A maturity model for the scientific review of clinical trial designs and their informativeness.

BACKGROUND: Informativeness, in the context of clinical trials, defines whether a study's results definitively answer its research questions with meaningful next steps. Many clinical trials end uninformatively. Clinical trial protocols are required to go through reviews in regulatory and ethical domains: areas that focus on specifics outside of trial design, biostatistics, and research methods. Private foundations and government funders rarely require focused scientific design reviews for these areas. There are no documented standards and processes, or even best practices, toward a capability for funders to perform scientific design reviews after their peer review process prior to a funding commitment. MAIN BODY: Considering the investment in and standardization of ethical and regulatory reviews, and the prevalence of studies never finishing or failing to provide definitive results, it may be that scientific reviews of trial designs with a focus on informativeness offer the best chance for improved outcomes and return-on-investment in clinical trials. A maturity model is a helpful tool for knowledge transfer to help grow capabilities in a new area or for those looking to perform a self-assessment in an existing area. Such a model is offered for scientific design reviews of clinical trial protocols. This maturity model includes 11 process areas and 5 maturity levels. Each of the 55 process area levels is populated with descriptions on a continuum toward an optimal state to improve trial protocols in the areas of risk of failure or uninformativeness. CONCLUSION: This tool allows for prescriptive guidance on next investments to improve attributes of post-funding reviews of trials, with a focus on informativeness. Traditional pre-funding peer review has limited capacity for trial design review, especially for detailed biostatistical and methodological review. Select non-industry funders have begun to explore or invest in post-funding review programs of grantee protocols, based on exemplars of such programs. Funders with a desire to meet fiduciary responsibilities and mission goals can use the described model to enhance efforts supporting trial participant commitment and faster cures.

Forensic genetic genealogy using microarrays for the identification of human remains: The need for good quality samples - A pilot study.

The successful application of forensic genetic genealogy (FGG) to identify Jane and John Doe cases in the United States has raised the prospect of using the technique in Australia to assist in the reconciliation of unidentified human remains (UHRs) with long term missing persons. A study was conducted to explore the feasibility of FGG using whole genome array (WGA) data from both pristine control samples as well as compromised casework samples, with the view to explore how DNA quantity and quality impacted on the ability to generate search results when compared to a genetic genealogy database, such as GEDmatch. From this study, several insights were gained as to the impact DNA quantity and degradation had on the percentage of SNPs genotyped and heterozygote/homozygote ratio - which are critical for successful matching outcomes. It was noted in this study (using a control sample) that successful matching occurred when genotyping errors were 5% or less. Two UHR cases were matched to kits on GEDmatch PRO, which provided investigative leads for identification purposes. The effectiveness of the FGG approach to match casework samples (as well as volunteer samples used in the study) is indicative of the usage of 'direct-to-consumer' (DTC) genetic testing by Australians. Given the (often) limited availability of casework samples, findings from this study will assist Australian agencies considering the use of FGG, to determine if WGA is a suitable method for their application.

Rapid DNA from a disaster victim identification perspective: Is it a game changer?

As an emerging technology, Rapid DNA has demonstrated its utility for law enforcement in the provision of DNA profiling data at the point of arrest, often not requiring analyst review of the profiles generated. Recently, efforts have centred on the evaluation of Rapid DNA (without analyst review) and modified Rapid DNA (requiring review by a trained analyst) for application to crime scene samples. In a broader forensic context, however, another application for Rapid DNA is its use to process post-mortem samples to assist with the identification of deceased persons; and while gaps in our knowledge remain as to how Rapid DNA instruments perform with these sample types (often compromised with regards to their yield and quality of DNA), they have been successfully deployed in the field to assist in the identification of disaster victims (as exemplified during the 2018 Californian wildfire). This review aims to provide the current research landscape for the forensic application of Rapid DNA as an emerging technology from a Disaster Victim Identification perspective.

Application of the Droplet Digital Polymerase Chain Reaction (ddPCR) Platform for Detection and Quantification of Vertebrate Host DNA in Engorged Mosquitoes.

Hematophagous arthropod bloodmeal identification has remained a challenge in the field of vector biology, but these studies are important to understand blood feeding patterns of arthropods, spatial, and temporal patterns in arbovirus transmission cycles, and risk of human and veterinary disease. We investigated the use of an existing vertebrate primer set for use on the droplet digital polymerase chain reaction (ddPCR) platform, to explore the use of this technology in the identification and quantification of vertebrate DNA in mosquito blood meals. Host DNA was detectable 48-h post-engorgement in some mosquitoes by ddPCR, compared with 24-h post-engorgement using traditional PCR. The capability of ddPCR for absolute quantification of template DNA offers unique potential applications of this new technology to field studies on the ecology of vector-borne diseases, but currently with limited scope.

