Biogenesis of specialized lysosomes in differentiated keratinocytes relies on close apposition with the Golgi apparatus.

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling of lysosomes and the Golgi apparatus. However, it was not known whether changes in Golgi and lysosome morphology and their redistribution were linked. Here, we show that disassembled Golgi is distributed in close physical apposition to lysosomes in differentiated keratinocytes. This atypical localization requires the Golgi tethering protein GRASP65, which is associated with both the Golgi and lysosome membranes. Depletion of GRASP65 results in the loss of Golgi-lysosome apposition and the malformation of lysosomes, defined by their aberrant morphology, size, and function. Surprisingly, a trans-Golgi enzyme and secretory Golgi cargoes are extensively localized to the lysosome lumen and secreted to the cell surface, contributing to total protein secretion of differentiated keratinocytes but not in proliferative precursors, indicating that lysosomes acquire specialization during differentiation. We further demonstrate that the secretory function of the Golgi apparatus is critical to maintain keratinocyte lysosomes. Our study uncovers a novel form of Golgi-lysosome cross-talk and its role in maintaining specialized secretory lysosomes in differentiated keratinocytes.

Celebrating the Birthday of AMPA Receptor Nanodomains: Illuminating the Nanoscale Organization of Excitatory Synapses with 10 Nanocandles.

A decade ago, in 2013, and over the course of 4 summer months, three separate observations were reported that each shed light independently on a new molecular organization that fundamentally reshaped our perception of excitatory synaptic transmission (Fukata et al., 2013; MacGillavry et al., 2013; Nair et al., 2013). This discovery unveiled an intricate arrangement of AMPA-type glutamate receptors and their principal scaffolding protein PSD-95, at synapses. This breakthrough was made possible, thanks to advanced super-resolution imaging techniques. It fundamentally changed our understanding of excitatory synaptic architecture and paved the way for a brand-new area of research. In this Progressions article, the primary investigators of the nanoscale organization of synapses have come together to chronicle the tale of their discovery. We recount the initial inquiry that prompted our research, the preceding studies that inspired our work, the technical obstacles that were encountered, and the breakthroughs that were made in the subsequent decade in the realm of nanoscale synaptic transmission. We review the new discoveries made possible by the democratization of super-resolution imaging techniques in the field of excitatory synaptic physiology and architecture, first by the extension to other glutamate receptors and to presynaptic proteins and then by the notion of trans-synaptic organization. After describing the organizational modifications occurring in various pathologies, we discuss briefly the latest technical developments made possible by super-resolution imaging and emerging concepts in synaptic physiology.

A conserved polar residue plays a critical role in mismatch detection in A-family DNA polymerases.

In A-family DNA polymerases (dPols), a functional 3'-5' exonuclease activity is known to proofread newly synthesized DNA. The identification of a mismatch in substrate DNA leads to transfer of the primer strand from the polymerase active site to the exonuclease active site. To shed more light regarding the mechanism responsible for the detection of mismatches, we have utilized DNA polymerase 1 from Aquifex pyrophilus (ApPol1). The enzyme synthesized DNA with high fidelity and exhibited maximal exonuclease activity with DNA substrates bearing mismatches at the -2 and - 3 positions. The crystal structure of apo-ApPol1 was utilized to generate a computational model of the functional ternary complex of this enzyme. The analysis of the model showed that N332 forms interactions with minor groove atoms of the base pairs at the -2 and - 3 positions. The majority of known A-family dPols show the presence of Asn at a position equivalent to N332. The N332L mutation led to a decrease in the exonuclease activity for representative purine-pyrimidine, and pyrimidine-pyrimidine mismatches at -2 and - 3 positions, respectively. Overall, our findings suggest that conserved polar residues located towards the minor groove may facilitate the detection of position-specific mismatches to enhance the fidelity of DNA synthesis.

A Case Report and Literature Review of Nonketotic Hyperglycemic Hemichorea.

Nonketogenic hyperglycemic hemichorea, also recognized as diabetic striatopathy, is a rare manifestation of diabetes mellitus. The diagnosis of nonketotic hyperglycemic hemichorea is usually made through imaging along with a corresponding clinical picture. Early identification and treatment can lead to complete resolution of the symptoms and better patient outcomes. Here we present a 49-year-old female patient, with a past medical history of poorly controlled type 2 diabetes mellitus and prior left index finger amputation as a complication of her diabetes, who presented for evaluation of a two-week duration of sudden-onset left upper extremity choreiform movements.

