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Purpose: The objective of this systematic review was to examine current evidence on the risks versus benefit of pre-hospital intubation when compared with in-hospital intubation in adult patients with traumatic brain injuries., Methods: We conducted electronic searches of PubMed, Medline, Embase, CIANHL and the Cochrane library up to March 2021. Data extracted compared mortality, length of hospital and intensive care stay, pneumonia and functional outcomes in traumatic brain injured patients undergoing pre-hospital intubation versus in-hospital intubation. The risk of bias was assessed using the Grading of Recommendations Assessment, Development and Evaluation., Results: Ten studies including 25,766 patients were analysed. Seven were retrospective studies, two prospective cohort studies and one randomised control study. The mean mortality rate in patients who underwent pre-hospital intubation was 44.5% and 31.98% for in-hospital intubation. The odds ratio for an effect of pre-hospital intubation on mortality ranged from 0.31 (favouring in-hospital intubation) to 3.99 (favouring pre-hospital). The overall quality of evidence is low; however, the only randomised control study showed an improved functional outcome for pre-hospital intubation at 6 months., Conclusions: The existing evidence does not support widespread pre-hospital intubation in all traumatic brain injured patients. This does not, however, contradict the need for the intervention when there is severe airway compromise; instead, it must be assessed by experienced personnel if a time critical transfer to hospital is more advantageous. Favourable neurological outcomes highlighted by the randomised control trial favours pre-hospital intubation, but further research is required in this field., (© 2022. Crown.)

Huntington's disease (HD) is a neurodegenerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phosphosites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein by proteomic and phosphoproteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phosphosites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together, these findings highlight categories of phosphosites that merit further study and provide a phosphosite kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Genome-wide loss-of-function screens are critical tools to identify novel genetic regulators of intracellular proteins. However, studying the changes in the organelle-specific expression profile of intracellular proteins can be challenging due to protein localization differences across the whole cell, hindering context-dependent protein expression and activity analyses. Here, we describe nuPRISM, a microfluidics chip specifically designed for large-scale isolated nuclei sorting. The new device enables rapid genome-wide loss-of-function phenotypic CRISPR-Cas9 screens directed at intranuclear targets. We deployed this technology to identify novel genetic regulators of β-catenin nuclear accumulation, a phenotypic hallmark of APC -mutated colorectal cancer. nuPRISM expands our ability to capture aberrant nuclear morphological and functional traits associated with distinctive signal transduction and subcellular localization-driven functional processes with substantial resolution and high throughput., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

High-throughput phenotypic cell sorting is critical to the development of cell-based therapies and cell screening discovery platforms. However, current cytometry platforms are limited by throughput, number of fractionated populations that can be isolated, cell viability, and cost. We present an ultrathroughput microfluidic cell sorter capable of processing hundreds of millions of live cells per hour per device based on protein expression. This device, a next-generation microfluidic cell sorter (NG-MICS), combines multiple technologies, including 3D printing, reversible clamp sealing, and superhydrophobic treatments to create a reusable and user-friendly platform ready for deployment. The utility of such a platform is demonstrated through the rapid isolation of mature natural killer cells from peripheral blood mononuclear cells, for use in CAR-NK therapies at clinically-relevant scale.

Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 10 8 cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by expansion of a CAG trinucleotide repeat in HTT, resulting in an extended polyglutamine tract in huntingtin. We and others have previously determined that the HD-causing expansion occurs on multiple different haplotype backbones, reflecting more than one ancestral origin of the same type of mutation. In view of the therapeutic potential of mutant allele-specific gene silencing, we have compared and integrated two major systems of HTT haplotype definition, combining data from 74 sequence variants to identify the most frequent disease-associated and control chromosome backbones and revealing that there is potential for additional resolution of HD haplotypes. We have used the large collection of 4078 heterozygous HD subjects analyzed in our recent genome-wide association study of HD age at onset to estimate the frequency of these haplotypes in European subjects, finding that common genetic variation at HTT can distinguish the normal and CAG-expanded chromosomes for more than 95% of European HD individuals. As a resource for the HD research community, we have also determined the haplotypes present in a series of publicly available HD subject-derived fibroblasts, induced pluripotent cells, and embryonic stem cells in order to facilitate efforts to develop inclusive methods of allele-specific HTT silencing applicable to most HD patients. Our data providing genetic guidance for therapeutic gene-based targeting will significantly contribute to the developments of rational treatments and implementation of precision medicine in HD.

