The Franciscan ‘Revolution’ Reconsidered

This paper reconsiders the assumption that the textual and visual output of the Franciscan Order is informed by a limpid naturalism which, in turn, enables viewers (and readers) a seamless immersive experience. By focusing on the rhetorical descriptions of Francis of Assisi’s stigmata, the paper reveals the modes whereby those unprecedented wounds were displayed and disseminated in texts and images in the first half of the duecento. The challenge facing the Order was the spectacularisation of a phenomenon that was deemed a profound enigma, and the details of which were guarded jealously as “secrets” by Francis. Consequently, the depiction of the alter Christus was implicated in a complex network of issues that subverted medieval notions of imitation, witness, participation, and representation

Establishing Larval Zebrafish as an In Vivo Model Organism for Characterizing the Roles of Otoferlin, a Sensory Hair Cell Protein Essential for Hearing

Auditory defects and disorders are prevalent at all ages and affect 8% of the population in developed nations including newborns and children. Congenital hearing loss is the most common birth defect and it is estimated that 1 in 1000 children are affected by deafness at birth or before the onset of speech. Most of these children suffer from non-syndromic hearing loss, where hearing loss is the sole symptom with no other associated symptoms. More than two-thirds of non-syndromic hearing loss cases are of genetic origin, with approximately 80% inherited in an autosomal recessive mode. Autosomal recessive deafness is attributed to DFNB loci. Different DFNB loci have been reported and more than 50 genes have been identified to date. One such gene that encodes otoferlin, OTOF is responsible for the DFNB9 nonsyndromic form of deafness, which accounts for up to 8% of all prelingual autosomal recessive nonsyndromic deafness. Patients with mutations in otoferlin suffer from profound sensorineural prelingual non-syndromic hearing loss. Otoferlin belongs to the ferlin family of proteins. Ferlins are a group of multi-C2 domain proteins with emerging roles in vesicle fusion, membrane trafficking and repair. Otoferlin has six C2 domains including a C-terminal trans-membrane domain and these C2 domains bind to calcium, phospholipids and SNARE proteins. Otoferlin has been proposed to play a role in exocytosis of synaptic vesicles at the auditory inner hair cell synapse and also contributes to the development of the auditory cell synapse. Ablation of this gene causes abolition of exocytosis and improper targeting of synaptic components in sensory hair cells. More than 60 pathogenic mutations have been reported and are distributed in different C2, non-C2, and transmembrane domains of otoferlin. Despite the fact that otoferlin is a major contributor to sensorineural deafness, very little is known about the actual role of this protein in sensory hair cells. Currently, the field is restricted by a lack of an in vivo model that could facilitate rapid analysis and easy characterization of otoferlin. Most studies have used mouse and rat models where, hair cells are few in number and require harvesting through time-consuming micro-dissections of the animal ear. Given the importance of otoferlin in hair cell development and function, and the lack of an easy genetically manipulatable model, the goal of this thesis was to develop a more tractable organismal model to establish the role of otoferlin. Over the past years, zebrafish has emerged as an ideal model to study vertebrate development. It allows rapid assessment of gene function in vivo because it is amenable to genetic manipulation. Most importantly, the transparency of the zebrafish embryos allows direct visualization of tissue morphogenesis as it occurs in a live organism. Moreover, zebrafish possesss the easily accessible lateral line system, comprised of clusters of sensory hair cells, which are similar to mammalian type II vestibular hair cells and have similar morphological and anatomical structure. These sensory cells possesss synaptic ribbon bodies composed of L-type voltage activated calcium channels, a VGlut3 transporter for glutamate loading in synaptic vesicles, and develop a post-synapse upon maturation. Despite all these structural similarities zebrafish hair cells possesss only 3-5 synapses per hair cell. The aim here is to characterize the role of otoferlin by generating zebrafish models for otoferlinopathy. Here we show for the first time that otoferlin is conserved in all species including zebrafish. However, due to a zebrafish genome duplication event, the otoferlin gene is paralogous, and there are two different subtypes which we named otoferlin a (encoded on chromosome 20) and otoferlin b (encoded on chromosome 17). Otoferlin a is the longer gene and has all six C2 domains, whereas otoferlin b is the shorter gene and lacks an N-terminal C2 domain. Otoferlin expression starts early during larval development and coincides with the onset of sensory morphogenesis and maturation. The otoferlin a isoform is strictly restricted to the hair cells of the developing otic region, whereas otoferlin b is distributed in the hair cells of the otic region and lateral line neuromasts. At the cellular level, otoferlin distribution is observed in the supranuclear and basolateral regions of hair cells. Depleting both isoforms of otoferlin results in auditory and vestibular defects in larval zebrafish, and larvae exhibit abnormal swimming behavior. Moreover, larvae fail to develop an inflated swim bladder and develop a curved spine phenotype that becomes more prominent with development and facilitates a circling swimming behavior. These otoferlin-depleted zebrafish morphants belong to a class of ‘circler’ mutants that possesss similar hearing and balance defects. At the molecular level, sensory hair cells of otoferlin-depleted zebrafish developed atypical synapses comprised of tightly coalesced synaptic ribbon bodies, diffusely distributed VGlut3 and a dense and enlarged post-synapse. However, these hair cells possesssed an intact calcium response to mechanical deflection of hair bundles indicating an absence of mechanotransduction defects upon otoferlin depletion. Furthermore, otoferlin- depleted zebrafish exhibited severe defects in endocytotic dye uptake predominantly in the basolateral region of neuromast hair cells. Studies have identified several otoferlin-interacting partners, however, a comprehensive study on otoferlin mutants is lacking. Also lacking is a more vivid description of the genes regulating the circler phenotype during development and maturation. For the first time we report a high-throughput transcriptomic analysis of otoferlin-depleted zebrafish in an attempt to characterize the circler phenotype in larvae that also exhibit abnormal synaptic development. This study validates zebrafish as a model for high-throughput studies of the auditory and vestibular circler motility mutants, including otoferlin mutants. In this high-throughput transcriptomic study we identify several novel transcripts that are up or downregulated due to otoferlin depletion in zebrafish larvae. Some of these transcripts are very specific to the lateral line neuromasts and otic vesicle and have been shown to play roles during morphogenesis and development of the zebrafish sensory and neuronal regions. Moreover, there is a correlation between altered levels of some of these novel transcripts in otoferlin-depleted sensory cells and their synaptic morphology; hair cells with reduced levels of identified transcripts show an abnormal synaptic morphology indicated by coalesced distribution of synaptic ribbons when compared with control siblings. Finally, this is also the first study that shows that the rescue of gross phenotypic loss due to otoferlin depletion is possible by co-injecting otoferlin-depleted larvae with the p5E-pmyo6b hair cell specific vector containing full length (FL) and truncated versions of mouse otoferlin constructs. This further reiterates that otoferlin is indeed conserved across species and that gross rescue of the phenotype can be achieved with either mouse otoferlin FL or truncated constructs that contains as little as just one C-terminal C2 domain with the trans-membrane region. This also provides a sense of which otoferlin domains are sufficient to rescue synaptic activity. Furthermore, we also see rescue in levels of reduced transcripts that are derived from the transcriptomic analysis when otoferlin-depleted larvae are coinjected with the p5E-pmyo6b mouse FL vector construct. Overall, these studies successfully establish zebrafish as an in vivo model organism to characterize defects associated with otoferlin loss, both at the molecular and transcriptomic levels in a much shorter span of time compared to mouse-based studies. They also show a promising application of otoferlin trans-species rescue using hair-cell-specific otoferlin rescue contructs that correct for loss of overall auditory and vestibular defects associated with otoferlin depletion in larval zebrafish

