bioRxiv Science⌕ Search

Biology subjects

Kallupurackal, V.

Publications and source records attributed to Kallupurackal, V..

2 recordsLinked to original sources

Integrated Histology and Molecular Profiling of Postmortem Human Auditory and Vestibular Organs via a Poly(Methyl Methacrylate)-Based Workflow

Hearing and balance disorders are the most prevalent sensory impairments, affecting hundreds of millions worldwide, yet their underlying cellular and molecular pathologies remain poorly understood. This knowledge gap stems from the inaccessibility of the ears sensory organs--embedded within the temporal bone (TB), the hardest bone in the body--which cannot be biopsied in living patients without causing irreversible damage. Conventional histopathology workflows rely on postmortem en bloc extraction of TBs, followed by lengthy decalcification, celloidin embedding, and manual serial sectioning of these large specimens--a process that takes one to two years, is labor- and cost-intensive, and lacks compatibility with most modern protein, DNA, and RNA assays. Here, we present a rapid, reversible polymethyl methacrylate (rPMMA) workflow that enables advanced molecular histopathology studies on formalin-fixed, calcified TBs. Our protocol uses low-temperature (-40 {degrees}C to +4 {degrees}C) resin embedding, precision near-serial sectioning (10-50 {micro}m) via femtosecond laser microtomy or precision diamond wire sawing, and subsequent deacrylation to fully restore tissue accessibility for high-fidelity histomorphology, multiplexed immunofluorescence, whole-genome sequencing, and in situ mRNA detection (RNAscope) assays. Compared to the gold-standard celloidin workflow, our method reduces processing time and costs by approximately 90% while integrating equivalent histomorphology with advanced molecular assays, providing a new benchmark for multidimensional studies in human hearing and balance pathologies.

pathology↗

The ReAct project: Analysis of data from 23 differentlaboratories to characterise DNA recovery given twosets of activity level propositions

The ReAct (Recovery, Activity) project is an ENFSI (European Network of Forensic Science Institutes) supported initiative comprising a large consortium of laboratories. Here, the results from more than 23 laboratories are presented. The primary purpose was to design experiments simulating typical casework circumstances; collect data and to implement Bayesian networks to assess the value (i.e., likelihood ratio) of DNA results given activity level propositions. Two different experimental designs were used to simulate a robbery, where a screwdriver was used to force a door or window. Propositions and case information were chosen following laboratory feedback listing typical casework circumstances (included in the paper). Whereas the proposition representing prosecutions view is applicable to many cases: the defendant forced the door/window with his screwdriver; the proposition representing defences view are more variable depending on what is alleged to have happened. In a direct transfer experiment, the defendant owned and used the screwdriver, but he did not force the door/window in question. An unknown person used the defendants stolen screwdriver. In an indirect transfer experiment, the defendant neither owned, saw, nor used the screwdriver, nor did they force the door or window. Instead, an unknown individual--someone with whom the defendant had interacted (e.g., by shaking hands or touching the same objects)--used the recovered tool to force entry. In the first experiment, if the defendant forced the door/window, then s/he is the last handler of the screw driver, whereas if the tool was stolen and used by an unknown person, the defendant is the first handler. For the second experiment, given the defence view, the defendant never held the screwdriver. We envisaged the situation where an object manipulated by the defendant (or the defendant himself/herself) would be touched by the unknown offender who would then force the window. The time delay between the touching of the object and the forcing of the window was 0, 1h, and 2h. This was accomplished by handshaking experiments, where the person forcing the door would shake a persons hand, and then use the screwdriver either immediately after hand-shake or after 1, 2 hours. It was found for the direct transfer experiment that unless a single contributor profile aligning with the known persons of interest profile was retrieved, the results did not allow to discriminate propositions. On the other hand, for the indirect transfer experiment, both single and major contributor profiles that aligned with the person of interest (POI) supported the proposition that the person used the tool rather than an unknown person who had touched an object, when indeed the former was true. There was considerable variation in median recoveries of DNA between laboratories (between 200pg - 5ng) for a given experiment if quantities are taken into account. These differences affect the likelihood ratios given activity level propositions. More than 2,700 samples were analysed in the course of this study. Two different Bayesian Networks are made available via an open source application written in Shiny R: Shiny_React(). For comparison, all datasets were analysed using a qualitative method categorised into absent, single, balanced or major given contributors. The need for standardisation of methods is highlighted, along with the need for new methods to assess probability of laboratory dependent DNA recovery. Open access databases that are made freely available are important adjuncts

genetics↗