Search Results (9)

Background: Host cell proteins (HCPs) are process-related impurities that must be monitored in biopharmaceutical products due to their potential impact on product quality and patient safety. Targeted LC–MS/MS approaches such as multiple reaction monitoring (MRM) enable protein-specific HCP quantification but are difficult to apply in highly multiplexed assays because of retention time (RT) variability across complex bioprocess matrices. Methods: Here, we show that conventional RT-scheduled MRM workflows lack transferability when applied to heterogeneous drug substances and process intermediates. Using a targeted assay comprising 240 peptides corresponding to 97 CHO-derived HCPs, RT shifts of several minutes resulted in truncated chromatographic peaks and peptide signal loss, even when wide scheduling windows were used. To overcome this limitation, a scout-triggered MRM (st-MRM) acquisition strategy based on event-driven monitoring was implemented. Results: This approach enabled robust peptide detection across diverse matrices within a single injection, without method re-optimization. Absolute quantification using stable isotope-labeled peptides spanned six orders of magnitude, with HCPs quantified down to 2.9 ppm in purified drug substances. Conclusion: Overall, st-MRM improves the robustness and transferability of highly multiplexed targeted proteomics workflows for HCP analysis. Full article

Introduction: ADT is routinely combined with radiotherapy (RT) for intermediate- and high-risk prostate cancer. While prostate shrinkage may facilitate planning, prospective CT-based, patient-level estimates over short, workflow-relevant intervals are scarce. Methods: We conducted a prospective study of 47 patients starting luteinizing hormone-releasing hormone agonist (LHRHa) therapy (leuprolide, 6-month depot). Prostate volumes were independently contoured by three blinded radiation oncologists on paired CT scans at baseline and ~8 weeks post-injection. The primary outcomes were the mean relative volume change and the proportion achieving a clinically relevant reduction (≥15%). PSA and testosterone were recorded at both time points; correlations and exploratory univariable logistic regression for ≥15% reduction were performed at the patient level. Results: Mean relative volume reduction ranged from −18.5% to −21.3% across observers; ≥60% of patients met the ≥15% threshold (RT-A 61.7%, RT-B 66.0%, RT-C 74.5%). PSA and testosterone decreased substantially (e.g., median PSA from 9.64 to 1.84 nmol/L) and were moderately correlated (Spearman ρ = 0.43, p = 0.002; Pearson r = 0.51, p < 0.001). No baseline clinical, histologic, or biochemical variables reached statistical significance for predicting ≥15% volume reduction; % PSA change showed a non-significant trend (OR 1.03; 95% CI 1.00–1.07; p = 0.076). Conclusions: Short-course LHRHa induced consistent CT-measured cytoreduction, with more than half of cases achieving ≥15% shrinkage within 8 weeks. Prostate downsizing was reproducible across readers and accompanied by marked PSA and testosterone declines, although biochemical responses did not predict volumetric change. These findings support incorporating a short neoadjuvant “window” before RT simulation and highlight the need for larger studies to refine predictors and compare agonist vs. antagonist trajectories. Full article

Objectives: This research paper proposes an innovative framework for developing adaptive and dedicated rehabilitation strategies based on the perceptions of specialists in sports rehabilitation (RT), sports training (AR) and with mixed expertise (RT+AR) regarding advanced resistance training methods, including Effort-Based Training (EBT-3/7), Cluster Training (CT), Rest-Pause Training (RPT) and Post-Activation Potentiation (PAP). The aim of this paper was to develop a tailored strategy for rehabilitation programs, grounded in a targeted selection of training methods, short-term periodization and exercises structured around key training variables such as frequency, intensity and volume. Methods: In order to reach this objective, a quantitative research method is proposed, aiming to identify the experts’ opinion on the way of managing and integrating Unilateral Resistance Training Exercise (URTE). Data processing and analysis were conducted by means of specific tests supplied by the SPSS Statistics for Windows (version 20.0, IBM Corp., Armonk, NY, USA). Results: The findings indicate that EBT-3/7 is perceived as the most effective method for rehabilitation with minimal injury risk, whereas CT and PAP are associated with performance benefits but higher perceived injury risk. RT+AR specialists reported more frequent use of these methods and higher perceived effectiveness. Additionally, they demonstrated superior operational and dynamic capabilities compared to single-domain specialists. Conclusions: According to specialists’ opinions, URTE is effective for post-injury rehabilitation, with combined rehabilitation and training expertise enhancing utilization, perceived effectiveness and implementation of personalized, performance-oriented strategies. Full article

