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Aim: For decades, the traditional approach for ligand-binding assays has been to generate two measurements from adjacent wells on the plate. In recent years, scientists have investigated the true benefit of this ‘duplicate analysis’ by looking back at previously generated bioanalytical data with the conclusion that the benefits are negligible. Materials & methods: We demonstrated a method development approach to determine the best number of replicate measurements of an immunogenicity assay. We used an anti-pembrolizumab immunogenicity assay on Gyrolab® to challenge the traditional use of duplicate measurements as we compare it to singlet measurement and show a balanced design for assessing the cut-point in singlet. Results & conclusion: We introduced the concept of calculating the maximum drug tolerance during method development. In this method, we found no practical benefit for duplicate analysis and go further in recommending that singlet analysis should be considered the default for all ligand-binding assays.

Brain capillary integrity is compromised during stroke (Hu 2017). Re-establishing the blood-brain barrier (BBB) post-stroke requires integration of cues from endothelial cells (EC), pericytes (PC), and the extracellular matrix (ECM) to promote vessel remodeling. Two ECM components, Types III and IV Collagen (Col-III and Col-IV, respectively), support vascular growth and maintenance. Here, we utilized a murine embryonic stem cell (ESC) model to investigate how ECM mis-regulation impacts PC and EC collagen synthesis. ECM stability was targeted by administering α,α-dipyridyl (iron chelator) (Ikeda 1992) or Type IV Collagenase to ESC-derived vessels. Our aim was to establish potential mechanistic roles of both Col-III and Col-IV in promoting vascular maintenance. We observed by qRT-PCR that α,α-dipyridyl administration decreased Col3a1 expression, but not Col4a1, in a dose-dependent manner. Analysis by immunostaining and confocal microscopy revealed increases in vascular-associated and interstitial Col-IV with the lower doses of α,α-dipyridyl, but decreases in both Col-IV regions at higher doses. Preliminary imaging data for Col-III, however, suggested decreases in both vascular-associated and interstitial Col-III with lower doses, but increases in these Col-III regions with the highest α,α-dipyridyl dose. In separate experiments, Type IV Collagenase exposure reduced both vascular and interstitial Col-IV despite apparent increases in Col3a1 and Col4a1 gene expression. Taken together, these data suggest that perturbations to the ECM microenvironment surrounding developing blood vessels can elicit various compensatory responses depending on the severity and specificity of the ECM component targeted. These observations, support the idea that dynamic remodeling of the microvessel wall, and specifically the surrounding ECM, may contribute to microvascular instability, as seen in the BBB following acute ischemic stroke.

Subsea HPHT components may be evaluated utilizing a fracture mechanics-based approach as per guidelines in API RP 17TR8 and ASME Section VIII Division 3. Typically, the assessment is performed based on methods described in API 579-1/ASME FFS-1 and BS7910. The analysis is performed to determine critical flaw sizes and estimate the fatigue life of a growing crack as a means of establishing inspection intervals for the equipment. In most cases, the assessment is based on a linear elastic fracture mechanics approach. The effect of plasticity is generally included via the use of a failure assessment diagram (FAD); however, even with this approach the effect of plastic strain around the crack is not explicitly considered. The assessment standards do not provide clear guidance for cases involving a crack which is located within a region of large-scale plastic strain. For these cases, API 579, Annex 9G.5 recommends utilizing a driving force method whereby the J-integral is directly evaluated from an elastic-plastic finite element model. This paper presents such an approach. A simplified representative geometry is considered for this study. A region of a stress concentration, such as is typically encountered near an internal radius is considered. Such a region can potentially show localized plasticity. J-integral is calculated by explicitly modeling a series of cracks of increasing depth through this zone of plasticity and the results are compared to the different methodologies described in API 579-1/ASME FFS-1 and BS7910. Cracks are modeled both completely and partially enveloped within the plastic zone. Results are summarized and compared, highlighting the key differences between different analysis approaches with the aim of identifying the most conservative assessment method for different crack sizes. Additionally, the effect of large-scale plasticity on the crack driving force is determined relative to similar conditions without plasticity. The results indicate that for cracks lying within the regions of localized plasticity, using an API579 Level 2 approach coupled with extracting elastic-plastic through wall stresses from an uncracked geometry may result in significant under prediction of the driving force. Conversely, extracting linear elastic stresses from an uncracked geometry may significantly over predict the driving force and may prove too conservative for determining acceptable crack sizes. This paper presents a comprehensive comparison of different analysis approaches used for evaluating cracks in subsea equipment. The results indicate that, for HPHT equipment with increased safety implications, a detailed elastic plastic fracture mechanics evaluation of the cracked geometry should be performed for cases in which localized plasticity is expected to occur.

Fracture mechanics assessments for pressure vessels are performed to determine critical flaw sizes and/or estimate the fatigue life of a growing crack as a means of establishing inspection intervals for the equipment. In most cases the evaluation is performed based on methods described in API 579-1/ASME FFS-1 and BS7910. The approaches described in these standards are mostly based on a linear elastic fracture mechanics approach. Even though plasticity can be accounted for by using a failure assessment diagram (FAD); however, even with this approach the effect of plastic strain around the crack is not explicitly considered. This paper presents an approach as per API 579, Annex 9G.5 which recommends utilizing a driving force method whereby the J-integral is directly evaluated from an elastic-plastic finite element model. The main goal is to study differences between the FAD approach against the elastic-plastic J-integral approach wherein the crack is modeled explicitly. Simplified representative geometries are considered for this study. Two scenarios for the plastic zone are considered a) crack present during initial loading with no residual plastic strain and b) crack in a residual stress zone. Different crack sizes are considered for this comparison study ranging from small cracks completely embedded within the plastic region and larger cracks with partial embedment. The paper presents comparison studies which highlight the key differences between different analysis approaches with the aim of identifying the most conservative assessment method for different crack geometries.

