A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Gonococcal PorB: a multifaceted modulator of host immune responses.

Neisseria gonorrhoeae is a human-specific pathogen responsible for the sexually transmitted infection, gonorrhoea. N. gonorrhoeae promotes its survival by manipulating both innate and adaptive immune responses. The most abundant gonococcal outer-membrane protein is PorB, an essential porin that facilitates ion exchange. Importantly, gonococcal PorB has several immunomodulatory properties. To subvert the innate immune response, PorB suppresses killing mechanisms of macrophages and neutrophils, and recruits negative regulators of complement to the gonococcal cell surface. For manipulation of adaptive immune responses, gonococcal PorB suppresses the capability of dendritic cells to stimulate proliferation of T cells. As gonococcal PorB is highly abundant in outer-membrane vesicles, consideration of the immunomodulatory properties of this porin is critical when designing gonococcal vaccines.

The molecular epidemiology and antimicrobial resistance of Staphylococcus pseudintermedius canine clinical isolates submitted to a veterinary diagnostic laboratory in South Africa.

Staphylococcus pseudintermedius is an important cause of clinical infections in small-animal-veterinary medicine. Evolutionary changes of strains using multilocus sequence typing (MLST) have been observed among S. pseudintermedius in European countries and the United States. However, there are limited or no studies on the detection of methicillin resistant Staphylococcus pseudintermedius (MRSP) and predominating MLST strains in South Africa. Therefore, this study aimed to determine the molecular epidemiology of S. pseudintermedius in South Africa. Twenty-six, non-duplicate, clinical isolates from dogs were obtained as convenience samples from four provinces in South Africa. The Kirby Bauer disk diffusion test was used to determine antimicrobial susceptibility. We used Resfinder and the Comprehensive Antibiotic Resistance Database (CARD) to detect antimicrobial resistance genes. Virulence genes were identified using the virulence factor database and Basic Local Alignment Search Tool (BLASTN) on Geneious prime. geoBURST analysis was used to study relationships between MLST. Finally, the maximum likelihood phylogeny was determined using Randomized Axelerated Maximum Likelihood (RAxML). Twenty-three isolates were confirmed as S. pseudintermedius of which 14 were MRSP. In addition to ß-lactam antimicrobials, MRSP isolates were resistant to tetracycline (85.7%), doxycycline (92.8%), kanamycin (92.8%), and gentamicin (85.7%). The isolates harbored antimicrobial resistance genes (tetM, ermB, drfG, cat, aac(6')-Ie-aph(2")-Ia, ant(6)-Ia, and aph(3')-III) and virulence genes (AdsA, geh, icaA, and lip). MLST analysis showed that ST2228, ST2229, ST2230, ST2231, ST2232, ST2318, ST2326 and ST2327 are unique sequence types in South Africa. Whereas, previously reported major STs including ST45, ST71, ST181, ST551 and ST496 were also detected. The geoBURST and phylogenetic analysis suggests that the isolates in South Africa are likely genetically related to isolates identified in other countries. Highly resistant MRSP strains (ST496, ST71, and ST45) were reported that could present challenges in the treatment of canine infections in South Africa. Hence, we have gained a better understanding of the epidemiology of MRSP in the African continent, the genes involved in resistance and virulence factors associated with these organisms.

Temporal changes in antibiotic resistance and population structure of methicillin-resistant Staphylococcus pseudintermedius between 2010 and 2021 in the United States.

The aim of this study was to perform a phenotypic and molecular epidemiological survey to determine temporal changes in the antimicrobial resistance and population structure of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in the United States. Samples from 200 S. pseudintermedius isolates were obtained from veterinary diagnostic facilities located in geographic regions sampled approximately ten years ago and compared to samples obtained in 2021. Kirby-Bauer disk diffusion was used to determine antimicrobial susceptibility. geoBURST analysis and MrBayes were used to infer relationships of isolates using MLST data. Almost all MRSP isolates (98%) in 2021 were multidrug-resistant with 21% of these isolates resistant to more than 16 antimicrobials. In 2010, 190 S. pseudintermedius isolates were collected and 141 of them were MRSP. From 2010-2021 there was a significant increase in resistance observed to all antibiotics tested except cephalothin and sulfonamides. Whereas ten years previously multilocus sequence types (ST) ST68 (35.7%), ST71 (10%), and ST84 (17.4%) predominated, these strains have been supplanted by other STs, notably ST45 (n = 14), ST155 (n = 9), ST181 (n = 13), ST496 (n = 9) and ST551 (n = 9). The newly prevalent STs are distantly related to ST68, ST71 and ST84 and most likely do not share any recent common ancestors. The population structure of MRSP is far more elastic than expected with new, highly resistant strains replacing the ones that predominated in the United States a decade ago. Antibiotic use may play a role in selection; however, the strains that were replaced were also multidrug-resistant and other factors are likely involved.

An epidemiological study of the predictors of multidrug resistance and methicillin resistance among Staphylococcus spp. isolated from canine specimens submitted to a diagnostic laboratory in Tennessee, USA.

Background: Understanding drivers of multidrug resistance (MDR) and methicillin resistance, which have increased among canine staphylococcal isolates, is essential for guiding antimicrobial use practices. Therefore, the objective of this study was to identify predictors of MDR and methicillin resistance among Staphylococcus spp. commonly isolated from canine clinical specimens. Methods: This retrospective study used records of canine specimens submitted to the University of Tennessee College of Veterinary Medicine Clinical Bacteriology Laboratory for bacterial culture and antimicrobial susceptibility testing between 2006 and 2017. Records from 7,805 specimens positive for the following Staphylococcus species were included for analysis: Staphylococcus pseudintermedius, Staphylococcus aureus, Staphylococcus coagulans (formerly Staphylococcus schleiferi subspecies coagulans), and Staphylococcus schleiferi (formerly S. schleiferi subsp. schleiferi). Generalized linear regression models were fit using generalized estimating equations (GEE) to identify predictors of MDR (defined as resistance to three or more antimicrobial classes) and methicillin resistance among these isolates. Results: Multidrug resistance (42.1%) and methicillin resistance (31.8%) were relatively common. Isolates from skeletal (joint and bone) specimens had the highest levels of MDR (51.3%) and methicillin resistance (43.6%), followed by cutaneous specimens (45.8% multidrug-resistant, 37.1% methicillin resistant). Staphylococcus species, specimen site, and clinical setting were significant (p < 0.01) predictors of both outcomes. Compared to S. pseudintermedius, S. schleiferi had higher odds of methicillin resistance, while S. coagulans and S. schleiferi had lower odds of MDR. The odds of both MDR and methicillin resistance for isolates from hospital patient specimens were significantly higher than those from referral patients for urine/bladder and otic specimens. Odds of MDR among isolates from skeletal specimens of hospital patients were also higher than those of referral patients. Conclusions: Staphylococcus isolates in this study had substantial levels of MDR and methicillin resistance. Differences in the odds of these outcomes between referral and hospital patient isolates did not persist for all specimen sites, which may reflect differences in diagnostic testing and antimicrobial use practices with respect to body site or system. Judicious antimicrobial use, informed by culture and susceptibility testing, is important to limit treatment failures and curb selection pressure.

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Like, it's important: The frequency and use of the discourse marker like in older autistic children.