Physics-Informed Machine Learning for Data Anomaly Detection, Classification, Localization, and Mitigation: A Review, Challenges, and Path Forward

Advancements in digital automation for smart grids have led to the installation of measurement devices like phasor measurement units (PMUs), micro-PMUs ($\mu$-PMUs), and smart meters. However, a large amount of data collected by these devices brings several challenges as control room operators need to use this data with models to make confident decisions for reliable and resilient operation of the cyber-power systems. Machine-learning (ML) based tools can provide a reliable interpretation of the deluge of data obtained from the field. For the decision-makers to ensure reliable network operation under all operating conditions, these tools need to identify solutions that are feasible and satisfy the system constraints, while being efficient, trustworthy, and interpretable. This resulted in the increasing popularity of physics-informed machine learning (PIML) approaches, as these methods overcome challenges that model-based or data-driven ML methods face in silos. This work aims at the following: a) review existing strategies and techniques for incorporating underlying physical principles of the power grid into different types of ML approaches (supervised/semi-supervised learning, unsupervised learning, and reinforcement learning (RL)); b) explore the existing works on PIML methods for anomaly detection, classification, localization, and mitigation in power transmission and distribution systems, c) discuss improvements in existing methods through consideration of potential challenges while also addressing the limitations to make them suitable for real-world applications

Spontaneous allelic variant in deafness–blindness gene \u3ci\u3eUsh1g\u3c/i\u3e resulting in an expanded phenotype

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bwnull mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell\u27s kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw/+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene

ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell’s cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed–TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience

Stereocilia Rootlets: Actin-Based Structures That Are Essential for Structural Stability of the Hair Bundle

Sensory hair cells of the inner ear rely on the hair bundle, a cluster of actin-filled stereocilia, to transduce auditory and vestibular stimuli into electrical impulses. Because they are long and thin projections, stereocilia are most prone to damage at the point where they insert into the hair cell’s soma. Moreover, this is the site of stereocilia pivoting, the mechanical movement that induces transduction, which additionally weakens this area mechanically. To bolster this fragile area, hair cells construct a dense core called the rootlet at the base of each stereocilium, which extends down into the actin meshwork of the cuticular plate and firmly anchors the stereocilium. Rootlets are constructed with tightly packed actin filaments that extend from stereocilia actin filaments which are wrapped with TRIOBP; in addition, many other proteins contribute to the rootlet and its associated structures. Rootlets allow stereocilia to sustain innumerable deflections over their lifetimes and exemplify the unique manner in which sensory hair cells exploit actin and its associated proteins to carry out the function of mechanotransduction

Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels