Stability and Dissociation of Adeno-Associated Viral Capsids by Variable Temperature-Charge Detection-Mass Spectrometry.

Adeno-associated viral (AAV) vectors have emerged as gene therapy and vaccine delivery systems. Differential scanning fluorimetry or differential scanning calorimetry is commonly used to measure the thermal stability of AAVs, but these global methods are unable to distinguish the stabilities of different AAV subpopulations in the same sample. To address this challenge, we combined charge detection-mass spectrometry (CD-MS) with a variable temperature (VT) electrospray source that controls the temperature of the solution prior to electrospray. Using VT-CD-MS, we measured the thermal stabilities of empty and filled capsids. We found that filled AAVs ejected their cargo first and formed intermediate empty capsids before completely dissociating. Finally, we observed that pH stress caused a major decrease in thermal stability. This new approach better characterizes the thermal dissociation of AAVs, providing the simultaneous measurement of the stabilities and dissociation pathways of different subpopulations.

New guidelines for the perioperative care of people living with frailty undergoing elective and emergency surgery-a commentary.

Frailty is common in the older population and is a predictor of adverse outcomes following emergency and elective surgery. Identification of frailty is key to enable targeted intervention throughout the perioperative pathway from contemplation of surgery to recovery. Despite evidence on how to identify and modify frailty, such interventions are not yet routine perioperative care. To address this implementation gap, a guideline was published in 2021 by the Centre for Perioperative Care and the British Geriatrics Society, working with patient representatives and all stakeholders involved in the perioperative care of patients with frailty undergoing surgery. The guideline covers all aspects of perioperative care relevant to adults living with frailty undergoing elective and emergency surgery. It is written for healthcare professionals, as well as for patients and their carers, managers and commissioners. Implementation of the guideline will require collaboration between all stakeholders, underpinned by an implementation strategy, workforce development with supporting education and training resources, and evaluation through national audit and research. The guideline is an important step in improving perioperative outcomes for people living with frailty and quality of healthcare services. This commentary provides a summary and discussion of the evidence informing the standards and recommendations in the published guideline.

The human visual cortex response to melanopsin-directed stimulation is accompanied by a distinct perceptual experience.

The photopigment melanopsin supports reflexive visual functions in people, such as pupil constriction and circadian photoentrainment. What contribution melanopsin makes to conscious visual perception is less studied. We devised a stimulus that targeted melanopsin separately from the cones using pulsed (3-s) spectral modulations around a photopic background. Pupillometry confirmed that the melanopsin stimulus evokes a response different from that produced by cone stimulation. In each of four subjects, a functional MRI response in area V1 was found. This response scaled with melanopic contrast and was not easily explained by imprecision in the silencing of the cones. Twenty additional subjects then observed melanopsin pulses and provided a structured rating of the perceptual experience. Melanopsin stimulation was described as an unpleasant, blurry, minimal brightening that quickly faded. We conclude that isolated stimulation of melanopsin is likely associated with a response within the cortical visual pathway and with an evoked conscious percept.

Coding of Object Size and Object Category in Human Visual Cortex.

A salient aspect of objects is their real-world size. Large objects tend to be fixed in the world and can act as navigational barriers and landmarks, whereas small objects tend to be moveable and manipulable. Previous work has identified regions of visual cortex that respond differentially to large versus small objects, but the role of size in organizing representations of object categories has not been fully explored. To address this issue, we scanned subjects while they viewed large and small objects drawn from 20 categories, with retinotopic extent equated across size classes. Univariate analyses replicated previous results showing a greater response to large than small objects in scene-responsive regions and the converse effect in the left occipitotemporal sulcus. Critically, multivariate analyses revealed organization-by-size both within and across functional regions, as evidenced by activation patterns that were more similar for object categories of the same size than for object categories of different size. This effect was observed in both scene- and object-responsive regions and across high-level visual cortex as a whole, but not in early visual cortex. We hypothesize that real-world size is an important dimension for object category organization because of the many ecologically significant differences between large and small objects.

Outside Looking In: Landmark Generalization in the Human Navigational System.

