Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.

Targeted proteomics strategies present a streamlined hypothesis-driven approach to analyze specific sets of pathways or disease related proteins. goDig is a quantitative, targeted tandem mass tag (TMT)-based assay that can measure the relative abundance differences for hundreds of proteins directly from unfractionated mixtures. Specific protein groups or entire pathways of up to 200 proteins can be selected for quantitative profiling, while leveraging sample multiplexing permits the simultaneous analysis of up to 18 samples. Despite these benefits, implementing goDig is not without challenges, as it requires access to an instrument application programming interface (iAPI), an elution order and spectral library, a web-based method builder, and dedicated companion software. In addition, the absence of an example test assay may dissuade researchers from testing or implementing goDig. Here, we repurpose the TKO11 standard─which is commercially available but may also be assembled in-lab─and establish it as a de facto test assay for goDig. We build a proteome-wide goDig yeast library, quantify protein expression across several gene ontology (GO) categories, and compare these results to a fully fractionated yeast gold-standard data set. Essentially, we provide a guide detailing the goDig-based quantification of TKO11, which can also be used as a template for user-defined assays in other species.

Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

Analyzing histone ChIP-seq data with a bin-based probability of being signal.

Histone ChIP-seq is one of the primary methods for charting the cellular epigenomic landscape, the components of which play a critical regulatory role in gene expression. Analyzing the activity of regulatory elements across datasets and cell types can be challenging due to shifting peak positions and normalization artifacts resulting from, for example, differing read depths, ChIP efficiencies, and target sizes. Moreover, broad regions of enrichment seen in repressive histone marks often evade detection by commonly used peak callers. Here, we present a simple and versatile method for identifying enriched regions in ChIP-seq data that relies on estimating a gamma distribution fit to non-overlapping 5kB genomic bins to establish a global background. We use this distribution to assign a probability of being signal (PBS) between zero and one to each 5 kB bin. This approach, while lower in resolution than typical peak-calling methods, provides a straightforward way to identify enriched regions and compare enrichments among multiple datasets, by transforming the data to values that are universally normalized and can be readily visualized and integrated with downstream analysis methods. We demonstrate applications of PBS for both broad and narrow histone marks, and provide several illustrations of biological insights which can be gleaned by integrating PBS scores with downstream data types.

Discovery of a DCAF11-dependent cyanoacrylamide-containing covalent degrader of BET-proteins.

Targeted protein degradation is mediated by small molecules that induce or stabilize protein-protein interactions between targets and the ubiquitin-proteasome machinery. Currently, there remains a need to expand the repertoire of viable E3 ligases available for hijacking. Notably, covalent chemistry has been employed to engage a handful of E3 ligases, including DCAF11. Here, we disclose a covalent PROTAC that enables DCAF11-dependent degradation, featuring a cyanoacrylamide warhead. Our findings underscore DCAF11 as an interesting candidate with a capacity to accommodate diverse electrophilic chemistries compatible with targeted protein degradation.

Proteomic Portraits Reveal Evolutionarily Conserved and Divergent Responses to Spinal Cord Injury.

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of cerebrospinal fluid and serum samples from 111 patients with acute SCI and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at 6 months with an area under the receiver operating characteristic curve of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response and establish the cerebrospinal fluid abundance of glial fibrillary acidic protein as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

Non-Invasive Treatment of Basal Cell Carcinoma: Photodynamic Therapy Following Curettage.

BACKGROUND: To investigate the effectiveness, safety, patient satisfaction, and cosmetic outcome of Methyl Aminolevulinate-Photodynamic Therapy (MAL-PDT) following curettage in order to make recommendations for its use in dermatology practices. METHODS: A retrospective chart review of patients who received MAL-PDT following curettage for the indication of basal cell carcinoma (BCC) between 2009 and 2016 at a single private clinic in Ontario, Canada. Two hundred and seventy-eight patients with 352 BCC lesions were included, consisting of 44.2% males (n=123) and 55.8% females (n=155) with a mean age of 57.24 years. The primary outcome measurement consisted of the cure rate. Secondary outcome measurements included side effects, patient satisfaction, and cosmetic outcome, as reported in the medical charts. RESULTS: The overall cure rate was 90.3% (n=318). After controlling for age, sex, and lesion type, nasal lesions were approximately 2.82 (95% CI: 1.24-6.40, P=0.01) times more likely to experience a recurrence. 18.3% of patients (n=51) reported side effects, the most common being burning (n=19). Of those who expressed satisfaction, 100% (n=25) reported being happy. Of lesions with cosmetic data, 90.3% displayed a good response (n=149). CONCLUSION: MAL-PDT following curettage is an effective and safe treatment option for BCC lesions with a good cosmetic outcome and suggested high patient satisfaction. J Drugs Dermatol. 2023;22(5): doi:10.36849/JDD.7133.

The impact of social connections and discrimination to HIV risk among Asian gay and bisexual men in Australia.

BACKGROUND: Asian gay, bisexual, and other men who have sex with men (GBMSM) are overrepresented in new HIV diagnoses in Australia. Social engagement with other GBMSM has been associated with HIV testing and pre-exposure prophylaxis (PrEP) uptake. Asian GBMSM may be socially disconnected from LGBTQ+ people, which may increase their HIV risk. This analysis assessed the contribution of social connection on HIV risk among Asian GBMSM. METHODS: Using an online cross-sectional survey of Asian GBMSM in Australia, we measured condomless anal intercourse (CLAI) in the last 6months without PrEP or an undetectable viral load (UVL), i.e. CLAI with a risk of HIV transmission. Bivariable and multivariable logistic regression models were performed to compare demographic characteristics and social engagement of participants who had CLAI without PrEP or UVL to those who had not. Analyses were restricted to participants who reported sex with casual partners in the last 6months. RESULTS: Among 509 participants who had casual partners in the last 6months, 151 (29.7%) reported CLAI without PrEP or UVL. CLAI without PrEP or UVL was negatively associated with full-time employment, and recently being tested for HIV and was positively associated with experiencing discrimination based on sexual orientation. Social engagement with LGBTQ+ people was not associated with CLAI without PrEP or UVL. CONCLUSIONS: CLAI without PrEP or UVL was not related to social connections with LGBTQ+ people but was more likely among Asian men who had experienced sexuality-related discrimination, suggesting that mitigating homophobia and biphobia may assist in improving HIV prevention among Asian GBMSM who live in Australia.

Prospective, Single-arm, Split-face Pain Management Evaluation of Nitrous Oxide System During Micro-Focused Ultrasound With Visualization.

BACKGROUND: Although micro-focused ultrasound with visualization (MFU-V) has great cosmetic results, patients can be hesitant or decline the treatment due to the pain experienced during the treatment. The primary objective is to assess the efficacy of pain management of the nitrous oxide system during MFU-V treatment. The secondary objectives include patient satisfaction and efficacy on reducing facial laxity and improving aesthetic appearance. PATIENTS AND METHODS: A prospective, single-arm, split-face, open-label study. Ten patients were treated with MFU-V without use of a self-administered nitrous oxide system on the right side of the face, followed by use of the nitrous oxide system on the left. A patient-reported Visual Analog Pain Intensity Scale was used to assess pain on the right and left sides at 3 different treatment time points. Patient satisfaction was measured using a 5-point Likert scale and efficacy was measured using the Investigator GAIS and the Facial Laxity Score. RESULTS: Clinically significant differences were seen between pain ratings for the treated and untreated sides at 2 of the 3 time points. 100.0% of patients (n=9) who had completed the study were very satisfied (55.6%) or somewhat satisfied (44.4%). Five patients (55.6%) were scored as Improved on the GAIS and 4 patients (44.4%) were scored as Much Improved. 90.00% (n=9) of patients had moderate lower face and neck laxity prior to treatment, compared with 11.11% (n=1) and 22.22% (n=2) with moderate laxity at follow-up. CONCLUSION: The nitrous oxide system provides clinically significant pain reduction, increases patient satisfaction, and improves cosmetic results of MFU-V treatment. J Drugs Dermatol. 2022;21(11):1228-1234. doi:10.36849/JDD.7030.