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of a ninth of diabetic patients. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges is observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood sugar, is rapidly reversed on a time scale of days, and often associated with a pericyte cell body located off-vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off-vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observe no pericyte loss relative to non-diabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy.

For biosimilar drug development programs, it is essential to demonstrate that there are no clinically significant differences between the proposed biosimilar therapeutic (biosimilar) and its reference product (originator). Based on a stepwise comprehensive comparability exercise, the biosimilar must demonstrate similarity to the originator in physicochemical characteristics, biological activity, pharmacokinetics, efficacy, and safety, including immunogenicity. The goal of the immunogenicity assessment is to evaluate potential differences between the proposed biosimilar product and the originator product in the incidence and severity of human immune responses. Establishing that there are no clinically meaningful differences in the immune response between the products is a key element in the demonstration of biosimilarity. An issue of practical, regulatory, and financial importance is to establish whether a two-assay (based on the biosimilar and originator respectively) or a one-assay approach (based on the biosimilar) is optimal for the comparative immunogenicity assessment. This paper recommends the use of a single, biosimilar-based assay for assessing immunogenic similarity in support of biosimilar drug development. The development and validation of an ADA assay used for a biosimilar program should include all the assessments recommended for an innovator program (10-16, 29). In addition, specific parameters also need to be evaluated, to gain confidence that the assay can detect antibodies against both the biosimilar and the originator. Specifically, the biosimilar and the originator should be compared in antigenic equivalence, to assess the ability of the biosimilar and the originator to bind in a similar manner to the positive control(s), as well as in the confirmatory assay and drug tolerance experiments. Practical guidance for the development and validation of anti-drug antibody (ADA) assays to assess immunogenicity of a biosimilar in comparison to the originator, using the one-assay approach, are described herein.

Marine Isotope Stage 11 (MIS 11) from ∼424,000 to 374,000 yrs ago included one of the longest and warmest interglacials of the last 800,000 yrs, and is a potential analogue for the Holocene due to the similarity of Earth's orbital configuration at this time. The question of how the El Nino–Southern Oscillation (ENSO) responds to warmer background climates remains unanswered and is critical to understand how the ENSO system will evolve under the influence of anthropogenic warming. In this study, we present a 35 yr-long, high-resolution record of MIS 11 climate variability in the Western Pacific Warm Pool (WPWP) based on coupled measurements of skeletal Mg/Ca and δ18O in giant Tridacna gigas clams from Huon Peninsula, Papua New Guinea. The δ18O of modern T. gigas from Huon Peninsula faithfully records sea surface temperature, salinity/rainfall and regional ENSO variability. The geochemical integrity of the MIS 11 T. gigas for recording paleo-ENSO events was established through trace element screening, detailed petrography and SEM analysis. The fossil T. gigas δ18O record indicates that ENSO was operating during a 35-yr window in MIS 11, but with fewer events of shorter duration compared to those experienced during the last 100 yrs. The suppressed ENSO variability in the MIS 11 T. gigas record corresponds with a reduction in the amplitude of the average annual cycle in δ18O values. Distinctive changes in local insolation seasonality, and T. gigas δ18O, brought about by changes in Earth's orbit, provide an additional geochronological constraint on the timing of reef growth at Huon Peninsula to around 402 ka during the MIS 11.3 sub-stage (∼424–395 ka).

Sediment-routing systems continuously transfer information and mass from eroding source areas to depositional sinks. Understanding how these systems alter environmental signals is critical when it comes to inferring source-area properties from the sedimentary record. We measure cosmogenic 10Be and 26Al along three large sediment-routing systems (~ 100,000 km2) in central Australia with the aim of tracking downstream variations in 26Al/10Be inventories and to identify the factors responsible. By comparing 56 new cosmogenic 10Be and 26Al measurements in stream sediments with matching data (n = 55) from source areas, we show that 26Al/10Be inventories in hillslope bedrock and soils set the benchmark for relative downstream modifications. Lithology is the primary determinant of erosion-rate variations in source areas and despite sediment mixing over hundreds of kilometres downstream a distinct lithological signal is retained. Postorogenic ranges yield catchment erosion rates of ~ 6–11 m/m.y. and silcrete-dominant areas erode as slow as ~ 0.2 m/m.y. 26Al/10Be inventories in stream-sediments reveal overall downstream-increasing minimum cumulative burial terms up to ~ 1.1 m.y. but more generally ~ 400–800 k.y. The magnitude of the burial signal correlates with increasing sediment cover downstream and reflects assimilation from storages with long exposure histories, such as alluvial fans, desert pavements, alluvial plains, and aeolian dunes. We propose that the tendency for large alluvial rivers to mask their 26Al/10Be source-area signal differs according to geomorphic setting. Signal preservation is favoured by i) high sediment supply rates, ii) high mean runoff, and iii) a thick sedimentary basin pile. Conversely, signal masking prevails in landscapes of i) low sediment supply, ii) discontinuous sediment flux, and iii) juxtaposition of sediment storages with notably different exposure histories.