Onset of neurodegenerative disorders, including Huntington's disease, is strongly influenced by aging. Hallmarks of aged cells include compromised nuclear envelope integrity, impaired nucleocytoplasmic transport, and accumulation of DNA double-strand breaks. We show that mutant huntingtin markedly accelerates all of these cellular phenotypes in a dose- and age-dependent manner in cortex and striatum of mice. Huntingtin-linked polyglutamine initially accumulates in nuclei, leading to disruption of nuclear envelope architecture, partial sequestration of factors essential for nucleocytoplasmic transport (Gle1 and RanGAP1), and intranuclear accumulation of mRNA. In aged mice, accumulation of RanGAP1 together with polyglutamine is shifted to perinuclear and cytoplasmic areas. Consistent with findings in mice, marked alterations in nuclear envelope morphology, abnormal localization of RanGAP1, and nuclear accumulation of mRNA were found in cortex of Huntington's disease patients. Overall, our findings identify polyglutamine-dependent inhibition of nucleocytoplasmic transport and alteration of nuclear integrity as a central component of Huntington's disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A comprehensive genetics-based precision medicine strategy to selectively and permanently inactivate only mutant, not normal allele, could benefit many dominantly inherited disorders. Here, we demonstrate the power of our novel strategy of inactivating the mutant allele using haplotype-specific CRISPR/Cas9 target sites in Huntington's disease (HD), a late-onset neurodegenerative disorder due to a toxic dominant gain-of-function CAG expansion mutation. Focusing on improving allele specificity, we combined extensive knowledge of huntingtin (HTT) gene haplotype structure with a novel personalized allele-selective CRISPR/Cas9 strategy based on Protospacer Adjacent Motif (PAM)-altering SNPs to target patient-specific CRISPR/Cas9 sites, aiming at the mutant HTT allele-specific inactivation for a given diplotype. As proof-of-principle, simultaneously using two CRISPR/Cas9 guide RNAs (gRNAs) that depend on PAM sites generated by SNP alleles on the mutant chromosome, we selectively excised ∼44 kb DNA spanning promoter region, transcription start site, and the CAG expansion mutation of the mutant HTT gene, resulting in complete inactivation of the mutant allele without impacting the normal allele. This excision on the disease chromosome completely prevented the generation of mutant HTT mRNA and protein, unequivocally indicating permanent mutant allele-specific inactivation of the HD mutant allele. The perfect allele selectivity with broad applicability of our strategy in disorders with diverse disease haplotypes should also support precision medicine through inactivation of many other gain-of-function mutations.

Huntington's disease (HD) is an autosomal dominant, neurodegenerative disorder that can be characterized by the presence of protein inclusions containing mutant huntingtin within a subset of neurons in the brain. Since their discovery, the relevance of inclusions to disease pathology has been controversial. We show using super-resolution fluorescence imaging and Förster resonance energy transfer (FRET) in live cells, that mutant huntingtin fragments can form two morphologically and conformationally distinct inclusion types. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that the two huntingtin inclusion types have unique dynamic properties. The ability to form one or the other type of inclusion can be influenced by the phosphorylation state of serine residues at amino acid positions 13 and 16 within the huntingtin protein. We can define two types of inclusions: fibrillar, which are tightly packed, do not exchange protein with the soluble phase, and result from phospho-modification at serines 13 and 16 of the N17 domain, and globular, which are loosely packed, can readily exchange with the soluble phase, and are not phosphorylated in N17. We hypothesize that the protective effect of N17 phosphorylation or phospho-mimicry seen in animal models, at the level of protein inclusions with elevated huntingtin levels, is to induce a conformation of the huntingtin amino-terminus that causes fragments to form tightly packed inclusions that do not exit the insoluble phase, and hence exert less toxicity. The identification of these sub-types of huntingtin inclusions could allow for drug discovery to promote protective inclusions of mutant huntingtin protein in HD.

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.

Two serine residues within the first 17 amino acid residues of huntingtin (N17) are crucial for modulation of mutant huntingtin toxicity in cell and mouse genetic models of Huntington's disease. Here we show that the stress-dependent phosphorylation of huntingtin at Ser13 and Ser16 affects N17 conformation and targets full-length huntingtin to chromatin-dependent subregions of the nucleus, the mitotic spindle and cleavage furrow during cell division. Polyglutamine-expanded mutant huntingtin is hypophosphorylated in N17 in both homozygous and heterozygous cell contexts. By high-content screening in live cells, we identified kinase inhibitors that modulated N17 phosphorylation and hence huntingtin subcellular localization. N17 phosphorylation was reduced by casein kinase-2 inhibitors. Paradoxically, IKKβ kinase inhibition increased N17 phosphorylation, affecting huntingtin nuclear and subnuclear localization. These data indicate that huntingtin phosphorylation at Ser13 and Ser16 can be modulated by small-molecule drugs, which may have therapeutic potential in Huntington's disease.

Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin-actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin-actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement-Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin-cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases.