Barriers to HIV testing in hospital settings within a culturally diverse urban district of Sydney, Australia.

Background Eleven percent of people living with HIV in Australia remain unaware of their diagnosis, and there are missed opportunities for HIV testing in priority settings in New South Wales. HIV testing remains low outside of sexual health clinics with the exception of antenatal settings where HIV testing is routine. To understand why HIV testing rates are low, we sought to identify health worker-related barriers to HIV testing. METHODS: We conducted an anonymous online survey to health workers in Western Sydney Local Health District (WSLHD) in September 2019. Tick-box, Likert scale responses were analysed using Chi-square and Kruskal-Wallis statistical tests, and free text responses were analysed with thematic analysis. RESULTS: Three percent (n = 420) of WSLHD's estimated 14 000 health workers responded. These included 317 clinicians (171 nurses, 65 doctors, 56 allied health professionals (AHPs), 25 midwives, and 103 health workers in non-clinical roles). Health workers were from a variety of in-patient/out-patient settings. Many health workers (291/420, 69%; 95%CI = 64.9-73.7%) were unaware that HIV testing is offered in their areas; doctors (82%) and midwives (80%) were more aware than nurses (23%) and AHPs (11%) (P < 0.0001). Doctors (Likert score = 3.62; 3.45/5) and midwives (2.84; 2.76) were significantly more comfortable discussing and confidently offering HIV testing than nurses (2.42; 1.81) or AHPs (1.83; 0.91) (P < 0.0001 for both). The top five barriers to HIV testing were (1) procedural knowledge, (2) identification of at-risk patients, (3) HIV knowledge, (4) positive result management, and (5) privacy concerns. Free text responses highlighted perceived stigma, testing/result responsibilities and resource challenges as barriers to HIV testing. CONCLUSIONS: Clinicians working in priority settings and with priority populations require more education and support to increase targeted HIV testing.

PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation.

PURPOSE: Radiotherapy (RT) constitutes an important part of breast cancer treatment. However, triple negative breast cancers (TNBC) exhibit remarkable resistance to most therapies, including RT. Developing new ways to radiosensitize TNBC cells could result in improved patient outcomes. The M2 isoform of pyruvate kinase (PK-M2) is believed to be responsible for the re-wiring of cancer cell metabolism after oxidative stress. The aim of the study was to determine the effect of ionizing radiation (IR) on PK-M2-mediated metabolic changes in TNBC cells, and their survival. In addition, we determine the effect of PK-M2 activators on breast cancer stem cells, a radioresistant subpopulation of breast cancer stem cells. METHODS: Glucose uptake, lactate production, and glutamine consumption were assessed. The cellular localization of PK-M2 was evaluated by western blot and confocal microscopy. The small molecule activator of PK-M2, TEPP46, was used to promote its pyruvate kinase function. Finally, effects on cancer stem cell were evaluated via sphere forming capacity. RESULTS: Exposure of TNBC cells to IR increased their glucose uptake and lactate production. As expected, PK-M2 expression levels also increased, especially in the nucleus, although overall pyruvate kinase activity was decreased. PK-M2 nuclear localization was shown to be associated with breast cancer stem cells, and activation of PK-M2 by TEPP46 depleted this population. CONCLUSIONS: Radiotherapy can induce metabolic changes in TNBC cells, and these changes seem to be mediated, at least in part by PK-M2. Importantly, our results show that activators of PK-M2 can deplete breast cancer stem cells in vitro. This study supports the idea of combining PK-M2 activators with radiation to enhance the effect of radiotherapy in resistant cancers, such as TNBC.

Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing.

Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, despite the fact that this KMTase is required for H3K9 dimethylation (H3K9me2) and efficient DNA methylation of these retroelements. Transcription of the exogenous retrovirus (XRV) Moloney murine leukemia virus is rapidly extinguished after integration in mESCs, concomitant with de novo DNA methylation. However, the role that H3K9 KMTases play in this process has not been addressed. Here, we demonstrate that G9a, but not Suv39h1 or Suv39h2, is required for silencing of newly integrated Moloney murine leukemia virus-based vectors in mESCs. The silencing defect in G9a(-/-) cells is accompanied by a reduction of H3K9me2 at the proviral LTR, indicating that XRVs are direct targets of G9a. Furthermore, de novo DNA methylation of newly integrated proviruses is impaired in the G9a(-/-) line, phenocopying proviral DNA methylation and silencing defects observed in Dnmt3a-deficient mESCs. Once established, however, maintenance of silencing of XRVs, like ERVs, is dependent exclusively on the KMTase Eset. Taken together, these observations reveal that in mESCs, the H3K9 KMTase G9a is required for the establishment, but not for the maintenance, of silencing of newly integrated proviruses.

Human saliva modifies growth, biofilm architecture, and competitive behaviors of oral streptococci.

The bacteria within supragingival biofilms participate in complex exchanges with other microbes inhabiting the same niche. One example is the mutans group streptococci (Streptococcus mutans), implicated in the development of tooth decay, and other health-associated commensal streptococci species. Previously, our group transcriptomically characterized intermicrobial interactions between S. mutans and several species of oral bacteria. However, these experiments were carried out in a medium without human saliva. To better mimic their natural environment, we first evaluated how inclusion of saliva affected growth and biofilm formation of eight Streptococcus species individually and found saliva to positively benefit growth rates while negatively influencing biofilm biomass accumulation and altering spatial arrangement. These results carried over during evaluation of 29 saliva-derived isolates of various species. Surprisingly, we also found that addition of saliva increased the competitive behaviors of S. mutans in coculture competitions against commensal streptococci that led to increases in biofilm microcolony volumes. Through transcriptomically characterizing mono- and cocultures of S. mutans and Streptococcus oralis with and without saliva, we determined that each species developed a nutritional niche under mixed-species growth, with S. mutans upregulating carbohydrate uptake and utilization pathways while S. oralis upregulated genome features related to peptide uptake and glycan foraging. S. mutans also upregulated genes involved in oxidative stress tolerance, particularly manganese uptake, which we could artificially manipulate by supplementing in manganese leading to an advantage over its opponent. Our report highlights observable changes in microbial behaviors through leveraging environmental- and host-supplied resources over their competitors. IMPORTANCE: Dental caries (tooth decay) is the most prevalent disease for both children and adults nationwide. Caries are initiated from demineralization of the enamel due to organic acid production through the metabolic activity of oral bacteria growing in biofilm communities attached to the tooth's surface. Mutans group streptococci are closely associated with caries development and initiation of the cariogenic cycle, which decreases the amount of acid-sensitive, health-associated commensal bacteria while selecting for aciduric and acidogenic species that then further drives the disease process. Defining the exchanges that occur between mutans group streptococci and oral commensals in a condition that closely mimics their natural environment is of critical need toward identifying factors that can influence odontopathogen establishment, persistence, and outgrowth. The goal of our research is to develop strategies, potentially through manipulation of microbial interactions characterized here, that prevent the emergence of mutans group streptococci while keeping the protective flora intact.

Alkylamine-tethered molecules recruit FBXO22 for targeted protein degradation.

Targeted protein degradation (TPD) relies on small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation by the proteasome. Only a few of the approximately 600 human E3 ligases are currently amenable to this strategy. This limits the actionable target space and clinical opportunities and thus establishes the necessity to expand to additional ligases. Here we identify and characterize SP3N, a specific degrader of the prolyl isomerase FKBP12. SP3N features a minimal design, where a known FKBP12 ligand is appended with a flexible alkylamine tail that conveys degradation properties. We found that SP3N is a precursor and that the alkylamine is metabolized to an active aldehyde species that recruits the SCFFBXO22 ligase for FKBP12 degradation. Target engagement occurs via covalent adduction of Cys326 in the FBXO22 C-terminal domain, which is critical for ternary complex formation, ubiquitylation and degradation. This mechanism is conserved for two recently reported alkylamine-based degraders of NSD2 and XIAP, thus establishing alkylamine tethering and covalent hijacking of FBXO22 as a generalizable TPD strategy.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation.