Background & Aims: Discourse markers, such as well or like, serve a variety of functions to support conversational reciprocity: filling pauses, aiding word-finding, and modulating turn-taking by holding the conversational floor. Previous research shows that autistic individuals use discourse markers less frequently than non-autistic (NonAu) peers; however, the discourse marker like has not been included in that research, despite its ubiquitous use by NonAu individuals, and despite the fact that like serves important pragmatic functions that are not encoded by any other discourse marker. Specifically, like signals to the listener that the content of upcoming speech is 1) Important/new; 2) Loose/approximate; 3) Reformulative; or 4) Quotative. The current study addresses this gap in the literature by comparing the frequency of discourse marker like use between older autistic and non-autistic children as well as exploring patterns of usage between the four like functions. Methods: Twenty-one 10-to-17-year-old children on the autism spectrum and 20 NonAu peers-statistically matched on age, sex, IQ and language scores-engaged in a semi-structured interview with a researcher. Uses of discourse-marker like were identified from written transcripts of interviews and each use was categorized into one of the four functions. Results: There were no significant differences in like frequencies between groups, nor were there differences in relative proportions of functions used by each group. Conclusions: Research consistently indicates that autistic individuals use discourse markers significantly less often than their NonAu counterparts, but the findings from our study suggest that this pattern does not persist to all such markers. This group of older autistic children use like as often as their peers and use it to signify similar information about upcoming speech to their listener.

Membrane lipid renovation in Pseudomonas aeruginosa - implications for phage therapy?

Pseudomonas aeruginosa is an important Gram-negative pathogen with intrinsic resistance to many clinically used antibiotics. It is particularly troublesome in nosocomial infections, immunocompromised patients, and individuals with cystic fibrosis. Antimicrobial resistance (AMR) is a huge threat to global health, with a predicted 10 million people dying from resistant infections by 2050. A promising therapy for combatting AMR infections is phage therapy. However, more research is required to investigate mechanisms that may influence the efficacy of phage therapy. An important overlooked aspect is the impact of membrane lipid remodelling on phage binding ability. P. aeruginosa undergoes changes in membrane lipids when it encounters phosphorus stress, an environmental perturbation that is likely to occur during infection. Lipid changes include the substitution of glycerophospholipids with surrogate glycolipids and the over-production of ornithine-containing aminolipids. Given that membrane lipids are known to influence the structure and function of membrane proteins, we propose that changes in the composition of membrane lipids during infection may alter phage binding and subsequent phage infection dynamics. Consideration of such effects needs to be urgently prioritised in order to develop the most effective phage therapy strategies for P. aeruginosa infections.

Markerless gene editing in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the gonococcus, is a pathogen of major public health concern, but sophisticated approaches to gene manipulation are limited for this species. For example, there are few methods for generating markerless mutations, which allow the generation of precise point mutations and deletions without introducing additional DNA sequence. Markerless mutations are central to studying pathogenesis, the spread of antimicrobial resistance (AMR) and for vaccine development. Here we describe the use of galK as a counter-selectable marker that can be used for markerless mutagenesis in N. gonorrhoeae. galK encodes galactokinase, an enzyme that metabolizes galactose in bacteria that can utilize it as a sole carbon source. GalK can also phosphorylate a galactose analogue, 2-deoxy-galactose (2-DOG), into a toxic, non-metabolisable intermediate, 2-deoxy-galactose-1-phosphate. We utilized this property of GalK to develop a markerless approach for mutagenesis in N. gonorrhoeae. We successfully deleted both chromosomally and plasmid-encoded genes, that are important for gonococcal vaccine development and studies of AMR spread. We designed a positive-negative selection cassette, based on an antibiotic resistance marker and galK, that efficiently rendered N. gonorrhoeae susceptible to growth on 2-DOG. We then adapted the galK-based counter-selection and the use of 2-DOG for markerless mutagenesis, and applied biochemical and phenotypic analyses to confirm the absence of target genes. We show that our markerless mutagenesis method for N. gonorrhoeae has a high success rate, and should be a valuable gene editing tool in the future.

Cutaneous Mycobacterium goodii infection in an immunocompetent cat in Louisiana: clinical presentation, molecular identification, antimicrobial susceptibility and management.

