Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

Evaluation of inter- and intra-observer variations in prostate gland delineation using CT-alone versus CT/TPUS.

Background: This study aims to explore the role of four-dimensional (4D) transperineal ultrasound (TPUS) in the contouring of prostate gland with planning computed tomography (CT) images, in the absence of magnetic resonance imaging (MRI). Materials and methods: Five radiation oncologists (ROs) performed two rounds of prostate gland contouring (single-blinded) on CT-alone and CT/TPUS datasets obtained from 10 patients who underwent TPUS-guided external beam radiotherapy. Parameters include prostate volume, DICE similarity coefficient (DSC) and centroid position. Wilcoxon signed-rank test assessed the significance of inter-modality differences, and the intraclass correlation coefficient (ICC ) reflected inter- and intra-observer reliability of parameters. Results: Inter-modality analysis revealed high agreement (based on DSC and centroid position) of prostate gland contours between CT-alone and CT/TPUS. Statistical significant difference was observed in the superior-inferior direction of the prostate centroid position (p = 0.011). All modalities yielded excellent inter-observer reliability of delineated prostate volume with ICC > 0.9, mean DSC > 0.8 and centroid position: CT-alone (ICC = 1.000) and CT/TPUS (ICC = 0.999) left-right (L/R); CT-alone (ICC = 0.999) and CT/TPUS (ICC = 0.998) anterior-posterior (A/P); CT-alone (ICC = 0.999) and CT/TPUS (ICC = 1.000) superior-inferior (S/I). Similarly, all modalities yielded excellent intra-observer reliability of delineated prostate volume, ICC > 0.9 and mean DSC > 0.8. Lastly, intra-observer reliability was excellent on both imaging modalities for the prostate centroid position, ICC > 0.9. Conclusion: TPUS does not add significantly to the amount of anatomical information provided by CT images. However, TPUS can supplement planning CT to achieve a higher positional accuracy in the S/I direction if access to CT/MRI fusion is limited.

An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide.

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product's distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

Duration-dependent margins for prostate radiotherapy-a practical motion mitigation strategy.

BACKGROUND AND OBJECTIVE: The magnitude of intra-fractional prostate displacement (change from initial position over time) is associated with the duration of the patient lying on the radiotherapy treatment couch. This study reports a minute-by-minute association and calculates the impact of this displacement on duration-dependent margins using real-time intra-fractional position data monitored by four-dimensional transperineal ultrasound (4D TPUS). MATERIALS AND METHODS: A total of 55 patients were recruited prospectively. Intra-fractional position of the prostate was monitored in real-time using a 4D TPUS Clarity® system. A total of 1745 monitoring sessions were analysed. Van Herk's margin recipe (2.5∑ + 1.64((σ2 + σp2)1/2 - σp)) was used to estimate the duration-dependant margins for every minute, up to the 15th minute. Linear regression analysis was then performed on the overall margins against time and direction. RESULTS: The mean intra-fractional position was 0.76 mm Inferior (Inf), 0 mm Lateral (Lat) and 0.94 mm Posterior (Post) at the 15th minute. A minimum margin expansion of 2.42 mm (Superior/Inf), 1.02 mm (Left/Right) and 2.65 mm (Anterior/Post) was required for an 8­minute treatment compared to 4.29 mm (Sup/Inf), 1.84 mm (Lt/Rt) and 4.63 mm (Ant/Post) for a 15-minute treatment. The required margin expansion increased linearly (R2 = 0.99) in all directions (p < 0.01). However, while there was no statistically significant difference (p = 0.10) in the required margin expansion in the Sup/Inf and Ant/Post directions respective of the time duration, the margins were much bigger compared to those in the Lt/Rt direction (p < 0.01). CONCLUSION: We report our experience in deriving the minimum duration-dependant margin to generate the required planning target volume for prostate radiotherapy. The required margin increases linearly in all directions within the 15-min duration; thus, the margin will depend on the duration of the technique chosen (IMRT/VMAT/3DCRT/proton).