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

'I want him to tell me he loves me': A smart audio device, Tochie, for resident-family connection in long-term care.

BACKGROUND: Older people living in long-term care homes are particularly susceptible to loneliness and social isolation, which the COVID-19 pandemic has exacerbated further. 'Tochie' is a smart audio device that allows family members to remotely record and send messages, such as daily reminders and comforting recordings, to their loved ones in LTC settings. The purpose of this study was to assess the feasibility and acceptability of using Tochie to improve resident-family connections, and to investigate user experience, impact and lessons learned. METHODS: Participants included 10 residents, nine family members and six care staff from two LTC homes in British Columbia, Canada. A Tochie was provided to each resident to use with their family members over a 4-week intervention period. The research team provided support and gathered feedback from family members and care staff through weekly phone and email correspondence. Qualitative descriptive design was used, including pre- and post-intervention focus groups and interviews held via Zoom and phone to gather participants' experiences with Tochie. Themes were identified through thematic analysis. RESULTS: Three themes were identified: (1) Facilitating emotional connection, (2) Using the device in creative and personalised ways and (3) Structural challenges and supports. Based on these findings, recommendations for future research and practice are provided. CONCLUSION: The COVID-19 pandemic has prompted a rethinking of what it means to 'stay in touch' with loved ones in LTC settings. This study found that Tochie has opened up new opportunities for family connection and provided emotional support for residents. The results of this study offer valuable insights into the implementation of assistive technology in LTC homes to support resident care.

Human Saliva Modifies Growth, Biofilm Architecture and Competitive Behaviors of Oral Streptococci.

The bacteria within supragingival biofilms participate in complex exchanges with other microbes inhabiting the same niche. One example are the mutans group streptococci (Streptococcus mutans), implicated in the development of tooth decay, and other health-associated commensal streptococci species. Previously, our group transcriptomically characterized intermicrobial interactions between S. mutans and several species of oral bacteria. However, these experiments were carried out in a medium that was absent of human saliva. To better mimic their natural environment, we first evaluated how inclusion of saliva affected growth and biofilm formation of eight streptococci species individually, and found saliva to positively benefit growth rates while negatively influencing biomass accumulation and altering spatial arrangement. These results carried over during evaluation of 29 saliva-derived isolates of various species. Surprisingly, we also found that addition of saliva increased the competitive behaviors of S. mutans in coculture competitions against commensal streptococci that led to increases in biofilm microcolony volumes. Through transcriptomically characterizing mono- and cocultures of S. mutans and Streptococcus oralis with and without saliva, we determined that each species developed a nutritional niche under mixed-species growth, with S. mutans upregulating carbohydrate uptake and utilization pathways while S. oralis upregulated genome features related to peptide uptake and glycan foraging. S. mutans also upregulated genes involved in oxidative stress tolerance, particularly manganese uptake, which we could artificially manipulate by supplementing in manganese to give it an advantage over its opponent. Our report highlights observable changes in microbial behaviors via leveraging environmental- and host-supplied resources over their competitors.

Large-scale map of RNA binding protein interactomes across the mRNA life-cycle.

Messenger RNAs (mRNAs) interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific RNA substrates, there has been considerably less exploration leveraging protein-protein interaction (PPI) methodologies to identify and study the role of proteins in mRNA life-cycle stages. To address this gap, we generated an RNA-aware RBP-centric PPI map across the mRNA life-cycle by immunopurification (IP-MS) of ~100 endogenous RBPs across the life-cycle in the presence or absence of RNase, augmented by size exclusion chromatography (SEC-MS). Aside from confirming 8,700 known and discovering 20,359 novel interactions between 1125 proteins, we determined that 73% of our IP interactions are regulated by the presence of RNA. Our PPI data enables us to link proteins to life-cycle stage functions, highlighting that nearly half of the proteins participate in at least two distinct stages. We show that one of the most highly interconnected proteins, ERH, engages in multiple RNA processes, including via interactions with nuclear speckles and the mRNA export machinery. We also demonstrate that the spliceosomal protein SNRNP200 participates in distinct stress granule-associated RNPs and occupies different RNA target regions in the cytoplasm during stress. Our comprehensive RBP-focused PPI network is a novel resource for identifying multi-stage RBPs and exploring RBP complexes in RNA maturation.

