Characterising the fluctuation of microRNA expression throughout a full menstrual cycle

When processing a crime scene, obtaining a DNA profile that can identify an individual is extremely important. However, the identification of the body fluid that the sample was obtained from could provide key information of the events that occurred. microRNA (miRNA) expression analysis is a technique that has the potential to differentiate body fluids. The presence and expression of body fluid specific miRNA would provide a fast and effective tool for progressing crime scene investigation, especially alleged sexual assault cases. Forensic case work lacks methods for identifying vaginal material, venous blood, menstrual blood and aspermatozoic seminal fluid within samples. A large screening study followed by a 31 day study on five female volunteers was performed utilising RT-qPCR on a large panel of body fluid markers. Screening showed a selection of markers were suitable to differentiate each body fluid, in some cases however, expression fluctuated when analysed over a 31 day period. The data shows that hsa-miR-412 may be suitable for identifying menstrual blood, expression from markers hsa-miR-124 and hsa-miR-205 varied significantly over the 31 day period and between individuals and therefore were less suitable for body fluid identification. The data supports the use of miRNA markers for the identification of certain body fluids such as menstrual and venous blood; however markers for the identification of body fluids such as vaginal material and saliva require further investigation

Forensic MicroRNA Analysis of Body Fluids Relating to Sexual Assaults

DNA profiling has become a universal technique for identifying individuals for evidential use in courts of law. In more complex cases such as sexual assaults, identifying the origin of a stain or sample offers valuable information as to the events that occurred. Currently, many ‘in service’ body fluid identification (BFID) techniques are presumptive, require significant sample volumes and generate false positives. As such, the development of a highly specific and reliable BFID technique would be highly beneficial to forensic scientists in provide more informative and reliable evidence. MicroRNAs (miRNA) are short, stable, non-coding RNA’s which modulate gene expression. Expression of some of these miRNA are body fluid specific, making them a potentially robust tool for BFID. The possibility for the integration of a robust, miRNA based BFID technology for forensic casework employing stem-loop reverse transcription and qPCR analysis was the theme of the research presented here. To be incorporated into the workflow of current forensic laboratories, the protocol must be able to be carried out alongside current techniques with limited addition of cost, equipment, analysts and time. A range of custom designed miRNA markers were analysed on vaginal material, menstrual blood, saliva, venous blood, semen, seminal fluid and skin. Screening indicated specificity of hsa mir-124 to vaginal material, hsa-mir-10a, 135a and 888 to semen, hsa-mir-412 and 507 to menstrual blood, hsa-mir-144-3, 144-5, 142 and 451 to blood and although highly expressed in saliva, hsa-mir-205 was also observed in vaginal material. Universal expression was observed in hsa-mir-93, 508, 1260b and SNORD 47 providing a means of normalisation through the designation of these markers as endogenous controls. A combined panel of markers are presented which were capable of identifying all body fluids, excluding skin from single stains. The panel was successful at identifying certain mixtures such as semen within vaginal material but was unable to confirm saliva presence within vaginal material. Screening of hsa-mir 205 within vaginal material uncovered the observation that hsa-mir-205 was impacted by the use of female contraception. Once a full BFID panel was generated the robustness of the markers was further analysed across the menstrual cycle. No significant difference (p>0.001) was seen in markers highly expressed in vaginal material during screening (hsa-mir-124, 203a, 205). Expression of non specific markers highlighted the importance of the optimisation of input miRNA. Differential extraction of genetic material was found to be detrimental to miRNA sample integrity. As such, total DNA extraction was employed for vaginal swabs obtained from volunteers following unprotected sexual intercourse, markers hsa mir-10a, 135a and 888 were able to successfully detect semen presence for up to 96 hours. The data generated to date has highlighted a number of miRNA markers that provide a platform for BFID. The developed protocol is reliable and robust; requiring minimal optimisation and is capable of integration with current laboratory workflow with minimum implications to time and cost. The markers identified for BFID have been implemented within studies that are representative of real case scenarios, and have demonstrated their ability to be stable, specific and successful in the identification of certain body fluids. Overall, this research showcases a reliable and body fluid specific protocol capable of being performed alongside DNA profiling

Differentiating between monozygotic twins through methylation specific high resolution melt curve analysis