After the successful cloning of the first gene for a polyglutamine disease in 1991, the expanded polyglutamine tract in the nine polyglutamine disease proteins became an obvious therapeutic target. Early hypotheses were that misfolded, precipitated protein could be a universal pathogenic mechanism. However, new data are accumulating on Huntington's disease and other polyglutamine diseases that appear to contradict the toxic aggregate hypothesis. Recent data suggest that the toxic species of protein in these diseases may be soluble mutant conformers, and that the protein context of expanded polyglutamine is critical to understanding disease specificity. Here we discuss recent publications that define other important therapeutic targets for polyglutamine-mediated neurodegeneration related to the context of the expanded polyglutamine tract in the disease protein.

We have recently published the precise definition of an aminoterminal membrane association domain in huntingtin, capable of targeting to the endoplasmic reticulum and late endosomes as well as autophagic vesicles. In response to ER stress induced by several pathways, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin expressing cells have a perturbed ER and an increase in autophagic vesicles. Here, we discuss the potential function of the huntingtin protein as an ER sentinel, potentially regulating autophagy in response to ER stress. We compare these recent findings to the well characterized mammalian target of rapamycin, mTor, a protein described over a decade ago as related to huntingtin structurally by leucine-rich, repetitive HEAT sequences. Since then, the described functional similarities between Huntingtin and mTor are striking, and this new information about huntingtin's direct association with autophagic vesicles indicates that this structural similarity may extend to functional similarities having an impact upon ER functionality and autophagy.

There are nine genetic neurodegenerative diseases caused by a similar genetic defect, a CAG DNA triplet-repeat expansion in the disease gene's open reading frame resulting in a polyglutamine expansion in the disease proteins. Despite the commonality of polyglutamine expansion, each of the polyglutamine diseases manifest as unique diseases, with some similarities, but important differences. These differences suggest that the context of the polyglutamine expansion is important to the mechanism of pathology of the disease proteins. Therefore, it is becoming increasingly paramount to understand the normal functions of these polyglutamine disease proteins, which include huntingtin, the polyglutamine-expanded protein in Huntington's disease (HD). Transcriptional dysregulation is seen in HD. Here we discuss the role of normal huntingtin in transcriptional regulation and misregulation in Huntington's disease in relation to potentially analogous model systems, and to other polyglutamine disease proteins. Huntingtin has functional roles in both the cytoplasm and the nucleus. One commonality of activity of polyglutamine disease proteins is at the level of protein dynamics and ability to import and export to and from the nucleus. Knowing the temporal location of huntingtin protein in response to signaling and neuronal communication could lead to valuable insights into an important trigger of HD pathology.

Huntington's disease is caused by an expanded polyglutamine tract in huntingtin protein, leading to accumulation of huntingtin in the nuclei of striatal neurons. The 18 amino-acid amino-terminus of huntingtin is an amphipathic alpha helical membrane-binding domain that can reversibly target to vesicles and the endoplasmic reticulum (ER). The association of huntingtin to the ER is affected by ER stress. A single point mutation in huntingtin 1-18 predicted to disrupt this helical structure displayed striking phenotypes of complete inhibition of polyglutamine-mediated aggregation, increased huntingtin nuclear accumulation and greatly increased mutant huntingtin toxicity in a striatal-derived mouse cell line. Huntingtin vesicular interaction mediated by 1-18 is specific to late endosomes and autophagic vesicles. We propose that huntingtin has a normal biological function as an ER-associated protein that can translocate to the nucleus and back out in response to ER stress or other events. The increased nuclear entry of mutant huntingtin due to loss of ER-targeting results in increased toxicity.

Since the early 1990s, DNA triplet repeat expansions have been found to be the cause in an ever increasing number of genetic neurologic diseases. A subset of this large family of genetic diseases has the expansion of a CAG DNA triplet in the open reading frame of a coding exon. The result of this DNA expansion is the expression of expanded glutamine amino acid repeat tracts in the affected proteins, leading to the term, Polyglutamine Diseases, which is applied to this sub-family of diseases. To date, nine distinct genes are known to be linked to polyglutamine diseases, including Huntington's disease, Machado-Joseph Disease and spinobulbar muscular atrophy or Kennedy's disease. Most of the polyglutamine diseases are characterized clinically as spinocerebellar ataxias. Here we discuss recent successes and advancements in polyglutamine disease research, comparing these different diseases with a common genetic flaw at the level of molecular biology and early drug design for a family of diseases where many new research tools for these genetic disorders have been developed. Polyglutamine disease research has successfully used interdisciplinary collaborative efforts, informative multiple mouse genetic models and advanced tools of pharmaceutical industry research to potentially serve as the prototype model of therapeutic research and development for rare neurodegenerative diseases.