Isolated Infratentorial Posterior Reversible Encephalopathy Syndrome (PRES) in Nephrotic Syndrome: A Case Report.

We present a case of infratentorial variant posterior reversible encephalopathy syndrome (PRES), which is a very rare presentation of PRES. Atypical PRES is more common than the typical parieto-occipital PRES. We present a 43-year-old male who presented with acute change in mentation, left gaze deviation, and paraparesis with initial blood pressures of 230/120 with anasarca. In the present admission, his CT showed diffuse infratentorial hypodensity. Computed tomography angiography (CTA) was negative for large vessel occlusion. MRI of the brain without contrast showed fluid-attenuated inversion recovery (FLAIR) change diffusely in the brainstem but also extended to the cerebellum and occipital lobe, along with diffusion restriction seen in different regions, including the brainstem and cortex. The patient improved clinically with the improvement of blood pressure and follow-up imaging in five weeks showed improvement of imaging findings. This presentation helps understand the approach to patients presenting with brainstem edema in the acute phase.

SRF-deficient astrocytes provide neuroprotection in mouse models of excitotoxicity and neurodegeneration.

Reactive astrogliosis is a common pathological hallmark of CNS injury, infection, and neurodegeneration, where reactive astrocytes can be protective or detrimental to normal brain functions. Currently, the mechanisms regulating neuroprotective astrocytes and the extent of neuroprotection are poorly understood. Here, we report that conditional deletion of serum response factor (SRF) in adult astrocytes causes reactive-like hypertrophic astrocytes throughout the mouse brain. These SrfGFAP-ERCKO astrocytes do not affect neuron survival, synapse numbers, synaptic plasticity or learning and memory. However, the brains of Srf knockout mice exhibited neuroprotection against kainic-acid induced excitotoxic cell death. Relevant to human neurodegenerative diseases, SrfGFAP-ERCKO astrocytes abrogate nigral dopaminergic neuron death and reduce ß-amyloid plaques in mouse models of Parkinson's and Alzheimer's disease, respectively. Taken together, these findings establish SRF as a key molecular switch for the generation of reactive astrocytes with neuroprotective functions that attenuate neuronal injury in the setting of neurodegenerative diseases.

Real-time heterogeneity of supramolecular assembly of amyloid precursor protein is modulated by an endocytic risk factor PICALM.

Recently, the localization of amyloid precursor protein (APP) into reversible nanoscale supramolecular assembly or "nanodomains" has been highlighted as crucial towards understanding the onset of the molecular pathology of Alzheimer's disease (AD). Surface expression of APP is regulated by proteins interacting with it, controlling its retention and lateral trafficking on the synaptic membrane. Here, we evaluated the involvement of a key risk factor for AD, PICALM, as a critical regulator of nanoscale dynamics of APP. Although it was enriched in the postsynaptic density, PICALM was also localized to the presynaptic active zone and the endocytic zone. PICALM colocalized with APP and formed nanodomains with distinct morphological properties in different subsynaptic regions. Next, we evaluated if this localization to subsynaptic compartments was regulated by the C-terminal sequences of APP, namely, the "Y682ENPTY687" domain. Towards this, we found that deletion of C-terminal regions of APP with partial or complete deletion of Y682ENPTY687, namely, APP-Δ9 and APP-Δ14, affected the lateral diffusion and nanoscale segregation of APP. Lateral diffusion of APP mutant APP-Δ14 sequence mimicked that of a detrimental Swedish mutant of APP, namely, APP-SWE, while APP-Δ9 diffused similar to wild-type APP. Interestingly, elevated expression of PICALM differentially altered the lateral diffusion of the APP C-terminal deletion mutants. These observations confirm that the C-terminal sequence of APP regulates its lateral diffusion and the formation of reversible nanoscale domains. Thus, when combined with autosomal dominant mutations, it generates distinct molecular patterns leading to onset of Alzheimer's disease (AD).

Golgi fragmentation - One of the earliest organelle phenotypes in Alzheimer's disease neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aß) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aß secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.

Pembrolizumab induced remission of recurrent and metastatic sinonasal squamous cell carcinoma after overcoming checkpoint-inhibitor pneumonitis: A case report and literature review.