Enterprise Implementation of Digital Pathology: Feasibility, Challenges, and Opportunities.

Digital pathology is becoming technically possible to implement for routine pathology work. At our institution, we have been using digital pathology for second opinion intraoperative consultations for over 10 years. Herein, we describe our experience in converting to a digital pathology platform for primary pathology diagnosis. We implemented an incremental rollout for digital pathology on subspecialty benches, beginning with cases that contained small amounts of tissue (biopsy specimens). We successfully scanned over 40,000 slides through our digital pathology system. Several lessons (both challenges and opportunities) were learned through this implementation. A successful conversion to digital pathology requires pre-imaging adjustments, integrated software and post-imaging evaluations.

Adaptive Biomedical Innovation: Evolving Our Global System to Sustainably and Safely Bring New Medicines to Patients in Need.

The current system of biomedical innovation is unable to keep pace with scientific advancements. We propose to address this gap by reengineering innovation processes to accelerate reliable delivery of products that address unmet medical needs. Adaptive biomedical innovation (ABI) provides an integrative, strategic approach for process innovation. Although the term "ABI" is new, it encompasses fragmented "tools" that have been developed across the global pharmaceutical industry, and could accelerate the evolution of the system through more coordinated application. ABI involves bringing stakeholders together to set shared objectives, foster trust, structure decision-making, and manage expectations through rapid-cycle feedback loops that maximize product knowledge and reduce uncertainty in a continuous, adaptive, and sustainable learning healthcare system. Adaptive decision-making, a core element of ABI, provides a framework for structuring decision-making designed to manage two types of uncertainty - the maturity of scientific and clinical knowledge, and the behaviors of other critical stakeholders.

Citrulline as a Biomarker for Gastrointestinal-Acute Radiation Syndrome: Species Differences and Experimental Condition Effects.

Animal models of hematopoietic and gastrointestinal acute radiation syndromes (ARS) have been characterized to develop medical countermeasures. Acute radiation-induced decrease of intestinal absorptive function has been correlated to a decrease in the number of intestinal crypt cells resulting from apoptosis and enterocyte mass reduction. Citrulline, a noncoded amino acid, is produced almost exclusively by the enterocytes of the small intestine. Citrullinemia has been identified as a simple, sensitive and suitable biomarker for radiation-induced injury associated with gastrointestinal ARS (GI-ARS). Here we discuss the effect of radiation on plasma citrulline levels in three different species, C57BL/6 mice, Göttingen minipigs and rhesus nonhuman primates (NHPs), measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of experimental study conditions such as feeding and anesthesia were also examined on plasma citrulline levels in the NHPs. Both the mice and Göttingen minipigs were partial-body irradiated (PBI) with doses from 13-17 Gy and 8-16 Gy, respectively, whereas NHPs were total-body irradiated (TBI) with doses from 6.72-13 Gy. Blood samples were taken at different time points and plasma citrulline levels were measured in the three species at baseline and after irradiation. Basal plasma citrulline concentrations (mean ± SEM) in mice and minipigs were 57.8 ± 2.8 µM and 63.1 ± 2.1 µM, respectively. NHPs showed a basal plasma citrulline concentration of 32.6 ± 0.7 µM, very similar to that of humans (∼40 µM). Plasma citrulline progressively decreased after irradiation, reaching nadir values between day 3.5 and 7. The onset of citrulline recovery was observed earlier at lower radiation doses, while only partial citrulline recovery was noted at higher radiation doses in minipigs and NHPs, complete recovery was noted in mice at all doses. Plasma citrulline levels in NHPs anesthetized with ketamine and acepromazine significantly decreased by 35.5% (P = 0.0017), compared to unanesthetized NHPs. In the postprandial state, citrulline concentrations in NHPs were slightly but significantly decreased by 12.2% (P = 0.0287). These results suggest that plasma citrulline is affected by experimental conditions such as anesthesia and feeding.

The contributions of anthropology and mitochondrial DNA analysis to the identification of the human skeletal remains of the Australian outlaw Edward 'Ned' Kelly.