The use of landmarks is central to many navigational strategies. Here we use multivoxel pattern analysis of fMRI data to understand how landmarks are coded in the human brain. Subjects were scanned while viewing the interiors and exteriors of campus buildings. Despite their visual dissimilarity, interiors and exteriors corresponding to the same building elicited similar activity patterns in the parahippocampal place area (PPA), retrosplenial complex (RSC), and occipital place area (OPA), three regions known to respond strongly to scenes and buildings. Generalization across stimuli depended on knowing the correspondences among them in the PPA but not in the other two regions, suggesting that the PPA is the key region involved in learning the different perceptual instantiations of a landmark. In contrast, generalization depended on the ability to freely retrieve information from memory in RSC, and it did not depend on familiarity or cognitive task in OPA. Together, these results suggest a tripartite division of labor, whereby PPA codes landmark identity, RSC retrieves spatial or conceptual information associated with landmarks, and OPA processes visual features that are important for landmark recognition. SIGNIFICANCE STATEMENT: A central element of spatial navigation is the ability to recognize the landmarks that mark different places in the world. However, little is known about how the brain performs this function. Here we show that the parahippocampal place area (PPA), a region in human occipitotemporal cortex, exhibits key features of a landmark recognition mechanism. Specifically, the PPA treats different perceptual instantiations of the same landmark as representationally similar, but only when subjects have enough experience to know the correspondences among the stimuli. We also identify two other brain regions that exhibit landmark generalization, but with less sensitivity to familiarity. These results elucidate the brain networks involved in the learning and recognition of navigational landmarks.

Preoperative assessment and optimisation prior to planned aortic aneurysm repair: a UK survey examining current practice and attitudes of vascular surgeons and vascular anaesthetists.

BACKGROUND: The majority of those diagnosed with aortic aneurysm in the UK are older, multi-morbid patients. Decision-making as to who may benefit from intervention (open or endovascular aneurysm repair) is highly variable across the NHS (as is the mode of intervention), in part because there are no detailed guidelines or consensus on preoperative assessment. Thus, there is likely to be significant variation in the pre-operative assessment and optimisation of these patients. METHODS: A survey was designed to understand current practice and attitudes of vascular surgeons and vascular anaesthetists in the UK regarding preoperative assessment and optimisation of patients undergoing elective aortic aneurysm repair. The survey was reviewed and validated by an expert panel, then distributed electronically to all vascular surgical and vascular anaesthetic leads in the UK. RESULTS: Overall, the response rate was 68%. The responses were varied between surgeons and anaesthetists, with differences reported in the preoperative assessment and optimisation of patients, the approach to shared decision-making, and the perioperative pathway. CONCLUSIONS: Despite initiatives such as Getting It Right First Time (GIRFT) and National Institute for Health and Care Excellence (NICE) guidelines, variation still exists between centres with some differences in opinion observed between surgeons and anaesthetists. These differences may be leading to duplication of work in the perioperative pathway, inconsistencies in how risk is assessed and communicated with consequent variation in patient care. Addressing these issues requires awareness and implementation of existing guidelines, transdisciplinary working, efficient data-driven pathways, and structured aortic aneurysm multi-disciplinary team to promote meaningful shared decision-making.

Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods.

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein (VP) sequences with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for the correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivity for viral protein (VP) subunits due to their sequence similarities, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainty in distinguishing empty and filled capsids. To address these challenges, we combined two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, a selective temporal overview of resonant ion (STORI)-based charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids. Liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were then used for the capsid denaturing measurements. This multimethod combination was applied to three AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.

Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry.

Native mass spectrometry (MS) and charge detection-mass spectrometry (CD-MS) have become versatile tools for characterizing a wide range of proteins and macromolecular complexes. Both commonly use nanoelectrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb to the glass, which may lower sensitivity and limit the quality of the data. To improve the sensitivity of native MS and CD-MS, we modified the surface of nESI needles with inert surface modifiers, including polyethylene-glycol. We found that the surface modification improved the signal intensity for native MS of proteins and for CD-MS of adeno-associated viral capsids. Based on mechanistic comparisons, we hypothesize that the improvement is more likely due to an increased flow rate with coated ESI needles rather than less nonspecific adsorption. In any case, these surface-modified needles provide a simple and inexpensive method for improving the sensitivity of challenging analytes.

The association between neurodegeneration and local complement activation in the thalamus to progressive multiple sclerosis outcome.