The Relationship Between the Tensile and the Torsional Properties of the Native Scapholunate Ligament and Carpal Kinematics.

PURPOSE: The purpose of this exploratory study was to examine the relationship between the tensile and the torsional properties of the native scapholunate interosseous ligament (SLIL) and kinematics of the scaphoid and lunate of an intact wrist during passive radioulnar deviation. METHODS: Eight fresh-frozen cadaveric specimens were transected at the elbow joint and loaded into a custom jig. Kinematic data of the scaphoid and lunate were acquired in a simulated resting condition for 3 wrist positions-neutral, 10° radial deviation, and 30° ulnar deviation-using infrared-emitting rigid body trackers. The SLIL bone-ligament-bone complex was then resected and loaded on a materials testing machine. Specimens underwent cyclic torsional and tensile testing and SLIL tensile and torsional laxity were evaluated. Correlations between scaphoid and lunate rotations and SLIL tensile and torsional properties were determined using Pearson correlation coefficients. RESULTS: Ulnar deviation of both the scaphoid and the lunate were found to decrease as the laxity of SLIL in torsion increased. In addition, the ratio of lunate flexion-extension to radial-ulnar deviation was found to increase with increased SLIL torsional rotation. CONCLUSIONS: Our findings support the theory that there is a relationship between scapholunate kinematics and laxity at the level of the interosseous ligaments. CLINICAL RELEVANCE: Laxity and, specifically, the tensile and torsional properties of an individual's native SLIL should guide reconstruction using a graft material that more closely replicates the individual's native SLIL properties.

An NMR Protocol for In Vitro Paclitaxel Release from an Albumin-Bound Nanoparticle Formulation.

The paclitaxel protein-bound particles for injectable suspension (marketed under the brand name Abraxane®) contains nanosized complexes of paclitaxel and albumin. The molecular interaction between paclitaxel and albumin within the higher-order nanostructure is analytically challenging to assess, as is any correlation of differences to differences in therapeutic effect. However, because the higher-order nanostructures may affect the paclitaxel release, a suitable in vitro assay to detect potential differences in paclitaxel release between comparator lots and products is desirable. Herein, solution NMR spectroscopy with a T2-filtering technique was developed to detect paclitaxel signal while suppressing albumin signals to follow the released paclitaxel in the NMR tube upon dilution. The non-invasive nature of NMR allows for precise measurement of a full range of dilution-induced drug release percentage from 14 to 92% without any sample extraction. The critical concentration of the drug product (DP) at 50% of release was 0.63 ± 0.04 mg/mL in PBS buffer. In addition, 2D diffusion ordered NMR spectroscopy (DOSY) results revealed that the released paclitaxel experiencing slightly slowed diffusion rates than free paclitaxel, which was attributed to paclitaxel in equilibrium with albumin-bound states. Collectively, the dilution-based NMR method offered an analytical approach to investigate physicochemical attributes of complex injectable products with minimal needed sample preparation and perturbation to nanoparticle formulation.

An Electrochemical Biosensor Architecture Based on Protein Folding Supports Direct Real-Time Measurements in Whole Blood.

The ability to monitor drug and biomarker concentrations in the body with high frequency and in real time would revolutionize our understanding of biology and our capacity to personalize medicine. The few in vivo molecular sensors that currently exist, however, all rely on the specific chemical or enzymatic reactivity of their targets and thus are not generalizable. In response, we demonstrate here an electrochemical sensing architecture based on binding-induced protein folding that is 1) independent of the reactivity of its targets, 2) reagentless, real-time, and with a resolution of seconds, and 3) selective enough to deploy in undiluted bodily fluids. As a proof of principle, we use the SH3 domain from human Fyn kinase to build a sensor that discriminates between the protein's peptide targets and responds rapidly and quantitatively even when challenged in whole blood. The resulting sensor architecture could drastically expand the chemical space accessible to continuous, real-time biosensors.

The role of mass spectrometry in the characterization of biologic protein products.

