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.

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.

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.

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.

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.

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.

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.

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.

'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.

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).

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.

Long-range phasing of dynamic, tissue-specific and allele-specific regulatory elements.

Epigenomic maps identify gene regulatory elements by their chromatin state. However, prevailing short-read sequencing methods cannot effectively distinguish alleles, evaluate the interdependence of elements in a locus or capture single-molecule dynamics. Here, we apply targeted nanopore sequencing to profile chromatin accessibility and DNA methylation on contiguous ~100-kb DNA molecules that span loci relevant to development, immunity and imprinting. We detect promoters, enhancers, insulators and transcription factor footprints on single molecules based on exogenous GpC methylation. We infer relationships among dynamic elements within immune loci, and order successive remodeling events during T cell stimulation. Finally, we phase primary sequence and regulatory elements across the H19/IGF2 locus, uncovering primate-specific features. These include a segmental duplication that stabilizes the imprinting control region and a noncanonical enhancer that drives biallelic IGF2 expression in specific contexts. Our study advances emerging strategies for phasing gene regulatory landscapes and reveals a mechanism that overrides IGF2 imprinting in human cells.

Concomitant Therapy of Oral Isotretinoin with Multiplex Pulsed Dye Laser and Nd: YAG Laser for Acne.

Objective: To evaluate concomitant therapy of oral isotretinoin with multiplex pulsed dye laser and Nd:YAG laser. Methods: A retrospective chart review of patients who received treatment of oral isotretinoin and non-ablative laser therapy to treat acne vulgaris at a single outpatient dermatology clinic site in Ontario, Canada between 2009 and 2017. Results: 187 patients were included, consisting of 45.5 percent males (n=85) and 54.5 percent females (n=102) with a mean age of 21.4 years. 31.6 percent (n=59) of patients reported experiencing side effects from concomitant isotretinoin and NAL therapy, the most common being eczema (n=14), erythema (n=11), significant dry skin/lips/eyes (n=8), flushing (n=6), and bruising (n=6). 99.2 percent of patients achieved clear or almost clear at treatment completion. Of those who expressed satisfaction, 65.2 percent (n=122) reported being satisfied with the treatment and the remaining patients did not report satisfaction nor dissatisfaction. Limitations: Limitations exist mainly due to the absence of standardized lesion counts and a comparator cohort. Thus, it is not possible to comment on whether the combination of isotretinoin and NAL is more efficacious that either treatment alone. Conclusion: Concomitant use of isotretinoin and non-ablative laser therapy is a safe and effective treatment option for acne vulgaris that provides patient satisfaction.

International Spinal Cord Injury Biobank: A Biorepository and Resource for Translational Research.

Over the past few decades, tremendous advances have been made in our understanding of the biological changes underpinning the devastating impairment of traumatic spinal cord injury (SCI). Much of this scientific research has focused on animal models of SCI, and comparatively little has been done in human SCI, largely because biospecimens from human SCI patients are not readily available. This paucity of scientific enquiry in human SCI represents an important void in the spectrum of translational research, as biological differences between animal models and the human condition need to be considered in the pre-clinical development of therapeutic approaches. The International Spinal Cord Injury Biobank (ISCIB) is a multi-user biorepository with the mission of accelerating therapeutic development in traumatic SCI through improved biological understanding of human injury, and the vision of serving as a global research resource where human SCI biospecimens are shared with researchers around the world. Aligned with internationally recognized best practices, ISCIB's formal governance structure and standard operating procedures have earned it official biobank certification through the Canadian Tissue Repository Network. Herein, we describe the translational research gap that ISCIB is helping to fill; its structure, governance and certification; how data and samples are accrued, processed and stored; and finally, the process through which samples and data are shared with global researchers. The purpose of this paper describing ISCIB is to serve as an introductory guidance document for the wider community of SCI researchers. By helping researchers understand the contents of ISCIB and the process of accessing biospecimens, we seek to further ISCIB's vision as being a resource for human and translational research in SCI, with the ultimate goal of finding disease-modifying therapies for this disabling condition.

Concomitant therapy of surgical shave excision and intralesional injections for ear keloids: Early results from a retrospective cohort study.

Introduction: Keloids are hypertrophic scars that commonly arise in the ear region. The authors' objectives were to (1) evaluate effectiveness of surgical shave excision followed by intralesional triamcinolone acetonide and onabotulinumtoxinA injections; and (2) evaluate safety and patient satisfaction. Methods and Materials: This study was a retrospective chart review of patients who received treatment of extralesional surgical shave excision followed by intralesional triamcinolone acetonide and onabotulinumtoxinA injections to treat ear keloids at a single outpatient dermatology clinic. A prospective patient questionnaire was administered to the same patient population to collect recurrence and patient satisfaction. Results: A total of 45 patients were included, consisting of 84.4% females (n = 38) and 15.6% males (n = 7) with a mean age of 25.5 years. Through retrospective chart review, early recurrence was seen in 6.7% of patients (n = 3), and via the prospective patient questionnaire, 11.1% of patients noted early keloid recurrence (n = 5). Of the patients who expressed their level of satisfaction in-clinic, 96.0% (n = 24) reported being satisfied or very satisfied and 4.0% (n = 1) were dissatisfied. Satisfaction was also assessed through the prospective patient questionnaire; of those who consented to the questionnaire, 100.0% (n = 24) were satisfied or very satisfied. Only 20.0% (n = 9) of all patients reported experiencing side effects, consisting of pruritus (11.1%; n = 5), tenderness (4.4%; n = 2), pain (2.2%; n = 1), and mild atrophy (2.2%; n = 1). Conclusion: Extralesional surgical shave excision followed by intralesional triamcinolone acetonide and onabotulinumtoxinA injections may represent a promising treatment option for ear keloids.Evidence Level: 3 retrospective cohort study. Lay Summary: Keloids are a type of raised scar, which can be painful and itchy for patients. Keloids can occur on various part of the body, including on the ear. They are challenging to treat and tend to come back. There are many treatment options, however, there is not one universal best treatment for keloids on the ear. We hoped to discover if shave excision followed by intralesional triamcinolone acetonide and onabotulinumtoxinA injections is effective at treating keloids on the ear. In order to answer this we completed a chart review of clinic patients, who have already completed the following combination treatment for keloids on the ear. The keloids were treated first by physically removing the bulk of the keloid with a scalpel, which is called shave excision. After the removal, triamcinolone acetonide and onabotulinumtoxinA were injected directly into the keloid. The rate of patient satisfaction and the rate of the keloid returning were collected during in-clinic visits and an optional post-clinic patient questionnaire. The treatment effectiveness and side effects experienced were reported during in-clinic visits. This indicated that with the low rate of side effects, high patient satisfaction, and low rate of keloid return, this treatment combination should be considered as an option for keloids on the ear. However, since this review was completed at one clinic with a small population of patients, it is not fully known if this treatment combination will work for all patients.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Crosstalk between CRISPR-Cas9 and the human transcriptome.

CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas9 can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas9 in human cells and find that Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Critically, transcriptome-wide Cas9 binding sites do not appear to correlate with published genome-wide Cas9 DNA binding or cut-site loci under gRNA co-expression. However, even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Our findings do not support the hypothesis that human RNAs can broadly guide Cas9 to bind and cleave human genomic DNA, but they illustrate a cellular and RNA impact likely inherent to CRISPR-Cas systems.

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.

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.