carcinoma cervix treated radically with EBRT of 50 Gy in 25# and at least 4 cycles of concurrent weekly Cisplatin having a complete or partial response to EBRT were taken for study. All patients had completed CT based Intracavitary brachytherapy to 21 Gy in 3# of 7 Gy per # with dose prescription at point A and optimizing dose to reduce bladder and rectal toxicity. Follow up data on locoregional recurrence was obtained. HR-CTV delineation was done retrospectively on the treatment plan following guidelines by Viswanathan et al. EQD2 doses for EBRT+BT were calculated for point A and HR-CTV D90. The dosimetric data to HR-CTV and to Point A were then compared with patients with locoregional control and with local recurrence. RESULTS: 48 patients were taken, all had squamous cell carcinoma. The median age was 48 years. 33.33% were stage IIA, the rest were stage IIB. Median follow-up was 30 months with 25% developing recurrence of the disease. HR-CTV D90 EQD2 dose was significantly higher in patients with locoregionally controlled disease than in patients with local recurrence (83.97 Gy10 vs. 77.96 Gy10, p = 0.002). Patients with HR-CTV D90 EQD2 dose greater than or equal to 79.75 Gy 10 had better locoregional control than patients receiving dose less than 79.75 Gy10 (p = 0.015). Kaplan Meier plot for PFS showed significantly improved PFS for patients receiving HR-CTV D90 dose of at least 79.75 Gy10 (log-rank p-value = 0.007). Three year progression free survival was 87.1% in patients receiving HR-CTV D90 dose of at least 79.75 Gy10. CONCLUSION: CT based HR-CTV volume delineation with the help of pre brachytherapy clinical diagrams and MRI imaging may be feasible in a select subgroup of patients with complete or near-complete response to external beam radiation.

Fast-track breeding system to introduce CTV resistance of trifoliate orange into citrus germplasm, by integrating early flowering transgenic plants with marker-assisted selection.

BACKGROUND: Global warming will expand the range of new and invasive pathogens in orchards, and subsequently increase the risk of disease epidemics and economic losses. The development of new resistant plant varieties can help to reduce the impact of pathogens, however, the breeding speed can be extremely slow, due to the growth rates of the plants, and the availability of resistance genes. Citrus trees are suffering immense damage from serious diseases such as citrus canker (XCC), huanglongbing (HLB), and citrus tristeza virus (CTV). A fast-track breeding system, that aimed at shortening the duration for disease resistance breeding by incorporating the resistance genes from related species to commercial varieties, has been developed using the integration of precocious transgenic trifoliate orange with the overexpression of CiFT and MAS. It was applied here to incorporate CTV resistance of trifoliate orange into citrus germplasm. RESULTS: One generation of backcrossed breeding, that would normally take at least 5 years, was achieved in a single year by fast-track breeding system. Linkage analysis using the corresponding DNA markers revealed that CTV resistance and T-DNA integrated regions were found in different linkage groups, and they were independently segregated in the BC progenies. The CTV resistant null segregants, in which the T-DNA integrated region was removed from their genome, were feasibly obtained by MAS in each generation of the BC progenies, and their CTV resistance was confirmed by immunological analysis. Several BC3 null segregants, whose genetic backgrounds had been substituted into citrus germplasm, except for the haplotype block of CTV resistance, were successfully obtained. CGH and NGS analyses revealed that the T-DNA integrated region was safely segregated out in null segregants. CONCLUSION: Fast-track breeding systems are expected to shorten the required breeding time by more than one-fifth in comparison with conventional cross breeding techniques. Using this system, we obtained BC3-8, whose genetic background was successfully substituted except for the CTV resistance locus, and could be a novel mandarin breeding material. The fast-track breeding system will be useful to introduce important traits from related species to citrus germplasm while also drastically reducing the time required for breeding.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus.

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Citrus Tristeza Virus Isolates of the Same Genotype Differ in Stem Pitting Severity in Grapefruit.

Two isolates of the T68 genotype of citrus tristeza virus (CTV) were derived from a common source, GFMS12, by single aphid transmission. These isolates, named GFMS12-8 and GFMS12-1.3, induced stem pitting with differing severity in 'Duncan' grapefruit (Citrus × paradisi [Macfad.]). Full-genome sequencing of these isolates showed only minor nucleotide sequence differences totaling 45 polymorphisms. Numerous nucleotide changes, in relatively close proximity, were detected in the p33 open reading frame (ORF) and the leader protease domains of ORF1a. This is the first report of full-genome characterization of CTV isolates of a single genotype, derived from the same source, but showing differences in pathogenicity. The results demonstrate the development of intragenotype heterogeneity known to occur with single-stranded RNA viruses. Identification of genetic variability between isolates showing different pathogenicity will enable interrogation of specific genome regions for potential stem pitting determinants.

Dosimetric impact on changes in target volumes during intensity-modulated radiotherapy for nasopharyngeal carcinoma.