INTRODUCTION: Mass spectrometry (MS) is widely used in the characterization of biomolecules including peptide and protein therapeutics. These biotechnology products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Advances in MS instrumentation and techniques have enhanced protein characterization capabilities and supported an increased development of biopharmaceutical products. Areas covered: This review describes recent developments in MS-based biotherapeutic analysis including sequence determination, post-translational modifications (PTMs) and higher order structure (HOS) analysis along with improvements in ionization and dissociation methods. An outlook of emerging applications of MS in the lifecycle of product development such as comparability, biosimilarity and quality control practices is also presented. Expert commentary: MS-based methods have established their utility in the analysis of new biotechnology products and their lifecycle appropriate implementation. In the future, MS will likely continue to grow as one of the leading protein identification and characterization techniques in the biopharmaceutical industry landscape.

Tensile and Torsional Structural Properties of the Native Scapholunate Ligament.

PURPOSE: The ideal material for reconstruction of the scapholunate interosseous ligament (SLIL) should replicate the mechanical properties of the native SLIL to recreate normal kinematics and prevent posttraumatic arthritis. The purpose of our study was to evaluate the cyclic torsional and tensile properties of the native SLIL and load to failure tensile properties of the dorsal SLIL. METHODS: The SLIL bone complex was resected from 10 fresh-frozen cadavers. The scaphoid and lunate were secured in polymethylmethacrylate and mounted on a test machine that incorporated an x-y stage and universal joint, which permitted translations perpendicular to the rotation/pull axis as well as nonaxial angulations. After a 1 N preload, specimens underwent cyclic torsional testing (±0.45 N m flexion/extension at 0.5 Hz) and tensile testing (1-50 N at 1 Hz) for 500 cycles. Lastly, the dorsal 10 mm of the SLIL was isolated and displaced at 10 mm/min until failure. RESULTS: During intact SLIL cyclic torsional testing, the neutral zone was 29.7° ± 6.6° and the range of rotation 46.6° ± 7.1°. Stiffness in flexion and extension were 0.11 ± 0.02 and 0.12 ± 0.02 N m/deg, respectively. During cyclic tensile testing, the engagement length was 0.2 ± 0.1 mm, the mean stiffness was 276 ± 67 N/mm, and the range of displacement was 0.4 ± 0.1 mm. The dorsal SLIL displayed a 0.3 ± 0.2 mm engagement length, 240 ± 65 N/mm stiffness, peak load of 270 ± 91 N, and displacement at peak load of 1.8 ± 0.3 mm. CONCLUSIONS: We report the torsional properties of the SLIL. Our novel test setup allows for free rotation and translation, which reduces out-of-plane force application. This may explain our observation of greater dorsal SLIL load to failure than previous reports. CLINICAL RELEVANCE: By matching the natural ligament with respect to its tensile and torsional properties, we believe that reconstructions will better restore the natural kinematics of the wrist and lead to improved outcomes. Future clinical studies should aim to investigate this further.

Treatment Trends in Older Adults With Midshaft Clavicle Fractures.

PURPOSE: We present a retrospective administrative claims database review examining the effect of recent literature supporting surgical clavicle fixation in a primarily young male population, on the treatment of midshaft clavicle fractures in patients older than 65 years. We tested the null hypothesis that there is no change in trends in surgical fixation of midshaft clavicle fractures in patients older than 65 years. Secondary analysis examined overall trends and trends based on sex. METHODS: Data from 2007 to 2012 were extracted using the Medicare Standard Analytic File and Humana administrative claim databases contained within the PearlDiver Patient Records Database. Patients with clavicle shaft fractures and their treatments were identified by International Classification of Disease, Ninth Revision, and Current Procedural Terminology codes. The primary response variable was the proportion of surgical to nonsurgical cases per year, and explanatory variables included age and sex. Data were analyzed using a trend in proportions test with significance set at P less than .05. RESULTS: A total of 32,929 patients with clavicle shaft fractures were identified. During the study period, the proportion of clavicle shaft fractures treated surgically in patients older than 65 years (2.4%-4.6%) and younger than 65 years (11.2%-16.4%) showed a significant increasing trend. When analyzed by both sex and age, there was also an increasing trend in the proportion of surgically treated males in the older than 65 years (3.3%-6.2%) and the younger than 65 years groups (10.9%-19.5%). Lastly, there was an increase in the proportion of surgically treated females older than 65 years (1.7%-3.4%) and younger than 65 years (12.1%-14.3%). CONCLUSIONS: Our analysis demonstrates an overall increase in the proportion of surgically treated clavicle shaft fractures, including in the male and female population older than 65 years. In the setting of an aging population, future research evaluating possible benefits of surgical intervention in this population is needed prior to adopting this practice pattern. TYPE OF STUDY/LEVEL OF EVIDENCE: II.