Background: Radiotherapy (RT) is a mainstay treatment for prostate cancer (PC). Accurate delineation of organs at risk (OARs) is crucial for optimizing the therapeutic window by minimizing side effects. Manual segmentation is time-consuming and prone to inter-operator variability. This study investigates the performance of Limbus^® Contour^® (LC), a deep learning-based auto-contouring software, in delineating pelvic structures in PC patients. Methods: We evaluated LC’s performance on key structures (bowel bag, bladder, rectum, sigmoid colon, and pelvic lymph nodes) in 52 patients. We compared auto-contoured structures with those manually delineated by radiation oncologists using different metrics. Results: LC achieved good agreement for the bladder (median Dice: 0.95) and rectum (median Dice: 0.83). However, limitations were observed for the bowel bag (median Dice: 0.64) and sigmoid colon (median Dice: 0.6), with inclusion of irrelevant structures. While the median Dice for pelvic lymph nodes was acceptable (0.73), the software lacked sub-regional differentiation, limiting its applicability in certain other oncologic settings. Conclusions: LC shows promise for automating OAR delineation in prostate radiotherapy, particularly for the bladder and rectum. Improvements are needed for bowel bag, sigmoid colon, and lymph node sub-regionalization. Further validation with a broader and larger patient cohort is recommended to assess generalizability. Full article

The evaluation of the reactivity and distress of cattle during corral management, by means of subjective scores, aims at the standardization of behavioral indicators, through non-invasive methods, in addition to enabling the development of more appropriate management practices, thus promoting the comfort and well-being of these animals. Therefore, in this study, we aimed to characterize the temperament and distress of cattle managed in a corral using behavioral indicators during the rainiest period. For this, the experiment was conducted on a property located in the municipality of Mojuí dos Campos, during the rainiest quarter (February–April). Thus, 30 male cattle, not castrated, approximately 29 months of age, clinically healthy, and weighing 310 + 20 kg, were divided into three rearing systems: silvopastoral (SP), traditional (SS), and integrated (SI) systems. There were 10 animals per system. Physiological parameters were collected to evaluate rectal temperature (RT) and respiratory rate (RR), as well as body surface temperature (BST), through thermal windows (head and flank infrared temperature and rump infrared temperature). To evaluate temperament and reactivity, scores indicative of corral behavior were used, namely escape speed (ES), tension score (SS_1), tension score (SS_2), reactivity scale (RS), movement score (MS), and temperament scale (TS). The results showed that there was a thermal amplitude of 5.9 °C on average and 8.6 °C at maximum when comparing the structure of the corral and the trees. In addition, the comparisons between the production systems for the behavioral variables did not differ at the 5% significance level, except for ES, where the traditional system differed from the integrated system and the silvopastoral system, showing intermediate average values for both. In addition, there was a positive correlation between the variables RT and RR (r = 0.72; p < 0.01), RR and SS_2 (r = 0.38; p = 0.04), flank infrared temperature and MS (r = 0.47; p = 0.01), rump infrared temperature and RS (r = 0.37; p = 0.04), SS_1 and RS (r = 0.41; p = 0.02), SS_1 and SS_2 (r = 0.39; p = 0.03), RS and SS_2 (r = 0.58; p = 0.00), RS and MS (r = 0.50; p = 0.01), RS and TS (r = 0.61; p = 0.00), SS_2 and MS (r = 0.51; p = 0.00), SS_2 and TS (r = 0.47; p = 0.01), and MS and TS (r = 0.44; p = 0.02), and a negative correlation between ES and TS (r = −0.42; p = 0.02). The rainy season had a major influence on the evaluation of temperature and distress levels during handling in the corral, as evidenced by the association between physiological and behavioral parameters. Full article

Healthy tissue-sparing effects of FLASH (≥40 Gy/s, ≥4–8 Gy/fraction) radiotherapy (RT) make it potentially useful for whole breast irradiation (WBI), since there is often a lot of normal tissue within the planning target volume (PTV). We investigated WBI plan quality and determined FLASH-dose for various machine settings using ultra-high dose rate (UHDR) proton transmission beams (TBs). While five-fraction WBI is commonplace, a potential FLASH-effect might facilitate shorter treatments, so hypothetical 2- and 1-fraction schedules were also analyzed. Using one tangential 250 MeV TB delivering 5 × 5.7 Gy, 2 × 9.74 Gy or 1 × 14.32 Gy, we evaluated: (1) spots with equal monitor units (MUs) in a uniform square grid with variable spacing; (2) spot MUs optimized with a minimum MU-threshold; and (3) splitting the optimized TB into two sub-beams: one delivering spots above an MU-threshold, i.e., at UHDRs; the other delivering the remaining spots necessary to improve plan quality. Scenarios 1–3 were planned for a test case, and scenario 3 was also planned for three other patients. Dose rates were calculated using the pencil beam scanning dose rate and the sliding-window dose rate. Various machine parameters were considered: minimum spot irradiation time (minST): 2 ms/1 ms/0.5 ms; maximum nozzle current (maxN): 200 nA/400 nA/800 nA; two gantry-current (GC) techniques: energy-layer and spot-based. For the test case (PTV = 819 cc) we found: (1) a 7 mm grid achieved the best balance between plan quality and FLASH-dose for equal-MU spots; (2) near the target boundary, lower-MU spots are necessary for homogeneity but decrease FLASH-dose; (3) the non-split beam achieved >95% FLASH for favorable (not clinically available) machine parameters (SB GC, low minST, high maxN), but <5% for clinically available settings (EB GC, minST = 2 ms, maxN = 200 nA); and (4) splitting gave better plan quality and higher FLASH-dose (~50%) for available settings. The clinical cases achieved ~50% (PTV = 1047 cc) or >95% (PTV = 477/677 cc) FLASH after splitting. A single UHDR-TB for WBI can achieve acceptable plan quality. Current machine parameters limit FLASH-dose, which can be partially overcome using beam-splitting. WBI FLASH-RT is technically feasible. Full article

Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and structural diversity making their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based lipidomics screening method using a combination of variable retention time window and relative dwell time weightage. Using this method, we identified more than 1000 lipid species within 24-min. The limit of detection varied from the femtomolar to the nanomolar range. About 883 lipid species were detected with a coefficient of variance <30%. We used this method to identify plasma lipids altered due to vitamin B₁₂ deficiency and found a total of 18 lipid species to be altered. Some of the lipid species with ω-6 fatty acid chains were found to be significantly increased while ω-3 decreased in vitamin B₁₂ deficient samples. This method enables rapid screening of a large number of lipid species in a single experiment and would substantially advance our understanding of the role of lipids in biological processes. Full article

Metastasis is the leading cause of cancer-related deaths worldwide. The epithelial-mesenchymal plasticity (EMP) status of primary tumours has relevance to metastatic potential and therapy resistance. Circulating tumour cells (CTCs) provide a window into the metastatic process, and molecular characterisation of CTCs in comparison to their primary tumours could lead to a better understanding of the mechanisms involved in the metastatic cascade. In this study, paired blood and tumour samples were collected from four prostate cancer patient-derived xenograft (PDX) models (BM18, LuCaP70, LuCaP96, LuCaP105) and assessed using an EMP-focused, 42 gene human-specific, nested quantitative RT-PCR assay. CTC burden varied amongst the various xenograft models with LuCaP96 having the highest number of CTCs per mouse (mean: 704; median: 31) followed by BM18 (mean: 101; median: 21), LuCaP70 (mean: 73; median: 16) and LuCaP105 (mean: 57; median: 6). A significant relationship was observed between tumour size and CTC number (p = 0.0058). Decreased levels of kallikrein-related peptidase 3 (KLK3) mRNA (which encodes prostate-specific antigen; PSA) were observed in CTC samples from all four models compared to their primary tumours. Both epithelial- and mesenchymal-associated genes were commonly expressed at higher levels in CTCs compared to the bulk primary tumour, although some common EMT-associated genes (CDH1, VIM, EGFR, EPCAM) remained unchanged. Immunofluorescence co-staining for pan-cytokeratin (KRT) and vimentin (VIM) indicated variable proportions of CTCs across the full EMP axis, even in the same model. EMP hybrids predominated in the BM18 and LuCaP96 models, but were not detected in the LuCaP105 model, and variable numbers of KRT⁺ and human VIM⁺ cells were observed in each model. SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was enriched at the RNA level in CTCs compared to primary tumours and was the most commonly expressed mesenchymal gene in the CTCs. Co-staining for SERPINE1 and KRT revealed SERPINE1⁺ cells in 7/11 samples, six of which had SERPINE⁺KRT⁺ CTCs. Cell size variation was observed in CTCs. The majority of samples (8/11) contained larger CTCs ranging from 15.3 to 37.8 µm, whilst smaller cells (10.7 ± 4.1 µm, similar in size to peripheral blood mononuclear cells (PBMCs)) were identified in 6 of 11 samples. CTC clusters were also identified in 9/11 samples, containing 2–100 CTCs per cluster. Where CTC heterogeneity was observed in the clusters, epithelial-like cells (KRT⁺VIM⁻) were located on the periphery of the cluster, forming a layer around hybrid (KRT⁺VIM⁺) or mesenchymal-like (KRT⁻VIM⁺) cells. The CTC heterogeneity observed in these models emphasises the complexity in CTC isolation and classification and supports the increasingly recognised importance of the epithelial-mesenchymal hybrid state in cancer progression and metastasis. Full article

A hybridization-based point-of-care (POC) assay for HIV-1 drug resistance would be useful in low- and middle-income countries (LMICs) where resistance testing is not routinely available. The major obstacle in developing such an assay is the extreme genetic variability of HIV-1. We analyzed 27,203 reverse transcriptase (RT) sequences from the Stanford HIV Drug Resistance Database originating from six LMIC regions. We characterized the variability in a 27-nucleotide window surrounding six clinically important drug resistance mutations (DRMs) at positions 65, 103, 106, 181, 184, and 190. The number of distinct codons at each DRM position ranged from four at position 184 to 11 at position 190. Depending on the mutation, between 11 and 15 of the 24 flanking nucleotide positions were variable. Nonetheless, most flanking sequences differed from a core set of 10 flanking sequences by just one or two nucleotides. Flanking sequence variability was also lower in each LMIC region compared with overall variability in all regions. We also describe an online program that we developed to perform similar analyses for mutations at any position in RT, protease, or integrase. Full article