BACKGROUND: For programmed death-ligand-1 (PD-L1) positive recurrent and metastatic head and neck squamous cell carcinoma (R/M-HNSCC), KEYNOTE-048 and KEYNOTE-040 clinical trials recently approved pembrolizumab monotherapy as first-line treatment. However, recurrent and metastatic sinonasal squamous cell carcinoma (R/M-SNSCC) was excluded from these clinical trials and treatment reports of immune-checkpoint inhibitor (ICI) in R/M-SNSCC are sparse. Immune-related adverse events (irAEs) are known to occur during ICI treatment and some of these such as checkpoint-inhibitor pneumonitis (CIP) can be fatal. ICI rechallenge after severe irAEs is debated. CASE: We describe a case of a 65-year-old male with R/M-SNSCC who is currently in remission with pembrolizumab monotherapy. He developed high-grade pneumonitis during the course of treatment warranting ICI discontinuation but has since tolerated full-dose pembrolizumab for 10 months now which is holding his disease stable. Our approach toward restarting full-dose pembrolizumab was by monitoring the patient's response to an initial low dose of pembrolizumab with concomitant oral steroid immunosuppression to control CIP. CONCLUSION: Clinicians should weigh the risk-to-reward ratio of ICI rechallenge after improvement of high-grade CIP, particularly for selected patients with aggressive tumors such as R/M-SNSCC and prior treatment response. Under close monitoring, ICI resumption at a low dose and assessing patient tolerance with concomitant immunosuppression may be a reasonable approach to reintroducing ICI after high-grade CIP in these patients.

Achieving nanoscale precision using neuromorphic localization microscopy.

Neuromorphic cameras are a new class of dynamic-vision-inspired sensors that encode the rate of change of intensity as events. They can asynchronously record intensity changes as spikes, independent of the other pixels in the receptive field, resulting in sparse measurements. This recording of such sparse events makes them ideal for imaging dynamic processes, such as the stochastic emission of isolated single molecules. Here we show the application of neuromorphic detection to localize nanoscale fluorescent objects below the diffraction limit, with a precision below 20 nm. We demonstrate a combination of neuromorphic detection with segmentation and deep learning approaches to localize and track fluorescent particles below 50 nm with millisecond temporal resolution. Furthermore, we show that combining information from events resulting from the rate of change of intensities improves the classical limit of centroid estimation of single fluorescent objects by nearly a factor of two. Additionally, we validate that using post-processed data from the neuromorphic detector at defined windows of temporal integration allows a better evaluation of the fractalized diffusion of single particle trajectories. Our observations and analysis is useful for event sensing by nonlinear neuromorphic devices to ameliorate real-time particle localization approaches at the nanoscale.

Thoracolumbar Sacral Orthosis for Spinal Fractures: What's the Evidence and Do Patients Use Them?

Introduction The general consensus regarding the non-operative management of thoracolumbar (TL) spine fractures revolves around the use of thoracolumbar spine orthosis (TLSO). The efficacy of TLSO bracing remains controversial within the current literature, with several studies showing that prolonged brace use is associated with diminished lung capacity, skin breakdown, and paraspinal muscular atrophy, with no significant difference in pain and functional outcomes between patients treated with or without TLSO. Aims The aim of this study was to identify the number of braces issued over the duration of the study and understand the cost implication, added length of stay, and patient satisfaction based on a questionnaire and reflect on whether we need to change our practice on the use of TLSO. Methods Data was collected retrospectively over an 18-month period with 75 patients being initially identified for the study. A total of 42 records were included in the final study after the application of inclusion/exclusion criteria. Patient-related outcomes were recorded through a questionnaire. Results Of the patients, 60% report not receiving adequate advice regarding the duration of treatment, 43% reported that the brace interfered with their activities of daily living (ADLs), and 73% came off the brace earlier than advised, with 60% of patients reporting that they would rather be without the brace if given the option. The average increase in length of stay waiting for bracing was three days, with the estimated cost incurred being approximately £114,000. Conclusion With equivalence between treatment with or without a brace, there is a need to rethink current practice and move toward a case-by-case, patient-centered approach to minimize costs and improve patient satisfaction.

A broadly neutralizing monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV-2 variants of concern.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Antibody multispecificity: A necessary evil?