Case summary: A 9-year-old spayed female domestic shorthair cat was presented to a referral hospital for management of recurring non-healing ulcerations and a subcutaneous mass on the ventral abdomen. Prior treatment included antibiotics (cefovecin followed by clindamycin), wound cleaning and surgical debulking, but the ulcerations and mass recurred 1 month after surgical removal. At this point, the cat was started on doxycycline and pradofloxacin and referred for further work-up. The culture of skin biopsy specimens obtained at the time of referral revealed a population of bacterial colonies with two distinctly different phenotypes. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene sequencing identified both colonies as Mycobacterium goodii. A diagnosis of a cutaneous infection of rapidly growing mycobacteria was made, and treatment with oral pradofloxacin and doxycycline was initiated. The ulcerations resolved within 4 months, and the subcutaneous mass gradually decreased in size until it was no longer palpable, even 4 months after the cessation of antibiotics. Relevance and novel information: This is the second reported feline cutaneous M goodii infection in North America. The organism was not visualized on histopathology but was successfully cultured from tissue obtained by skin punch biopsy. A phenotypic switching phenomenon affecting the susceptibility results was suspected, possibly explaining the presence of phenotypically different but genetically identical strains. This case highlights the importance of submitting aseptically obtained tissue, fluid or fine-needle aspirates for culture and species identification, as well as histopathology, when infection with higher bacteria, such as rapidly growing mycobacteria, is suspected.

Patterns of antimicrobial, multidrug and methicillin resistance among Staphylococcus spp. isolated from canine specimens submitted to a diagnostic laboratory in Tennessee, USA: a descriptive study.

BACKGROUND: Multidrug- and methicillin-resistant staphylococci are both veterinary and public health concerns due to their zoonotic potential. Therefore, the objective of this study was to investigate patterns of antimicrobial, multidrug, and methicillin resistance among four Staphylococcus spp. commonly isolated from canine clinical specimens submitted to the Clinical Bacteriology Laboratory at the University of Tennessee College of Veterinary Medicine (UTCVM). METHODS: Results of antimicrobial susceptibility testing and mecA polymerase chain reaction (PCR) for isolates of four common Staphylococcus spp. isolates were obtained from the Bacteriology Laboratory at the UTCVM between 01/01/2006 and 12/31/2017. Cochran-Armitage trend test was used to assess temporal trends of antimicrobial resistance (AMR), multidrug resistance (MDR), and methicillin resistance. Kappa test of agreement was used to assess agreement between the results of PCR and disk diffusion tests. RESULTS: Most of the 7805 isolates were S. pseudintermedius (6453 isolates), followed by S. coagulans (860), S. aureus (330), and S. schleiferi (162). Among S. pseudintermedius isolates, 45.5% were MDR, and 30.8% were methicillin-resistant (MRSP). There was a significant temporal increase in MRSP (p = 0.017). Chloramphenicol resistance increased among both MRSP and methicillin-susceptible (MSSP) isolates (p <  0.0001). Among S. aureus isolates, 40.9% were MDR, 37.4% were methicillin-resistant (MRSA), and the proportion of MRSA isolates increased significantly (p = 0.0480) over time. There was an increasing temporal trend in the proportion of MDR isolates among MSSP (p = 0.0022), but a decrease among MRSP (p <  0.0001) and MRSA (p = 0.0298). S. schleiferi had the highest percentage (56.9%) of methicillin-resistant isolates. Oxacillin disk diffusion was superior to cefoxitin for the detection of mecA-mediated resistance and had almost perfect agreement with mecA PCR assay for S. pseudintermedius (95.4% agreement, kappa (κ) = 0.904; p <  0.0001), S. coagulans (95.6%, κ = 0.913; p <  0.0001) and S. schleiferi (97.7%, κ = 0.945; p <  0.0001). However, cefoxitin disk diffusion was superior to oxacillin disk diffusion and had almost perfect agreement with mecA PCR assay for S. aureus (95.3%, κ = 0.834; p <  0.0001). CONCLUSIONS: The levels of resistance and increasing temporal trends are concerning. These findings have implications for treatment decisions and public health due to the zoonotic potential of staphylococci. Continued surveillance and use of antibiograms to guide clinical decisions will be critical.