Cost Minimization Analysis of Ganglion Cyst Excision.

PURPOSE: Cost minimization analysis can be employed to determine the least costly option when multiple treatments lead to equivalent outcomes. We present a cost minimization analysis from the payers' perspective, of the direct per patient cost of arthroscopic versus open ganglion cyst excision. We tested the null hypothesis that there is no difference in cost between the 2 procedures from the payer perspective. METHODS: We utilized data from a private payer administrative claims database comprising 16 million individuals from 2007 to 2015. Using Current Procedural Terminology codes to identify open and arthroscopic ganglion excisions, we extracted demographic data and fees paid to providers and facilities for the procedure. RESULTS: We identified 5,119 patients undergoing open ganglion cyst excision and 20 patients undergoing arthroscopic ganglion excision. The average cost of an open excision was significantly lower than an arthroscopic excision ($1,821 vs $3,668). CONCLUSIONS: Surgical costs from arthroscopic ganglion excision are significantly more than open excision. This data can inform health systems participating in value-based models. TYPE OF STUDY/LEVEL OF EVIDENCE: Economic and Decision Analysis IV.

Nonclinical evaluation of the potential for mast cell activation by an erythropoietin analog.

The erythropoietin analog peginesatide was withdrawn from marketing due to unexpected severe anaphylactic reactions associated with administration of the multi-use formulation. The adverse events occurred rapidly following the first ever administration of the drug with most affected patients becoming symptomatic in less than 30min. This is most consistent with an anaphylactoid reaction due to direct activation of mast cells. Laboratory evaluation was undertaken using rat peritoneal mast cells as the model system. Initial studies showed that high concentrations of the formulated drug as well as formulated vehicle alone could cause mast cell degranulation as measured by histamine release. The purified active drug was not able to cause histamine release whereas the vehicle filtrate and lab created drug vehicle were equally potent at causing histamine release. Individual formulations of vehicle leaving one component out showed that histamine release was due to phenol. Dose response studies with phenol showed a very sharp dose response curve that was similar in three buffer systems. Cellular analysis by flow cytometry showed that the histamine release was not due to cell death, and that changes in light scatter parameters consistent with degranulation were rapidly observed. Limited testing with primary human mast cells showed a similar dose response of histamine release with exposure to phenol. To provide in vivo confirmation, rats were injected with vehicle formulated with various concentrations of phenol via a jugular vein cannula. Significant release of histamine was detected in blood samples taken 2min after dosing at the highest concentrations tested.

Modern analytics for synthetically derived complex drug substances: NMR, AFFF-MALS, and MS tests for glatiramer acetate.

Glatiramer acetate (GA) is a mixture of synthetic copolymers consisting of four amino acids (glutamic acid, lysine, alanine, and tyrosine) with a labeled molecular weight range of 5000 to 9000 Da. GA is marketed as Copaxone™ by Teva for the treatment of multiple sclerosis. Here, the agency has evaluated the structure and composition of GA and a commercially available comparator, Copolymer-1. Modern analytical technologies which can characterize these complex mixtures are desirable for analysis of their comparability and structural "sameness." In the studies herein, a molecular fingerprinting approach is taken using mass-accurate mass spectrometry (MS) analysis, nuclear magnetic resonance (NMR) (1D-(1)H-NMR, 1D-(13)C-NMR, and 2D NMR), and asymmetric field flow fractionation (AFFF) coupled with multi-angle light scattering (MALS) for an in-depth characterization of three lots of the marketplace drug and a formulated sample of the comparator. Statistical analyses were applied to the MS and AFFF-MALS data to assess these methods' ability to detect analytical differences in the mixtures. The combination of multiple orthogonal measurements by liquid chromatography coupled with MS (LC-MS), AFFF-MALS, and NMR on the same sample set was found to be fit for the intended purpose of distinguishing analytical differences between these complex mixtures of peptide chains.

Pulsed low-dose rate radiotherapy for recurrent bone sarcomas: case reports and brief review.

PURPOSE: Re-irradiation for bulky recurrent sarcoma carries significant risks. Pulsed low-dose rate radiotherapy (PLDR) is an attractive option for re-irradiation due to inherent radiobiological advantages. MATERIALS AND METHODS: We present two patients who underwent reirradiation using PLDR technique, followed by a literature review. RESULTS: The first case is that of a 76-year-old male who developed an in-field recurrence of a bulky pelvic bone high-grade chondrosarcoma after he was treated with definitive radiotherapy using helical TomoTherapy with a total dose of 66 Gy. The patient was re-irradiated using PLDR with a shrinking field technique; 50 Gy in 2 Gy fractions followed by a boost of 20 Gy in 2 Gy fractions. The patient remains disease-free without significant toxicity 60 months post-irradiation. The second case is that of an 82-year-old female who was treated with a definitive irradiation of 66 Gy in 33 fractions for a right shoulder grade II chondrosarcoma. She developed an in-field recurrence 28 months later and presented with bulky disease causing brachial plexopathy and lymphedema. The patient was re-irradiated with a palliative intent to a total dose of 50 Gy in 2 Gy fractions over 5 weeks using PLDR. Brachial plexopathy resolved shortly after re-irradiation, but local progression near the surface was evident 8 months later. She passed away from unrelated causes 11 months later. CONCLUSION: We present two cases highlighting our early experience with PLDR, which was effective in the reirradiation of recurrent bony sarcoma. Our study highlights PLDR as an option for reirradiation in recurrent unresectable tumors.

Predicting dice similarity coefficient of deformably registered contours using Siamese neural network.

Objective. Automatic deformable image registration (DIR) is a critical step in adaptive radiotherapy. Manually delineated organs-at-risk (OARs) contours on planning CT (pCT) scans are deformably registered onto daily cone-beam CT (CBCT) scans for delivered dose accumulation. However, evaluation of registered contours requires human assessment, which is time-consuming and subjects to high inter-observer variability. This work proposes a deep learning model that allows accurate prediction of Dice similarity coefficients (DSC) of registered contours in prostate radiotherapy.Approach. Our dataset comprises 20 prostate cancer patients with 37-39 daily CBCT scans each. The pCT scans and planning contours were deformably registered to each corresponding CBCT scan to generate virtual CT (vCT) scans and registered contours. The DSC score, which is a common contour-based validation metric for registration quality, between the registered and manual contours were computed. A Siamese neural network was trained on the vCT-CBCT image pairs to predict DSC. To assess the performance of the model, the root mean squared error (RMSE) between the actual and predicted DSC were computed.Main results. The model showed promising results for predicting DSC, giving RMSE of 0.070, 0.079 and 0.118 for rectum, prostate, and bladder respectively on the holdout test set. Clinically, a low RMSE implies that the predicted DSC can be reliably used to determine if further DIR assessment from physicians is required. Considering the event where a registered contour is classified as poor if its DSC is below 0.6 and good otherwise, the model achieves an accuracy of 92% for the rectum. A sensitivity of 0.97 suggests that the model can correctly identify 97% of poorly registered contours, allowing manual assessment of DIR to be triggered.Significance. We propose a neural network capable of accurately predicting DSC of deformably registered OAR contours, which can be used to evaluate eligibility for plan adaptation.