Antibodies represent key effectors of the adaptive immune system. The specificity of antibodies is an established hallmark of the immune response. However, a certain proportion of antibodies exhibit limited promiscuity or multireactivity. Germline antibodies display plasticity which imparts multispecificity to enhance the antibody repertoire. Surprisingly, even affinity matured antibodies display such plasticity and multireactivity enabling their binding to more than one antigen. We propose that antibody multispecificity is a physiological requirement to expand the antibody repertoire at the germline level and to tolerate plasticity in antigens at the mature level. This property of the humoral immune response may attenuate the ability of infectious RNA viruses such as influenza, HIV and SARS-CoV-2 to acquire mutations that render resistance to neutralizing antibodies.

Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis.

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP's domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP's domains in translation regulation.

Were US Asian Indian decedents with atherosclerosis more likely to have concurrent diabetes mellitus? Analysis of national multiple cause of mortality data (2012-2019).

BACKGROUND: Asian Indians (AI) are at high risk for both atherosclerotic diseases (ATH) and diabetes mellitus (DM). We analyze the clustering of these two comorbidities as contributing causes of death in AI versus Non-AI populations in the US. METHODS: Using Mortality Multiple Cause-of-Death Files (2012-2019) from the National Center for Health Statistics, we included deaths at age ≥ 45 years among US residents where AI versus Non-AI status could be ascertained (n = 55,461 AI; n = 20,090,038 Non-AI) and identsified ATH (ICD10: I20-I25, I63, I70) and DM (ICD10: E10-E14) as contributing causes of death. We calculated the tetrachoric correlation (Rho) between these contributing causes and the difference in the fraction of deaths involving DM in those with versus without ATH. RESULTS: Among AI decedents, 29.9% of deaths included ATH as a contributing cause, 16.4% included DM as a contributing cause with 8.3% deaths being included in the overlap (Rho = 0.36, SE = 0.007) whereas, among Non-AI, 22.4% of deaths included ATH as a contributing cause, 10.0% included DM as a contributing cause with 4.1% deaths being included in the overlap (Rho = 0.31, SE = 0.001). Thus, DM and ATH as co-occurring causes correlated more strongly in AI versus Non-AI (p < 0.001). Further, this difference in clustering of DM with ATH was highest for younger AI women (age < 60 years) compared to comparable Non-AI women. CONCLUSIONS: The more frequent co-occurrence of DM and ATH as causes of death among AI compared to Non-AI suggest that the increased burden of these diseases among AI during life has vicious synergistic consequences in terms of mortality. Public health strategies targeted to AI should focus on prevention and clinical treatment of both conditions jointly, in all adults, and especially in women < 60 years.

Nanoscale regulation of Ca2+ dependent phase transitions and real-time dynamics of SAP97/hDLG.

Synapse associated protein-97/Human Disk Large (SAP97/hDLG) is a conserved, alternatively spliced, modular, scaffolding protein critical in regulating the molecular organization of cell-cell junctions in vertebrates. We confirm that the molecular determinants of first order phase transition of SAP97/hDLG is controlled by morpho-functional changes in its nanoscale organization. Furthermore, the nanoscale molecular signatures of these signalling islands and phase transitions are altered in response to changes in cytosolic Ca2+. Additionally, exchange kinetics of alternatively spliced isoforms of the intrinsically disordered region in SAP97/hDLG C-terminus shows differential sensitivities to Ca2+ bound Calmodulin, affirming that the molecular signatures of local phase transitions of SAP97/hDLG depends on their nanoscale heterogeneity and compositionality of isoforms.

Effect of Early vs Standard Approach to Tracheostomy on Functional Outcome at 6 Months Among Patients With Severe Stroke Receiving Mechanical Ventilation: The SETPOINT2 Randomized Clinical Trial.