This paper details the anthropological and genetic analyses that contributed to the identification of the notorious Australian outlaw ('bushranger') Edward ('Ned') Kelly. In 1880 at the age of 25, Kelly was hanged and buried at the former Melbourne Gaol in Victoria, Australia. In 1929, the remains of executed prisoners (including those of Kelly) were haphazardly disinterred following the demolition of parts of the Melbourne Gaol and haphazardly reinterred in three distinct "pits" at the Pentridge Prison. In 1999 the Pentridge Prison was sold for commercial development and subsequently in 2008 and 2009 the human remains of prisoners were recovered. A total of 41 cases of unidentified human skeletal remains from Pentridge were examined using traditional anthropological techniques. At least one representative sample from each of the remains (mostly clavicles) from all three pits was selected for DNA analysis. Comparative ante-mortem reference samples were also located. Given the antiquity and condition of remains recovered from Pentridge, and the 130 years that had passed since Kelly's execution, mitochondrial DNA analysis was chosen as a suitable DNA analysis tool to examine the Pentridge cases to assist in the inclusion or exclusion of remains as being those of Ned Kelly. Only one of the Pentridge cases (Pen14) matched the HV1/HV2 mitochondrial DNA haplotype of the reference sample. Additional anthropological analyses indicated a number of pathological features that provided support that the remains of Pen14 are those of Edward ("Ned") Kelly.

Roles of type VI collagen and decorin in human mesenchymal stem cell biophysics during chondrogenic differentiation.

Human mesenchymal stem cells (hMSCs) induced towards chondrogenesis develop a pericellular matrix (PCM), rich in type VI collagen (ColVI) and proteoglycans such as decorin (DCN). Individual PCM protein functions still need to be elucidated to fully understand the mechanobiological role of this matrix. In this study we identified ColVI and DCN as important contributors in the mechanical function of the PCM and as biochemical modulators during chondrogenesis through targeted knockdown using shRNA lentiviral vectors. Gene expression, western blotting, immunofluorescence and cell deformation analysis were examined at 7, 14 and 28 days post chondrogenic induction. ColVI and DCN knockdown each affected gene expression of acan, bgn, and sox9 during chondrogenesis. ColVI was found to be of central importance in resisting applied strains, while DCN knockdown had strain dependent effects on deformation. We demonstrate that by using genetic engineering to control the biophysical microenvironment created by differentiating cells, it may be possible to guide cellular mechanotransduction.

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers.

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E2 (PGE2) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3 µM) used alone or in combination with the P2Y12 antagonist cangrelor (1 µM), both without and with aspirin (100 µM). Platelet aggregation and P-selectin expression were measured in whole blood (n = 10) following stimulation with the thromboxane A2 (TXA2) mimetic U46619 (0.3 or 1 µM) in combination with either the EP3 agonist sulprostone (0.1 µM), or PGE2 (1 µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n = 42) were randomly assigned to receive no background treatment, clopidogrel (300 mg loading dose plus 75 mg daily) or clopidogrel and aspirin (75 mg daily) for 10 days alongside DG-041 (200 mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y12 antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y12 blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.

Small-world topology of functional connectivity in randomly connected dynamical systems.

Characterization of real-world complex systems increasingly involves the study of their topological structure using graph theory. Among global network properties, small-world property, consisting in existence of relatively short paths together with high clustering of the network, is one of the most discussed and studied. When dealing with coupled dynamical systems, links among units of the system are commonly quantified by a measure of pairwise statistical dependence of observed time series (functional connectivity). We argue that the functional connectivity approach leads to upwardly biased estimates of small-world characteristics (with respect to commonly used random graph models) due to partial transitivity of the accepted functional connectivity measures such as the correlation coefficient. In particular, this may lead to observation of small-world characteristics in connectivity graphs estimated from generic randomly connected dynamical systems. The ubiquity and robustness of the phenomenon are documented by an extensive parameter study of its manifestation in a multivariate linear autoregressive process, with discussion of the potential relevance for nonlinear processes and measures.

Mutant allele-specific imbalance modulates prognostic impact of KRAS mutations in colorectal adenocarcinoma and is associated with worse overall survival.

The prognostic impact of distinct KRAS mutations in colorectal carcinomas is not fully characterized. We hypothesized that the prognostic impact of KRAS mutations is modulated by KRAS mutant allele-specific imbalance (MASI). KRAS MASI was assessed by sequencing electropherograms in KRAS-mutated colorectal carcinomas (N = 394, prospectively tested). The mechanism of KRAS MASI was studied by fluorescence in situ hybridization (FISH; N = 50). FISH showed that KRAS MASI developed by chromosome 12 hyperploidy (9/18, 50%) or KRAS amplification (1/18, 5.5%). KRAS MASI was more common in tumors with KRAS codon 13 than with codon 12 mutations [24/81, 30% vs. 54/313, 17%; odds ratio (OR), 2.0, 95% confidence interval (CI), 1.2-3.5; p = 0.01]. KRAS MASI was correlated with overall survival (N = 358, median follow-up = 21 months). In a multivariate analysis, KRAS codon 13 MASI was an independent adverse prognostic factor (compared to codon 13 mutants without MASI combined with all codon 12 mutants; adjusted hazard ratio, 2.2, 95% CI: 1.2-3.9; p = 0.01). KRAS MASI arises through chromosome 12 hyperploidy or KRAS amplification and, when affects KRAS codon 13, is associated with worse overall survival.

Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse.

Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sperm lyophilized from fresh or frozen-thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze-thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.

The role of nonlinearity in computing graph-theoretical properties of resting-state functional magnetic resonance imaging brain networks.

In recent years, there has been an increasing interest in the study of large-scale brain activity interaction structure from the perspective of complex networks, based on functional magnetic resonance imaging (fMRI) measurements. To assess the strength of interaction (functional connectivity, FC) between two brain regions, the linear (Pearson) correlation coefficient of the respective time series is most commonly used. Since a potential use of nonlinear FC measures has recently been discussed in this and other fields, the question arises whether particular nonlinear FC measures would be more informative for the graph analysis than linear ones. We present a comparison of network analysis results obtained from the brain connectivity graphs capturing either full (both linear and nonlinear) or only linear connectivity using 24 sessions of human resting-state fMRI. For each session, a matrix of full connectivity between 90 anatomical parcel time series is computed using mutual information. For comparison, connectivity matrices obtained for multivariate linear Gaussian surrogate data that preserve the correlations, but remove any nonlinearity are generated. Binarizing these matrices using multiple thresholds, we generate graphs corresponding to linear and full nonlinear interaction structures. The effect of neglecting nonlinearity is then assessed by comparing the values of a range of graph-theoretical measures evaluated for both types of graphs. Statistical comparisons suggest a potential effect of nonlinearity on the local measures-clustering coefficient and betweenness centrality. Nevertheless, subsequent quantitative comparison shows that the nonlinearity effect is practically negligible when compared to the intersubject variability of the graph measures. Further, on the group-average graph level, the nonlinearity effect is unnoticeable.

Successful cryopreservation of expanded equine blastocysts.

Effective cryopreservation of expanded equine blastocysts (> 300 µm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 µm) were vitrified in fine-diameter microloader pipette tips using dimethylsulfoxide-containing medium (DM) or ethylene glycol-containing medium (EG). A third group was vitrified with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrification, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300-730 µm in diameter were then biopsied and vitrified; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P = 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele fluid after biopsy was associated with higher survival. Therefore, an altered ("Central") biopsy technique was used to aspirate blastocoele fluid, followed by vitrification in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P = 0.1). Finally, expanded blastocysts 407 to 565 µm in diameter were biopsied from the periphery, and blastocoele fluid was removed with gentle suction. After vitrification with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These findings demonstrated that blastocoele collapse and vitrification in fine-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts.

Examples of kinship analysis where Profiler Plus™ was not discriminatory enough for the identification of victims using DNA identification.

The identification of the victims of the 2009 Victorian bushfires disaster, as in other mass disasters, relied on a number of scientific disciplines - including DNA analysis. As part of the DVI response, DNA analysis was performed to assist in the identification of victims through kinship (familial matching to relatives) or direct (self source of sample) matching of DNA profiles. The majority of the DNA identifications made (82%) were achieved through kinship matching of familial reference samples to post mortem (PM) samples obtained from the victims. Although each location affected by the bushfires could be treated as a mini-disaster (having a small closed-set of victims), with many such sites spread over vast areas, DNA analysis requires that the short tandem repeat (STR) system used be able to afford enough discrimination between all the DVI cases to assign a match. This publication highlights that although a 9-loci multiplex was sufficient for a DVI of this nature, there were instances that brought to light the short comings of using a 9-loci multiplex for kinship matching--particularly where multiple family members are victims. Moreso it serves to reinforce the recommendation that a minimum of 12 autosomal STR markers (plus Amelogenin) be used for DNA identification of victims which relies heavily on kinship matching.

The contribution of DNA to the disaster victim identification (DVI) effort.

As part of the disaster victim identification (DVI) response to the 2009 Victorian bushfires disaster, a number of scientific disciplines contributed to the human identification process--forensic pathology, anthropology and odontology, as well as fingerprinting and DNA profiling. The DNA laboratory received 182 post-mortem (PM) samples from 120 DVI cases and 236 reference samples corresponding to 163 missing persons (and two non-DVI cases). DNA analysis yielded full DNA profiles for 102 DVI cases and 190 ante-mortem (AM) samples (relating to all 163 missing persons), respectively. Subsequent comparison of DNA profiles, through direct and kinship matching, resulted in the submission of 76 DNA reports to the DVI Reconciliation Centre which assisted in the identification of 67 deceased. This paper describes the contribution of DNA analysis towards the DVI response to the 2009 Victorian bushfires disaster.