The Development and Characterisation of ssDNA Aptamers via a Modified Cell-SELEX Methodology for the Detection of Human Red Blood Cells.

Blood is one of the most commonly found biological fluids at crime scenes, with the detection and identification of blood holding a high degree of evidential value. It can provide not only information about the nature of the crime but can also lead to identification via DNA profiling. Presumptive tests for blood are usually sensitive but not specific, so small amounts of the substrate can be detected, but false-positive results are often encountered, which can be misleading. Novel methods for the detection of red blood cells based on aptamer-target interactions may be able to overcome these issues. Aptamers are single-stranded DNA or RNA sequences capable of undergoing selective antigen association due to three-dimensional structure formation. The use of aptamers as a target-specific moiety poses several advantages and has the potential to replace antibodies within immunoassays. Aptamers are cheaper to produce, display no batch-to-batch variation and can allow for a wide range of chemical modifications. They can help limit cross-reactivity, which is a hindrance to current forensic testing methods. Within this study, a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process was used to generate aptamers against whole red blood cells. Obtained aptamer pools were analysed via massively parallel sequencing to identify viable sequences that demonstrate a high affinity for the target. Using bioinformatics platforms, aptamer candidates were identified via their enrichment profiles. Binding characterisation was also conducted on two selected aptamer candidates via fluorescent microscopy and qPCR to visualise and quantify aptamer binding. The potential for these aptamers is broad as they can be utilised within a range of bioassays for not only forensic applications but also other analytical science and medical applications. Potential future work includes the incorporation of developed aptamers into a biosensing platform that can be used at crime scenes for the real-time detection of human blood.

Generating aptamers towards human sperm cells using massively parallel sequencing.

Determining the presence of sperm cells on an item or swab is often a crucial component of sexual offence investigation. However, traditional histological staining techniques used for the morphological identification of spermatozoa lack both specificity and sensitivity, making analysis a complex and time-consuming process. New methods for the detection of sperm cells based on aptamer recognition may be able to overcome these issues. In this work, we present the selection of ssDNA aptamers against human sperm cells using Cell-SELEX and massively parallel sequencing technologies. A total of 14 rounds of selection were performed following a modified Cell-SELEX protocol, which included additional steps for the isolation of spermatozoa from seminal fluid. Massively parallel sequencing using the Illumina Miseq platform was conducted on enriched aptamer pools to elucidate the structure of potential binders. A custom bioinformatics pipeline was also developed using Galaxy for the automated processing of sequencing datasets. This data revealed several promising aptamer candidates, which were shown to selectively bind sperm cells through both microscale thermophoresis and enzyme-linked oligonucleotide assays. These aptamers have the potential to increase the efficiency of sexual offence casework by facilitating sperm detection.

Monitoring Criminal Activity through Invisible Fluorescent "Peptide Coding" Taggants.

Complementing the demand for effective crime reduction measures are the increasing availability of commercial forensic "taggants", which may be used to physically mark an object in order to make it uniquely identifiable. This study explores the use of a novel "peptide coding" reagents to establish evidence of contact transfer during criminal activity. The reagent, containing a fluorophore dispersed within an oil-based medium, also includes a unique synthetic peptide sequence that acts as a traceable "code" to identify the origin of the taggant. The reagent is detectable through its fluorescent properties, which then allows the peptide to be recovered by swabbing and extracted for electrospray ionization-mass spectrometry (ESI-MS) analysis via a simple liquid-liquid extraction procedure. The performance of the reagent in variable conditions that mimic the limits of a real world use are investigated.

INTEGRATING HEALTH INTO BUILDINGS OF THE FUTURE.