Importance: Many patients with severe stroke have impaired airway protective reflexes, resulting in prolonged invasive mechanical ventilation. Objective: To test whether early vs standard tracheostomy improved functional outcome among patients with stroke receiving mechanical ventilation. Design, Setting, and Participants: In this randomized clinical trial, 382 patients with severe acute ischemic or hemorrhagic stroke receiving invasive ventilation were randomly assigned (1:1) to early tracheostomy (≤5 days of intubation) or ongoing ventilator weaning with standard tracheostomy if needed from day 10. Patients were randomized between July 28, 2015, and January 24, 2020, at 26 US and German neurocritical care centers. The final date of follow-up was August 9, 2020. Interventions: Patients were assigned to an early tracheostomy strategy (n = 188) or to a standard tracheostomy (control group) strategy (n = 194). Main Outcomes and Measures: The primary outcome was functional outcome at 6 months, based on the modified Rankin Scale score (range, 0 [best] to 6 [worst]) dichotomized to a score of 0 (no disability) to 4 (moderately severe disability) vs 5 (severe disability) or 6 (death). Results: Among 382 patients randomized (median age, 59 years; 49.8% women), 366 (95.8%) completed the trial with available follow-up data on the primary outcome (177 patients [94.1%] in the early group; 189 patients [97.4%] in the standard group). A tracheostomy (predominantly percutaneously) was performed in 95.2% of the early tracheostomy group in a median of 4 days after intubation (IQR, 3-4 days) and in 67% of the control group in a median of 11 days after intubation (IQR, 10-12 days). The proportion without severe disability (modified Rankin Scale score, 0-4) at 6 months was not significantly different in the early tracheostomy vs the control group (43.5% vs 47.1%; difference, -3.6% [95% CI, -14.3% to 7.2%]; adjusted odds ratio, 0.93 [95% CI, 0.60-1.42]; P = .73). Of the serious adverse events, 5.0% (6 of 121 reported events) in the early tracheostomy group vs 3.4% (4 of 118 reported events) were related to tracheostomy. Conclusions and Relevance: Among patients with severe stroke receiving mechanical ventilation, a strategy of early tracheostomy, compared with a standard approach to tracheostomy, did not significantly improve the rate of survival without severe disability at 6 months. However, the wide confidence intervals around the effect estimate may include a clinically important difference, so a clinically relevant benefit or harm from a strategy of early tracheostomy cannot be excluded. Trial Registration: ClinicalTrials.gov Identifier: NCT02377167.

Society for Vascular Surgery appropriate use criteria for management of intermittent claudication.

The Society for Vascular Surgery appropriate use criteria (AUC) for the management of intermittent claudication were created using the RAND appropriateness method, a validated and standardized method that combines the best available evidence from medical literature with expert opinion, using a modified Delphi process. These criteria serve as a framework on which individualized patient and clinician shared decision-making can grow. These criteria are not absolute. AUC should not be interpreted as a requirement to administer treatments rated as appropriate (benefit outweighs risk). Nor should AUC be interpreted as a prohibition of treatments rated as inappropriate (risk outweighs benefit). Clinical situations will occur in which moderating factors, not included in these AUC, will shift the appropriateness level of a treatment for an individual patient. Proper implementation of AUC requires a description of those moderating patient factors. For scenarios with an indeterminate rating, clinician judgement combined with the best available evidence should determine the treatment strategy. These scenarios require mechanisms to track the treatment decisions and outcomes. AUC should be revisited periodically to ensure that they remain relevant. The panelists rated 2280 unique scenarios for the treatment of intermittent claudication (IC) in the aortoiliac, common femoral, and femoropopliteal segments in the round 2 rating. Of these, only nine (0.4%) showed a disagreement using the interpercentile range adjusted for symmetry formula, indicating an exceptionally high degree of consensus among the panelists. Post hoc, the term "inappropriate" was replaced with the phrase "risk outweighs benefit." The term "appropriate" was also replaced with "benefit outweighs risk." The key principles for the management of IC reflected within these AUC are as follows. First, exercise therapy is the preferred initial management strategy for all patients with IC. Second, for patients who have not completed exercise therapy, invasive therapy might provide net a benefit for selected patients with IC who are nonsmokers, are taking optimal medical therapy, are considered to have a low physiologic and technical risk, and who are experiencing severe lifestyle limitations and/or a short walking distance. Third, considering the long-term durability of the currently available technology, invasive interventions for femoropopliteal disease should be reserved for patients with severe lifestyle limitations and a short walking distance. Fourth, in the common femoral segment, open common femoral endarterectomy will provide greater net benefit than endovascular intervention for the treatment of IC. Finally, in the infrapopliteal segment, invasive intervention for the treatment of IC is of unclear benefit and could be harmful.