Although short tandem repeat profiling is extremely powerful in identifying individuals from crime scene stains, it is unable to differentiate between monozygotic (MZ) twins. Efforts to address this include mutation analysis through whole genome sequencing and through DNA methylation studies. Methylation of DNA is affected by environmental factors; thus, as MZ twins age, their DNA methylation patterns change. This can be characterized by bisulfite treatment followed by pyrosequencing. However, this can be time-consuming and expensive; thus, it is unlikely to be widely used by investigators. If the sequences are different, then in theory the melting temperature should be different. Thus, the aim of this study was to assess whether high-resolution melt curve analysis can be used to differentiate between MZ twins. Five sets of MZ twins provided buccal swabs that underwent extraction, quantification, bisulfite treatment, polymerase chain reaction amplification and high-resolution melting curve analysis targeting two markers, Alu-E2F3 and Alu-SP. Significant differences were observed between all MZ twins targeting Alu-E2F3 and in four of five MZ twins targeting Alu-SP (P<0.05). Thus, it has been demonstrated that bisulfite treatment followed by high-resolution melting curve analysis could be used to differentiate between MZ twins

A first-in-human phase 1 study of cavrotolimod, a TLR9 agonist spherical nucleic acid, in healthy participants: Evidence of immune activation

IntroductionTumor immunotherapy is designed to control malignancies through the host immune response but requires circumventing tumor-dysregulated immunomodulation through immunostimulation, relieving immunorepression, or a combination of both approaches. Here we designed and characterized cavrotolimod (formerly AST-008), an immunostimulatory spherical nucleic acid (SNA) compound targeting Toll-like receptor 9 (TLR9). We assessed the safety and pharmacodynamic (PD) properties of cavrotolimod in healthy participants in a first-in-human Phase 1 study under protocol AST-008-101 (NCT03086278; https://clinicaltrials.gov/ct2/show/NCT03086278).MethodsHealthy participants aged 18 to 40 years were enrolled to evaluate four dose levels of cavrotolimod across four cohorts. Each cohort included four participants, and all received a single subcutaneous dose of cavrotolimod. The dose levels were 5, 10, 12.5 and 18.8 µg/kg.Results and discussionCavrotolimod was well tolerated and elicited no serious adverse events or dose limiting toxicities at the doses tested. The results demonstrated that cavrotolimod is a potent innate immune activator, specifically stimulating Th1-type immune responses, and exhibits PD properties that may result in anti-tumor effects in patients with cancer. This study suggests that cavrotolimod is a promising clinical immunotherapy agent

Osteogenesis imperfecta: Ultrastructural and histological findings on examination of skin revealing novel insights into genotype-phenotype correlation

© 2016 Taylor & Francis. Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders of bone formation, resulting in low bone mass and an increased propensity to fracture. Over 90% of patients with OI have a mutation in COL1A1/COL1A2, which shows an autosomal dominant pattern of inheritance. In-depth phenotyping and in particular, studies involving manifestations in the skin connective tissue have not previously been undertaken in OI. The aims of the study were to perform histological and ultrastructural examination of skin biopsies in a cohort of patients with OI; to identify common and distinguishing features in order to inform genotype-phenotype correlation; and to identify common and distinguishing features between the different subtypes of OI. As part of the RUDY (Rare Diseases in Bone, Joints and/or Blood Vessels) study, in collaboration with the NIHR Rare Diseases Translational Research Collaboration, we undertook a national study of skin biopsies in patients with OI. We studied the manifestations in the skin connective tissue and undertook in-depth clinical and molecular phenotyping of 16 patients with OI. We recruited 16 patients: analyses have shown that in type 1 collagen mutation positive patients (COL1A1/ COL1A2) (n-4/16) consistent findings included: variable collagen fibril diameter (CFD) and presence of collagen flowers. Histological examination in these patients showed an increase in elastic fibers that are frequently fragmented and clumped. These observations provide evidence that collagen flowers and CFD variability are consistent features in OI due to type 1 collagen defects and reinforce the need for accurate phenotyping in conjunction with genomic analyses

Long-term efficacy, safety, and tolerability of Hizentra® for treatment of primary immunodeficiency disease

Hizentra® (20% subcutaneous immunoglobulin [SCIG]) was administered to subjects with primary immunodeficiency disease in two extension studies in the EU and US to assess long-term efficacy and tolerability. Subjects (aged 4–69 years) were treated for 148 weeks in the EU (N = 40; 5405 infusions) and 87 weeks in the US (N = 21; 1735 infusions). Weekly doses were 116.0 mg/kg (EU) and 193.2 mg/kg (US); IgG levels were 7.97 g/L (EU) and 11.98 g/L (US). Annualized rates of serious bacterial infections were 0.05 infections/subject/year (EU) and 0.06 infections/subject/year (US). Rates of any infection were 3.33 infections/subject/year (EU) and 2.38 infections/subject/year (US). The rate of bronchopulmonary infections was higher in the EU study. No treatment-related serious AEs occurred; no subject discontinued because of treatment-related AEs. Self-administered Hizentra afforded sustained effective protection from infections and favorable tolerability during an extended treatment period of up to 3 years