The health and wellbeing of building occupants should be a key priority in the design, building, and operation of new and existing buildings. Buildings can be designed, renovated, and constructed to promote healthy environments and behaviors and mitigate adverse health outcomes. This paper highlights health in terms of the relationship between occupants and buildings, as well as the relationship of buildings to the community. In the context of larger systems, smart buildings and green infrastructure strategies serve to support public health goals. At the level of the individual building, interventions that promote health can also enhance indoor environmental quality and provide opportunities for physical activity. Navigating the various programs that use metrics to measure a building's health impacts reveals that there are multiple co-benefits of a "healthy building," including those related to the economy, environment, society, transportation, planning, and energy efficiency.

Enabling fluorescent biosensors for the forensic identification of body fluids.

The search for body fluids often forms a crucial element of many forensic investigations. Confirming fluid presence at a scene can not only support or refute the circumstantial claims of a victim, suspect or witness, but may additionally provide a valuable source of DNA for further identification purposes. However, current biological fluid testing techniques are impaired by a number of well-characterised limitations; they often give false positives, cannot be used simultaneously, are sample destructive and lack the ability to visually locate fluid depositions. These disadvantages can negatively affect the outcome of a case through missed or misinterpreted evidence. Biosensors are devices able to transduce a biological recognition event into a measurable signal, resulting in real-time analyte detection. The use of innovative optical sensing technology may enable the highly specific and non-destructive detection of biological fluid depositions through interaction with several fluid-endogenous biomarkers. Despite considerable impact in a variety of analytical disciplines, biosensor application within forensic analyses may be considered extremely limited. This article aims to explore a number of prospective biosensing mechanisms and to outline the challenges associated with their adaptation towards detection of fluid-specific analytes.

Nanomaterials for optical biosensors in forensic analysis.

Biosensors are compact analytical devices capable of transducing a biological interaction event into a measurable signal outcome in real-time. They can provide sensitive and affordable analysis of samples without the need for additional laboratory equipment or complex preparation steps. Biosensors may be beneficial for forensic analysis as they can facilitate large-scale high-throughput, sensitive screening of forensic samples to detect target molecules that are of high evidential value. Nanomaterials are gaining attention as desirable components of biosensors that can enhance detection and signal efficiency. Biosensors that incorporate nanomaterials within their design have been widely reported and developed for medical purposes but are yet to find routine employment within forensic science despite their proven potential. In this article, key examples of the use of nanomaterials within optical biosensors designed for forensic analysis are outlined. Their design and mechanism of detection are both considered throughout, discussing how nanomaterials can enhance the detection of the target analyte. The critical evaluation of the optical biosensors detailed within this review article should help to guide future optical biosensor design via the incorporation of nanomaterials, for not only forensic analysis but alternative analytical fields where such biosensors may prove a valuable addition to current workflows.

Fingermarks in wildlife forensics: A review.

Wildlife forensics is defined as providing forensic evidence to support legal investigations involving wildlife crime, such as the trafficking and poaching of animals and/ or their goods. While wildlife forensics is an underexplored field of science, the ramifications of poaching can be catastrophic. The consequences of wildlife crime include disease spread, species and habitat loss, human injury, and cultural loss. Efforts to use forensic science to combat poaching are currently limited to DNA-based techniques. However, fingermark analysis for the identification of perpetrators of wildlife crimes has not been explored to the same extent, despite being a cost-effective, simple-to-use forensic method that is easy to deploy in-field. This review covers literature that has explored fingermark examination techniques used on wildlife-related samples, such as pangolin scales, ivory-based substances, bone, and eggs, as well as feathers and skins, among more obscure trafficked items. Useful preliminary work has been conducted in this subject area, demonstrating that commonly used fingermark analysis techniques can be applied to wildlife-based items. However, many of these studies suffer from limitations in terms of experimental design. More work should be done on creating studies with larger sample sizes and novel approaches should be validated under environmental conditions that mimic real crime scenes. Further research into determining the forensic fingermark analysis techniques that perform the most efficiently in the environmental conditions of the countries where they are needed would therefore benefit legal investigations and help to reduce instances of poaching.

Hypotony in uveitis: an overview of medical and surgical management.