Association of Lunate Morphology With Progression to Scaphoid Fracture Nonunion.

Background: The purpose of our study was to review a series of patients with scaphoid fractures to determine whether there was an association between lunate morphology and progression to delayed union or nonunion when treated operatively or nonoperatively. Secondary aims included evaluation of the relationship between lunate morphology and scaphoid fracture location. Methods: A retrospective review of all patients with a diagnosis of scaphoid fracture was performed at our institution between 2014 and 2017. Medical records and radiographs were evaluated to determine lunate morphology, scaphoid fracture location, treatment, and time to union. Differences between groups were determined using χ2 analysis with significance set at P <.05. Multiple logistic regression analyses were used to evaluate scaphoid union in the setting of lunate morphology when controlling for confounders. Results: A total of 169 patients were included; 45.0% (n = 76) of patients had type I lunate morphology, and 55.0% (n = 93) had type II. In all, 64.5% (n = 49) of patients with type I lunate and 68.8% (n = 64) with type II lunate had a fracture at the scaphoid waist. Among all patients with a scaphoid fracture, type II lunates were more likely than type I lunates to progress to nonunion when treated both operatively and nonoperatively (18.3% vs 4.0%, P = .0042). Lunate facet size was not shown to be a significant risk factor for nonunion among patients with a type II lunate (P = .4221). Conclusions: Patients with a scaphoid fracture and type II lunate morphology were more likely to progress to nonunion than patients with a type I lunate.

Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients.

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.

Cardiovascular toxicities of androgen deprivation therapy in Asian men with localized prostate cancer after curative radiotherapy: a registry-based observational study.

BACKGROUND: Androgen deprivation therapy (ADT) and radiotherapy (RT) are the mainstay treatment for localized prostate cancer and recurrence after surgery. Cardiovascular (CV) toxicity of ADT is increasingly recognized, and the risk relates to pre-existing risk factors and ADT modalities. Despite ethnic differences in the prevalence of CV risk factors and variations of CV mortality, data on ADT-related cardiotoxicities in the Asian population remain inconclusive. Our registry-based study investigated ADT-related major adverse cardiovascular events (MACE) after primary or salvage RT. METHODS: Our study combined two prospectively established registry databases from National Cancer Center Singapore and National Heart Center Singapore. The primary endpoint is time to first MACE after treatment. MACE is defined as myocardial infarction, stroke, unstable angina, or cardiovascular death. Two types of propensity score adjustments, including ADT propensity score as a covariate in the multivariable regression model and propensity score weighting, were applied to balance baseline features and CV risk factors between RT alone and RT + ADT groups. RESULTS: From 2000 to 2019, 1940 patients received either RT alone (n = 494) or RT + ADT (n = 1446) were included. After a median follow-up of 10 years (RT) and 7.2 years (RT+ ADT), the cumulative incidence of MACE at 1, 3 and 9 years was 1.2, 5 and 16.2% in RT group, and 1.1, 5.2 and 17.6% in RT + ADT group, respectively. There were no differences in the incidence of MACE between 2 groups (HR 1.01, 95% CI 0.78-1.30, p = 0.969). Pre-treatment CV risk factors were common (80%), and CV disease (15.9%) was the second leading cause of death after prostate cancer (21.1%). On univariate analysis, older age, Indians and Malays, pre-existing CV risk factors, and history of MACE were associated with higher MACE risk. After propensity score adjustments, there remained no significant differences in MACE risk between RT + ADT and RT group on multivariable analysis. CONCLUSIONS: In our registry-based study, ADT is not associated with increased risk of major cardiovascular events among Southeast Asian men with prostate cancer after curative radiotherapy.

Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF-II cells.

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.

Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins.

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid ß-protein (Aß) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aß at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aß oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.