BACKGROUND AND PURPOSE: To assess anatomic changes during intensity modulated radiotherapy (IMRT) for nasopharyngeal carcinoma (NPC) and to determine its dosimetric impact. PATIENTS AND METHODS: Twenty patients treated with IMRT for NPC were enrolled in this study. A second CT was performed at 38 Gy. Manual contouring of the macroscopic tumor volumes (GTV) and the planning target volumes (PTV) were done on the second CT. We recorded the volumes of the different structures, D98 %, the conformity, and the homogeneity indexes for each PTV. Volume percent changes were calculated. RESULTS: We observed a significant reduction in tumor volumes (58.56 % for the GTV N and 29.52 % for the GTV T). It was accompanied by a significant decrease in the D98 % for the 3 PTV (1.4 Gy for PTV H, p = 0.007; 0.3 Gy for PTV I, p = 0.03 and 1.15 Gy for PTV L, p = 0 0.0066). In addition, we observed a significant reduction in the conformity index in the order of 0.02 (p = 0.001) and 0.01 (p = 0.007) for PTV H and PTV I, respectively. The conformity variation was not significant for PTV L. Moreover, results showed a significant increase of the homogeneity index for PTV H (+ 0.03, p = 0.04) and PTV L (+ 0.04, p = 0.01). CONCLUSION: Tumor volume reduction during the IMRT of NPC was accompanied by deterioration of the dosimetric coverage for the different target volumes. It is essential that a careful adaptation of the treatment plan be considered during therapy for selected patients.

Radiotherapy for the treatment of pituitary adenomas: A dosimetric comparison of three planning techniques.

AIM: Our goal was to compare conformal 3D (C3D) radiotherapy (RT), modulated intensity RT (IMRT), and volumetric modulated arc therapy (VMAT) planning techniques in treating pituitary adenomas. BACKGROUND: RT is important for managing pituitary adenomas. Treatment planning advances allow for higher radiation dosing with less risk of affecting organs at risk (OAR). MATERIALS AND METHODS: We conducted a 5-year retrospective review of patients with pituitary adenoma treated with external beam radiation therapy (C3D with flattening filter, flattening filter-free [FFF], IMRT, and VMAT). We compared dose-volume histogram data. For OARs, we recorded D2%, maximum, and mean doses. For planning target volume (PTV), we registered V95%, V107%, D95%, D98%, D50%, D2%, minimum dose, conformity index (CI), and homogeneity index (HI). RESULTS: Fifty-eight patients with pituitary adenoma were included. Target-volume coverage was acceptable for all techniques. The HI values were 0.06, IMRT; 0.07, VMAT; 0.08, C3D; and 0.09, C3D FFF (p < 0.0001). VMAT and IMRT provided the best target volume conformity (CI, 0.64 and 0.74, respectively; p < 0.0001). VMAT yielded the lowest doses to the optic pathway, lens, and cochlea. The position of the neck in extreme flexion showed that it helps in planning mainly with VMAT by allowing only one arc to be used and achieving the desired conformity, decreasing the treatment time, while allowing greater protection to the organs of risk using C3D, C3DFFF. CONCLUSIONS: Our results confirmed that EBRT in pituitary adenomas using IMRT, VMAT, C3D, C3FFF provide adequate coverage to the target. VMAT with a single arc or incomplete arc had a better compliance with desired dosimetric goals, such as target coverage and normal structures dose constraints, as well as shorter treatment time. Neck extreme flexion may have benefits in treatment planning for better preservation of organs at risk. C3D with extreme neck flexion is an appropriate treatment option when other treatment techniques are not available.

Transitioning from conformal radiotherapy to intensity-modulated radiotherapy after radical prostatectomy: Clinical benefit, oncologic outcomes and incidence of gastrointestinal and urinary toxicities.

AIM: The purpose of this study was to review genitourinary (GU) and gastrointestinal (GI) toxicity associated with high-dose radiotherapy (RT) delivered with 3-dimensional conformal radiotherapy (3D-CRT) and intensity-modulated radiotherapy (IMRT) or volumetric arc therapy (VMAT) following radical prostatectomy (RP). BACKGROUND: RP is a therapeutic option for the management of prostate cancer (PrCa). When assessing postoperative RT techniques for PrCa, the published literature focuses on patients treated with 2-dimensional conventional methods without reflecting the implementation of 3D-CRT, IMRT, or VMAT. MATERIALS AND METHODS: A total of 83 patients were included in this analysis; 30 patients received 3D-CRT, and 53 patients received IMRT/VMAT. Acute and late symptoms of the GU and lower GI tract were retrospectively graded according to the Radiation Therapy Oncology Group and the European Organization for Research and Treatment of Cancer radiation toxicity grading systems. The relapse failure-free rate and overall survival were also evaluated. RESULTS: The rate of acute GU toxicity was 9.4% vs. 13.3% for the IMRT/VMAT and 3D-CRT groups (p = 0.583). The 5-year actuarial rates of late GI toxicity for IMRT/VMAT and 3D-CRT treatments were 1.9% and 6.7%, respectively. The rate of late GU toxicity for the IMRT/VMAT and 3D-CRT treatment groups was 7.5% and 16.6%, respectively (p = 0.199). We found no association between acute or late toxicity and the RT technique in univariate and multivariate analyses. CONCLUSION: Postprostatectomy IMRT/VMAT and 3D-CRT achieved similar morbidity and cancer control outcomes. The clinical benefit of highly conformal techniques in this setting is unclear although formal analysis is needed.

The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N. benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N. benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N. benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.