The extent of grey matter demyelination and neurodegeneration in the progressive multiple sclerosis (PMS) brains at post-mortem associates with more severe disease. Regional tissue atrophy, especially affecting the cortical and deep grey matter, including the thalamus, is prognostic for poor outcomes. Microglial and complement activation are important in the pathogenesis and contribute to damaging processes that underlie tissue atrophy in PMS. We investigated the extent of pathology and innate immune activation in the thalamus in comparison to cortical grey and white matter in blocks from 21 cases of PMS and 10 matched controls. Using a digital pathology workflow, we show that the thalamus is invariably affected by demyelination and had a far higher proportion of active inflammatory lesions than forebrain cortical tissue blocks from the same cases. Lesions were larger and more frequent in the medial nuclei near the ventricular margin, whilst neuronal loss was greatest in the lateral thalamic nuclei. The extent of thalamic neuron loss was not associated with thalamic demyelination but correlated with the burden of white matter pathology in other forebrain areas (Spearman r = 0.79, p < 0.0001). Only thalamic neuronal loss, and not that seen in other forebrain cortical areas, correlated with disease duration (Spearman r = -0.58, p = 0.009) and age of death (Spearman r = -0.47, p = 0.045). Immunoreactivity for the complement pattern recognition molecule C1q, and products of complement activation (C4d, Bb and C3b) were elevated in thalamic lesions with an active inflammatory pathology. Complement regulatory protein, C1 inhibitor, was unchanged in expression. We conclude that active inflammatory demyelination, neuronal loss and local complement synthesis and activation in the thalamus, are important to the pathological and clinical disease outcomes of PMS.

Schematic representations of local environmental space guide goal-directed navigation.

To successfully navigate to a target, it is useful to be able to define its location at multiple levels of specificity. For example, the location of a favorite coffee mug can be described in terms of which room it is in, or in terms of where it is within the room. An appealing hypothesis is that these levels of description are retrieved from memory by accessing the same representation at progressively finer levels of granularity-first remembering the general location of an object and then "zooming in." Here we provide evidence for an alternative view, in which navigational behavior is guided by independent representations at multiple spatial scales. Subjects learned the locations of objects that were positioned within four visually distinct but geometrically similar buildings, which were in turn positioned within a broader virtual park. They were then tested on their knowledge of object location by asking them to navigate to the remembered location of each object. We examined errors during the test phase for confusions among geometrically analogous locations in different buildings-that is, navigating to the right location in the wrong building. We observed that subjects frequently made these confusions, which are analogous to remembering a passage's location on the page of a book but not remembering the page that the passage is on. This suggests that subjects were recalling the object's local location without recalling its global location. Further manipulations across seven experiments indicated that geometric confusions were observed even between buildings that were not metrically identical as long as geometrical equivalence could be defined. However, removing the walls so that the larger environment was no longer divided into subspaces abolished these errors. Taken together, our results suggest that human spatial memory contains two separable representations of "where" an object can be found: (i) a schematic map of where an object lies with respect to local landmarks and boundaries; (ii) a representation of the identity and location of each local environment.

The Occipital Place Area Is Causally Involved in Representing Environmental Boundaries during Navigation.

Thirty years of research suggests that environmental boundaries-e.g., the walls of an experimental chamber or room-exert powerful influence on navigational behavior, often to the exclusion of other cues [1-9]. Consistent with this behavioral work, neurons in brain structures that instantiate spatial memory often exhibit firing fields that are strongly controlled by environmental boundaries [10-15]. Despite the clear importance of environmental boundaries for spatial coding, however, a brain region that mediates the perception of boundary information has not yet been identified. We hypothesized that the occipital place area (OPA), a scene-selective region located near the transverse occipital sulcus [16], might provide this perceptual source by extracting boundary information from visual scenes during navigation. To test this idea, we used transcranial magnetic stimulation (TMS) to interrupt processing in the OPA while subjects performed a virtual-reality memory task that required them to learn the spatial locations of test objects that were either fixed in place relative to the boundary of the environment or moved in tandem with a landmark object. Consistent with our prediction, we found that TMS to the right OPA impaired spatial memory for boundary-tethered, but not landmark-tethered, objects. Moreover, this effect was found when the boundary was defined by a wall, but not when it was defined by a marking on the ground. These results show that the OPA is causally involved in boundary-based spatial navigation and suggest that the OPA is the perceptual source of the boundary information that controls navigational behavior.

Anchoring the neural compass: coding of local spatial reference frames in human medial parietal lobe.

The neural systems that code for location and facing direction during spatial navigation have been investigated extensively; however, the mechanisms by which these quantities are referenced to external features of the world are not well understood. To address this issue, we examined behavioral priming and functional magnetic resonance imaging activity patterns while human subjects recalled spatial views from a recently learned virtual environment. Behavioral results indicated that imagined location and facing direction were represented during this task, and multivoxel pattern analyses indicated that the retrosplenial complex (RSC) was the anatomical locus of these spatial codes. Critically, in both cases, location and direction were defined on the basis of fixed elements of the local environment and generalized across geometrically similar local environments. These results suggest that RSC anchors internal spatial representations to local topographical features, thus allowing us to stay oriented while we navigate and retrieve from memory the experience of being in a particular place.