Unresolved stalled ribosome complexes restrict cell-cycle progression after genotoxic stress.

During the translation surveillance mechanism known as ribosome-associated quality control, the ASC-1 complex (ASCC) disassembles ribosomes stalled on the mRNA. Here, we show that there are two distinct classes of stalled ribosome. Ribosomes stalled by translation elongation inhibitors or methylated mRNA are short lived in human cells because they are split by the ASCC. In contrast, although ultraviolet light and 4-nitroquinoline 1-oxide induce ribosome stalling by damaging mRNA, and the ASCC is recruited to these stalled ribosomes, we found that they are refractory to the ASCC. Consequently, unresolved UV- and 4NQO-stalled ribosomes persist in human cells. We show that ribosome stalling activates cell-cycle arrest, partly through ZAK-p38MAPK signaling, and that this cell-cycle delay is prolonged when the ASCC cannot resolve stalled ribosomes. Thus, we propose that the sensitivity of stalled ribosomes to the ASCC influences the kinetics of stall resolution, which in turn controls the adaptive stress response.

Novel open reading frames in human accelerated regions and transposable elements reveal new leads to understand schizophrenia and bipolar disorder.

Schizophrenia (SCZ) and bipolar disorder are debilitating neuropsychiatric disorders arising from a combination of environmental and genetic factors. Novel open reading frames (nORFs) are genomic loci that give rise to previously uncharacterized transcripts and protein products. In our previous work, we have shown that nORFs can be biologically regulated and that they may play a role in cancer and rare diseases. More importantly, we have shown that nORFs may emerge in accelerated regions of the genome giving rise to species-specific functions. We hypothesize that nORFs represent a potentially important group of biological factors that may contribute to SCZ and bipolar disorder pathophysiology. Human accelerated regions (HARs) are genomic features showing human-lineage-specific rapid evolution that may be involved in biological regulation and have additionally been found to associate with SCZ genes. Transposable elements (TEs) are another set of genomic features that have been shown to regulate gene expression. As with HARs, their relevance to SCZ has also been suggested. Here, nORFs are investigated in the context of HARs and TEs. This work shows that nORFs whose expression is disrupted in SCZ and bipolar disorder are in close proximity to HARs and TEs and that some of them are significantly associated with SCZ and bipolar disorder genomic hotspots. We also show that nORF encoded proteins can form structures and potentially constitute novel drug targets.

In vitro and in vivo assessment of caprine origin Staphylococcus aureus ST398 strain UTCVM1 as an osteomyelitis pathogen.

Staphylococcus aureus (SA) is a significant and well-recognized causative organism of bacterial osteomyelitis. Osteomyelitis is an inflammatory bone disease characterized by progressive bone destruction and loss. This disease causes significant morbidity and mortality to the patient and poses therapeutic challenges for clinicians. To improve the efficacy of therapeutic strategies to combat bacterial osteomyelitis, there is a need to define the molecular epidemiology of bacterial organisms more clearly and further the understanding of the pathogenesis of SA osteomyelitis. We conducted in vitro characterization of the pathogenic capabilities of an isolate of SA ST398 derived from a clinical case of osteomyelitis in a goat. We also report a rodent mandibular defect model to determine the ability of ST398 to cause reproducible osteomyelitis. Our results indicate that ST398 can invade and distort pre-osteoblastic cells in culture, induce significant inflammation and alter expression of osteoregulatory cytokines. We also demonstrate the ability of ST398 to induce osteomyelitis in a rat mandibular model. When compiled, these data support ST398 as a competent osteomyelitis pathogen.

Corrigendum: In vitro and In vivo assessment of caprine origin Staphylococcus aureus ST398 strain UTCVM1 as an osteomyelitis pathogen.

[This corrects the article DOI: 10.3389/fcimb.2022.1015655.].

Phosphorus stress induces the synthesis of novel glycolipids in Pseudomonas aeruginosa that confer protection against a last-resort antibiotic.

Pseudomonas aeruginosa is a nosocomial pathogen with a prevalence in immunocompromised individuals and is particularly abundant in the lung microbiome of cystic fibrosis patients. A clinically important adaptation for bacterial pathogens during infection is their ability to survive and proliferate under phosphorus-limited growth conditions. Here, we demonstrate that P. aeruginosa adapts to P-limitation by substituting membrane glycerophospholipids with sugar-containing glycolipids through a lipid renovation pathway involving a phospholipase and two glycosyltransferases. Combining bacterial genetics and multi-omics (proteomics, lipidomics and metatranscriptomic analyses), we show that the surrogate glycolipids monoglucosyldiacylglycerol and glucuronic acid-diacylglycerol are synthesised through the action of a new phospholipase (PA3219) and two glycosyltransferases (PA3218 and PA0842). Comparative genomic analyses revealed that this pathway is strictly conserved in all P. aeruginosa strains isolated from a range of clinical and environmental settings and actively expressed in the metatranscriptome of cystic fibrosis patients. Importantly, this phospholipid-to-glycolipid transition comes with significant ecophysiological consequence in terms of antibiotic sensitivity. Mutants defective in glycolipid synthesis survive poorly when challenged with polymyxin B, a last-resort antibiotic for treating multi-drug resistant P. aeruginosa. Thus, we demonstrate an intriguing link between adaptation to environmental stress (nutrient availability) and antibiotic resistance, mediated through membrane lipid renovation that is an important new facet in our understanding of the ecophysiology of this bacterium in the lung microbiome of cystic fibrosis patients.

Acute neurological consequences of novel psychoactive substance use: a retrospective review in a large UK hospital.

BACKGROUND: Novel psychoactive substances (NPS) are a growing public health concern. We aimed to identify the acute neurological consequences of NPS. METHOD: We performed a retrospective case-note review of patients who presented to the emergency department after taking NPS. RESULTS: We identified 237 admissions from 190 patients, mostly young men. There were high rates of psychiatric comorbidity (43%), unemployment (39%), homelessness (24%) and incarceration (17%). Most reported use of synthetic cannabinoids (SC; 91%). Some took synthetic cathinones (SCath; 7%) or nitrous oxide (NOS; 2%). SC caused impaired consciousness (61%) and seizures (16%). SCath users presented with psychiatric disturbance or seizures (55%). Most patients were managed conservatively (67%) and a small proportion (14%) were referred to drug or psychology services. CONCLUSIONS: NPS users represent a vulnerable group in society. Certain clinical features may suggest the type of NPS used. Most patients require supportive management and onward referral to drug addiction services is recommended.

Proteomics insights into the Burkholderia cenocepacia phosphorus stress response.

The Burkholderia cepacia complex is a group of Burkholderia species that are opportunistic pathogens causing high mortality rates in patients with cystic fibrosis. An environmental stress often encountered by these soil-dwelling and pathogenic bacteria is phosphorus limitation, an essential element for cellular processes. Here, we describe cellular and extracellular proteins differentially regulated between phosphate-deplete (0 mM, no added phosphate) and phosphate-replete (1 mM) growth conditions using a comparative proteomics (LC-MS/MS) approach. We observed a total of 128 and 65 unique proteins were downregulated and upregulated respectively, in the B. cenocepacia proteome. Of those downregulated proteins, many have functions in amino acid transport/metabolism. We have identified 24 upregulated proteins that are directly/indirectly involved in inorganic phosphate or organic phosphorus acquisition. Also, proteins involved in virulence and antimicrobial resistance were differentially regulated, suggesting B. cenocepacia experiences a dramatic shift in metabolism under these stress conditions. Overall, this study provides a baseline for further research into the biology of Burkholderia in response to phosphorus stress.

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.