Hypotony is a well-recognised, sight-threatening complication of uveitis. It can also be the final common endpoint for a multitude of disease entities. Multiple mechanisms underlie hypotony, and meticulous clinical history alongside ocular phenotyping is necessary for choosing the best intervention and therapeutic management. In this narrative review, a comprehensive overview of medical and surgical treatment options for the management of non-surgically induced hypotony is provided. Management of ocular hypotony relies on the knowledge of the aetiology and mechanisms involved. An understanding of disease trajectory is vital to properly educate patients. Both anatomical and functional outcomes depend on the underlying pathophysiology and choice of treatment.

WireSafeTM - A pilot study of a novel safety engineered device designed to prevent guidewire retention and reduce sharps injuries during central venous catheter insertion.

Background: Guidewire retention and sharps injury during central venous catheter insertion are errors that cause patient and healthcare professional harm. The WireSafeTM is a novel procedure safety pack engineered to prevent guidewire retention and sharps injury during central venous catheter insertion. This is a pilot study aimed to determine its acceptability, usability and safety during clinical practice. Methods: An observational time and motion study was conducted comparing central venous catheter insertion and sharps disposal practice using standard versus WireSafeTM techniques. One-year following implementation, a structured survey was conducted to determine clinician opinion and experiences of using the WireSafeTM. Results: 15 procedures were observed using standard practice and 16 using the WireSafeTM technique. The WireSafeTM technique decreased the time taken from removal of the guidewire to disposal of sharps (standard 11.4 ± 5.6 min vs WireSafeTM 8.7 ± 1.4 min, p = 0.035), as well as total procedure time (standard 16 ± 7 min vs WireSafeTM 14.2 ± 2 min, p = 0.17), although this latter trend did not reach significance. Clinicians frequently practiced unsafe behaviour during sharps disposal in the standard group (53%), but when using the WireSafeTM technique, 100% exhibited safe practice by transferring sharps to the bin inside the sealed WireSafeTM box. One-year following implementation, 20 clinicians participated in the structured survey. Clinicians across three different departments used the WireSafeTM in varying clinical situations and reported that its use for central line insertion was either easier (10/20) or no different (10/20) compared to standard practice. All clinicians (20/20) felt that the WireSafeTM reduced the risk of guidewire retention and all stated that they approved of the WireSafeTM technique, and supported its use for convenience and safety benefits. Conclusion: Utilising the WireSafeTM for central line insertion facilitated earlier and safer sharps disposal, and the device was well supported by clinicians for its convenience and safety benefits.

The classification of handwriting features of the Kazakh language written in Latin script.

In 2017, the Republic of Kazakhstan began the phased transition of its alphabet from Cyrillic to Latin script. This transition has presented significant challenges to Kazakhstani document examiners, who have yet to develop appropriate methodologies for the analysis of handwriting samples written in the Kazakh language using Latin letters. This study aims to identify distinguishing macro and micro features of letters within Kazakh writing samples produced using the Latin alphabet and determine their frequencies of occurrence and discriminating power indices. Micro features were examined using the four most frequently appearing letters: "a", "y", "e" and "n". A comparative analysis of tested Latin letters with those of a similar configuration in Cyrillic demonstrated differences in the number of distinguishing features, as well as in the frequency of occurrence and discriminating power indices of similar features. These results show that separate statistical bases should be used for Latin and Cyrillic letters when analysing handwriting samples based on the frequencies of occurrence of micro and macro writing features.

The forensic investigation of vehicle-pedestrian collisions: A review.

Rapid urbanisation, a steady increase in the number of vehicles, speeding, negligence in road safety, and other factors have led to the inevitable worldwide growth of road traffic accidents involving pedestrians. According to the 'Global Status Report on Road Safety' released by the World Health Organization, road traffic collisions are one of the leading causes of death for people of all ages, with approximately 1.35 million road fatality deaths occurring globally each year. Figures from the report also highlight that a large part of road deaths involves pedestrians as the most vulnerable road users. Therefore, forensic examination of vehicle-pedestrian collisions has become increasingly important in the detection, investigation and reduction of road casualties and permanent development of this discipline is urgently needed. Thus, this article aims to review the capability and effectiveness of forensic examination in tackling road fatalities and explores the most important aspects of this discipline, such as nature of a vehicle-pedestrian collision, common issues resolved by this type of examination and typical physical evidence used in the reconstruction of vehicle-pedestrian collisions. Moreover, the paper outlines the latest advances and approaches in the field.

Feasibility of a handheld near infrared device for the qualitative analysis of bloodstains.

One of the most common tasks in criminal investigation is to determine from which tissue source a biological fluid stain originates. As a result, there are many tests that are frequently used to determine if a stain is blood, semen or saliva by exploiting the properties of certain molecules present within the fluids themselves. These include chemical reagents such as the Kastle-Meyer or Acid Phosphatase tests, as well as other techniques like the use of alternative light sources. However, most of the tests currently available have some major drawbacks. In this study, a handheld near-infrared spectrometer is investigated for the specific identification of deposited bloodstains. First, a calibration was carried out by scanning over 500 positive (blood present) and negative (blood absent) samples to train several predictive models based on machine learning principles. These models were then tested on over 100 new positive and negative samples to evaluate their performance. All models tested were able to correctly classify deposited stains as blood in at least 81% of tested samples, with some models allowing for even higher classification accuracy at over 94%. This suggests that handheld near infrared devices could offer great opportunity for the rapid, low cost and non-destructive screening of body fluids at scenes of crime.

Establishing evidence of contact transfer in criminal investigation by a novel 'peptide coding' reagent.

Forensic investigators are often faced with the challenge of forming a logical association between a suspect, object or location and a particular crime. This article documents the development of a novel reagent that may be used to establish evidence of physical contact between items and individuals as a result of criminal activity. Consisting of a fluorescent compound suspended within an oil-based medium, this reagent utilises the addition of short customisable peptide molecules of a specific known sequence as unique owner-registered 'codes'. This product may be applied onto goods or premises of criminal interest and subsequently transferred onto objects that contact target surfaces. Visualisation of the reagent is then achieved via fluorophore excitation, subsequently allowing rapid peptide recovery and analysis. Simple liquid-liquid extraction methods were devised to rapidly isolate the peptide from other reagent components prior to analysis by ESI-MS.

Application of fluorescent substrates to the in situ detection of prostate specific antigen.

The forensic identification of body fluids frequently presents an important source of genetic material and investigative interpretation. However, presumptive testing techniques presently employed in the discrimination of biological fluids are subject to criticism for poor specificity, lack of fluid localisation ability and detrimental effects on DNA recovery rates. The recognition of fluid-specific biomarkers by fluorogenic substrates may provide a novel resolution to these issues but research has yet to establish any pertinent in situ fluid detection applicability. This study therefore utilises a fluorogenic substrate (Mu-HSSKLQ-AFC) specific to the seminal protein prostate specific antigen in an effort to detect human semen deposited on a number of surfaces typical to criminal investigation. The ability of fluorescent fluorogenic substrates to simultaneously identify and visualise biological fluids in situ is demonstrated for the first time, whilst the production of complete STR profiles from fluid sources is also confirmed to be completely unaffected by substrate application.

An analysis with Bion: an interview with James Gooch. Interview by JoAnn Culbert-Koehn.

In 1968 Wilfred Bion moved to Los Angeles, escaping the perils of fame in London. He lived in Los Angeles until a few months before his death in Oxford in 1979. He made a deep impact on psychoanalysis in Los Angeles through those he analysed and what he wrote. James Gooch, psychiatrist and founding president of the Psychoanalytic Center of California describes in detail the transformative experience of his analysis with Bion in an interview with JoAnn Culbert-Koehn, Jungian analyst. Dr. Gooch describes important differences between his analysis with Bion and his classical Freudian analysis during his analytic training.