Association of CSF and Serum Neurofilament Light and Glial Fibrillary Acidic Protein, Injury Severity, and Outcome in Spinal Cord Injury.

BACKGROUND AND OBJECTIVES: Traumatic spinal cord injury (SCI) is highly heterogeneous, and tools to better delineate pathophysiology and recovery are needed. Our objective was to profile the response of 2 biomarkers, neurofilament light (NF-L) and glial fibrillary acidic protein (GFAP), in the serum and CSF of patients with acute SCI to evaluate their ability to objectively characterize injury severity and predict neurologic recovery. METHODS: Blood and CSF samples were obtained from prospectively enrolled patients with acute SCI through days 1-4 postinjury, and the concentration of NF-L and GFAP was quantified using Simoa technology. Neurologic assessments defined the ASIA Impairment Scale (AIS) grade and motor score (MS) at presentation and 6 months postinjury. RESULTS: One hundred eighteen patients with acute SCI (78 AIS A, 20 AIS B, and 20 AIS C) were enrolled, with 113 (96%) completing 6-month follow-up. NF-L and GFAP levels were strongly associated between paired serum and CSF specimens, were both increased with injury severity, and distinguished among baseline AIS grades. Serum NF-L and GFAP were significantly (p = 0.02 to <0.0001) higher in AIS A patients who did not improve at 6 months, predicting AIS grade conversion with a sensitivity and specificity (95% CI) of 76% (61, 87) and 77% (55, 92) using NF-L and 72% (57, 84) and 77% (55, 92) using GFAP at 72 hours, respectively. Independent of clinical baseline assessment, a serum NF-L threshold of 170 pg/mL at 72 hours predicted those patients who would be classified as motor complete (AIS A/B) compared with motor incomplete (AIS C/D) at 6 months with a sensitivity of 87% (76, 94) and specificity of 84% (69, 94); a serum GFAP threshold of 13,180 pg/mL at 72 hours yielded a sensitivity of 90% (80, 96) and specificity of 84% (69, 94). DISCUSSION: The potential for NF-L and GFAP to classify injury severity and predict outcome after acute SCI will be useful for patient stratification and prognostication in clinical trials and inform communication of prognosis. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that higher serum NF-L and GFAP are associated with worse neurological outcome after acute SCI. TRIAL REGISTRATION INFORMATION: Registered on ClinicalTrials.gov: NCT00135278 (March 2006) and NCT01279811 (January 2012).

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane.

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking ß-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.

DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity.

Histone H3K9 methylation is required for DNA methylation and silencing of repetitive elements in plants and filamentous fungi. In mammalian cells however, deletion of the H3K9 histone methyltransferases (HMTases) Suv39h1 and Suv39h2 does not affect DNA methylation of the endogenous retrovirus murine leukaemia virus, indicating that H3K9 methylation is dispensable for DNA methylation of retrotransposons, or that a different HMTase is involved. We demonstrate that embryonic stem (ES) cells lacking the H3K9 HMTase G9a show a significant reduction in DNA methylation of retrotransposons, major satellite repeats and densely methylated CpG-rich promoters. Surprisingly, demethylated retrotransposons remain transcriptionally silent in G9a(-/-) cells, and show only a modest decrease in H3K9me2 and no decrease in H3K9me3 or HP1alpha binding, indicating that H3K9 methylation per se is not the relevant trigger for DNA methylation. Indeed, introduction of catalytically inactive G9a transgenes partially 'rescues' the DNA methylation defect observed in G9a(-/-) cells. Taken together, these observations reveal that H3K9me3 and HP1alpha recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity.