A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution.

Frozen shoulder is a spontaneously self-resolving chronic inflammatory fibrotic human disease, which distinguishes the condition from most fibrotic diseases that are progressive and irreversible. Using single-cell analysis, we identify pro-inflammatory MERTKlowCD48+ macrophages and MERTK + LYVE1 + MRC1+ macrophages enriched for negative regulators of inflammation which co-exist in frozen shoulder capsule tissues. Micro-cultures of patient-derived cells identify integrin-mediated cell-matrix interactions between MERTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in frozen shoulder resolution. Cross-tissue analysis reveals a shared gene expression cassette between shoulder capsule MERTK+ macrophages and a respective population enriched in synovial tissues of rheumatoid arthritis patients in disease remission, supporting the concept that MERTK+ macrophages mediate resolution of inflammation and fibrosis. Single-cell transcriptomic profiling and spatial analysis of human foetal shoulder tissues identify MERTK + LYVE1 + MRC1+ macrophages and DKK3+ and POSTN+ fibroblast populations analogous to those in frozen shoulder, suggesting that the template to resolve fibrosis is established during shoulder development. Crosstalk between MerTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts could facilitate resolution of frozen shoulder, providing a basis for potential therapeutic resolution of persistent fibrotic diseases.

Intravenous anakinra for the treatment of haemophagocytic lymphohistiocytosis/macrophage activation syndrome: A systematic review.

BACKGROUND: Haemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) has a potentially high mortality rate. Anakinra, an interleukin-1 receptor antagonist, is now recommended early in HLH/MAS, with intravenous (IV) use proposed in critically unwell patients. This systematic review establishes the literature relating to IV anakinra in secondary HLH/MAS (sHLH/MAS). METHODS: We screened Embase, PubMed, and Medline, including all reports of IV anakinra for HLH or MAS. We extracted age, HLH/MAS trigger, continuous infusion or bolus dosing, and survival. RESULTS: Twenty-nine case reports/series identified 87 patients (median age 22 years, range 22 months to 84 years), all with sHLH. Amongst identifiable triggers, 43% were systemic infection, 33% rheumatological, 9% oncological. Children had predominantly a rheumatological trigger (48%), whilst adults were more commonly infection-driven (50%). Overall, rheumatologically triggered disease showed greater survival (83.3%), particularly compared with oncological triggers (42.9%). Children had a greater survival, particularly under 10 years (83%, vs. adults, 63%). CONCLUSIONS: Despite IV anakinra recipients likely to be critically unwell, this cohort had similar disease triggers and survival compared to large historical cohorts, and enhances awareness of age and trigger-specific survival patterns. IV anakinra had a wide therapeutic dosing range and tolerability, regardless of trigger, demonstrating substantial utility in severe sHLH/MAS.

Nanocellulose from Nata de Coco as a Bioscaffold for Cell-Based Meat.

The three-dimensional formation of bio-engineered tissue for applications such as cell-based meat requires critical interaction between the bioscaffold and cellular biomass. To explore the features underlying this interaction, we have assessed the commercially available bacterial nanocellulose (BNC) product from Cass Materials for its suitability to serve as a bioscaffold for murine myoblast attachment, proliferation, and differentiation. Rigorous application of both scanning electron microscopy and transmission electron microscopy reveals cellular details of this interaction. While the retention rate of myoblast cells appears low, BNC is able to provide effective surface parameters for the formation of anchor points to form mature myotubes. Understanding the principles that govern this interaction is important for the successful scaling of these materials into edible, commercially viable, and nutritious biomass.

Continuous intravenous anakinra for treating severe secondary haemophagocytic lymphohistiocytosis/macrophage activation syndrome in critically ill children.

The cytokine storm of secondary haemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) can cause life-threatening multiorgan failure. Interleukin-1 (IL-1) receptor blockade with anakinra can be effective in the management of sHLH/MAS. Subcutaneous (SC) dosing regimens are widely described; however, intravenous (IV) dosing is advantageous where time-critical intervention is vital and where SC oedema and/or hypoperfusion limits absorption. We review three critically ill children (aged 9, 11 and 17) with sHLH and rapidly progressive multiorgan dysfunction, successfully treated with continuous IV anakinra infusion. This case series significantly enhances the incipient knowledge regarding the safety and efficacy of IV anakinra for life-threatening sHLH.

Using ICD-10 diagnostic codes to identify 'missing' paediatric patients during nationwide COVID-19 lockdown in Oxfordshire, UK.

The study aims to identify 'missing' diagnoses amongst paediatric admissions during the UK's first national lockdown, compared with the previous 5 years. A retrospective observational cohort study of all children (0-15 years) attending for urgent care across Oxfordshire, during the first UK lockdown in 2020, compared to matched dates in 2015-2019, across two paediatric hospitals providing secondary care, including one with tertiary services. Our outcome measures were changes in numbers of patients attending and inpatient diagnoses (using ICD-10 classification) during the first 2020 lockdown, compared with the previous 5 years, were used. We found that total Emergency Department (ED) attendances (n = 4030) and hospital admissions (n = 1416) during the first UK lockdown were reduced by 56.8% and 59.4%, respectively, compared to 2015-2019 (5-year means n = 7446.8 and n = 2491.6, respectively). Proportions of patients admitted from ED and length of stay were similar across 2015-2020. ICD-10 diagnoses in lockdown of 2020 (n = 2843) versus matched 2015-2019 dates (n = 19,946) demonstrated significantly greater neoplasm diagnoses (p = 0.0123). Of diagnoses 'missing' in lockdown, 80% were categorised as infectious diseases or their sequelae and 20% were non-specific pains/aches/malaise and accidental injury/poisonings.Conclusions: Pandemic public health measures significantly altered paediatric presentations. Oxfordshire hospitals had a 58% reduction in ED attendances/inpatient admissions, with 'missing' diagnoses predominantly infection-related illnesses. These are likely driven by a combination of the following: (1) public health infection control measures successfully reducing disease transmission, (2) parents/carers keeping mild/self-limiting disease at home, and (3) pandemic-related healthcare anxieties. Prospective studies are needed to ensure referral pathways identify vulnerable children, those with social concerns, and avoid delayed presentation. What is Known: • Significant reductions of paediatric ED attendances and inpatient admissions are reported globally, throughout national and regional lockdowns for COVID-19. • Previous studies (supplemental table 5) examined only ED diagnoses or specific inpatient diagnoses during lockdown periods, demonstrating reductions of infectious diseases, accidents/injuries, and safeguarding referrals. What is New: • Using ICD-10 coding, robustly controlling for five historical years and adopting a hypothesis-independent analysis, demonstrating 80% of 'missing' inpatient diagnoses during national COVID-19 lockdown were infectious diseases or its sequelae, the remainder being non-specific aches/pains/malaise and accidental injuries/poisonings. • Greater numbers of neoplasms and other specific diagnoses were detected during lockdown, including greater documentation of co-morbidities and incidental findings.

Decreased ATM Function Causes Delayed DNA Repair and Apoptosis in Common Variable Immunodeficiency Disorders.

PURPOSE: Common variable immunodeficiency disorders (CVID) is characterized by low/absent serum immunoglobulins and susceptibility to bacterial infection. Patients can develop an infections-only phenotype or a complex disease course with inflammatory, autoimmune, and/or malignant complications. We hypothesized that deficient DNA repair mechanisms may be responsible for the antibody deficiency and susceptibility to inflammation and cancer in some patients. METHODS: Germline variants were identified following targeted sequencing of n = 252 genes related to DNA repair in n = 38 patients. NanoString nCounter PlexSet assay measured gene expression in n = 20 CVID patients and n = 7 controls. DNA damage and apoptosis were assessed by flow cytometry in n = 34 CVID patients and n = 11 controls. RESULTS: Targeted sequencing supported enrichment of rare genetic variants in genes related to DNA repair pathways with novel and rare likely pathogenic variants identified and an altered gene expression signature that distinguished patients from controls and complex patients from those with an infections-only phenotype. Consistent with this, flow cytometric analyses of lymphocytes following DNA damage revealed a subset of CVID patients whose immune cells have downregulated ATM, impairing the recruitment of other repair factors, delaying repair and promoting apoptosis. CONCLUSION: These data suggest that germline genetics and altered gene expression predispose a subset of CVID patients to increased sensitivity to DNA damage and reduced DNA repair capacity.

Archaea join the conversation: detection of AHL-like activity across a range of archaeal isolates.

Quorum sensing is a mechanism of genetic control allowing single cell organisms to coordinate phenotypic response(s) across a local population and is often critical for ecosystem function. Although quorum sensing has been extensively studied in bacteria comparatively less is known about this mechanism in Archaea. Given the growing significance of Archaea in both natural and anthropogenic settings, it is important to delineate how widespread this phenomenon of signaling is in this domain. Employing a plasmid-based AHL biosensor in conjunction with thin-layer chromatography (TLC), the present study screened a broad range of euryarchaeota isolates for potential signaling activity. Data indicated the presence of 11 new Archaeal isolates with AHL-like activity against the LuxR-based AHL biosensor, including for the first time putative AHL activity in a thermophile. The presence of multiple signals and distinct changes between growth phases were also shown via TLC. Multiple signal molecules were detected using TLC in Haloferax mucosum, Halorubrum kocurii, Natronococcus occultus and Halobacterium salinarium. The finding of multiple novel signal producers suggests the potential for quorum sensing to play an important role not only in the regulation of complex phenotypes within Archaea but the potential for cross-talk with bacterial systems.

Communication within East Antarctic Soil Bacteria.

Antarctica, being the coldest, driest, and windiest continent on Earth, represents the most extreme environment in which a living organism can survive. Under constant exposure to harsh environmental threats, terrestrial Antarctica remains home to a great diversity of microorganisms, indicating that the soil bacteria must have adapted a range of survival strategies that require cell-to-cell communication. Survival strategies include secondary metabolite production, biofilm formation, bioluminescence, symbiosis, conjugation, sporulation, and motility, all of which are often regulated by quorum sensing (QS), a type of bacterial communication. Until now, such mechanisms have not been explored in terrestrial Antarctica. In this study, LuxI/LuxR-based quorum sensing (QS) activity was delineated in soil bacterial isolates recovered from Adams Flat, in the Vestfold Hills region of East Antarctica. Interestingly, we identified the production of potential homoserine lactones (HSLs) with chain lengths ranging from medium to long in 19 bacterial species using three biosensors, namely, Agrobacterium tumefaciens NTL4, Chromobacterium violaceum CV026, and Escherichia coli MT102, in conjunction with thin-layer chromatography (TLC). The majority of detectable HSLs were from Gram-positive species not previously known to produce HSLs. This discovery further expands our understanding of the microbial community capable of this type of communication, as well as provides insights into physiological adaptations of microorganisms that allow them to survive in the harsh Antarctic environment.IMPORTANCE Quorum sensing, a type of bacterial communication, is widely known to regulate many processes, including those that confer a survival advantage. However, little is known about communication by bacteria residing within Antarctic soils. Employing a combination of bacterial biosensors, analytical techniques, and genome mining, we found a variety of Antarctic soil bacteria speaking a common language, via LuxI/LuxR-based quorum sensing, thus potentially supporting survival in a mixed microbial community. This study reports potential quorum sensing activity in Antarctic soils and has provided a platform for studying physiological adaptations of microorganisms that allow them to survive in the harsh Antarctic environment.

Isolation of novel quorum-sensing active bacteria from microbial mats in Shark Bay Australia.

Quorum sensing is a potent system of genetic control allowing phenotypes to be coordinated across localized communities. In this study, quorum sensing systems in Shark Bay microbial mats were delineated using a targeted approach analyzing whole mat extractions as well as the creation of an isolate library. A library of 165 isolates from different mat types were screened using the AHL biosensor E. coli MT102. Based on sequence identity 30 unique isolates belonging to Proteobacteria, Actinobacteria and Firmicutes were found to activate the AHL biosensor, suggesting AHLs or analogous compounds were potentially present. Several of the isolates have not been shown previously to produce signal molecules, particularly the members of the Actinobacteria and Firmicutes phyla including Virgibacillus, Halobacillius, Microbacterium and Brevibacterium. These active isolates were further screened using thin-layer chromatography (TLC) providing putative identities of AHL molecules present within the mat communities. Nine isolates were capable of producing several spots of varying sizes after TLC separation, suggesting the presence of multiple signalling molecules. This study is the first to delineate AHL-based signalling in the microbial mats of Shark Bay, and suggests quorum sensing may play a role in the ecosphysiological coordination of complex phenotypes across microbial mat communities.

Disentangling the drivers of functional complexity at the metagenomic level in Shark Bay microbial mat microbiomes.

The functional metagenomic potential of Shark Bay microbial mats was examined for the first time at a millimeter scale, employing shotgun sequencing of communities via the Illumina NextSeq 500 platform in conjunction with defined chemical analyses. A detailed functional metagenomic profile has elucidated key pathways and facilitated inference of critical microbial interactions. In addition, 87 medium-to-high-quality metagenome-assembled genomes (MAG) were assembled, including potentially novel bins under the deep-branching archaeal Asgard group (Thorarchaetoa and Lokiarchaeota). A range of pathways involved in carbon, nitrogen, sulfur, and phosphorus cycles were identified in mat metagenomes, with the Wood-Ljungdahl pathway over-represented and inferred as a major carbon fixation mode. The top five sets of genes were affiliated to sulfate assimilation (cysNC cysNCD, sat), methanogenesis (hdrABC), Wood-Ljungdahl pathways (cooS, coxSML), phosphate transport (pstB), and copper efflux (copA). Polyhydroxyalkanoate (PHA) synthase genes were over-represented at the surface, with PHA serving as a potential storage of fixed carbon. Sulfur metabolism genes were highly represented, in particular complete sets of genes responsible for both assimilatory and dissimilatory sulfate reduction. Pathways of environmental adaptation (UV, hypersalinity, oxidative stress, and heavy metal resistance) were also delineated, as well as putative viral defensive mechanisms (core genes of the CRISPR, BREX, and DISARM systems). This study provides new metagenome-based models of how biogeochemical cycles and adaptive responses may be partitioned in the microbial mats of Shark Bay.

Effects of placebos without deception compared with no treatment: A systematic review and meta-analysis.

OBJECTIVE: To investigate the clinical efficacy of open-label placebos compared with no treatment in a systematic review and meta-analysis. METHODS: We searched the Cochrane Injuries Group's Specialised Register, The Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations (OvidSP), EMBASE (OvidSP), and clinical trials registers and screened reference lists. The search was run on 27th April 2015. We included all randomized controlled trials of any medical condition with open-label placebo and no-treatment groups. Authors independently assessed records and extracted data. We excluded nonrandomized trials and nonclinical studies. Risk of bias was assessed using Cochrane criteria. We used random-effects model for meta-analysis. RESULTS: We screened 348 publications, assessed 24 articles for eligibility and identified five trials (260 participants) that met inclusion criteria. The clinical conditions were: irritable bowel syndrome, depression, allergic rhinitis, back pain, and attention deficit hyperactivity disorder. The risk of bias was moderate. We found a positive effect for nondeceptive placebos (standardized mean difference 0.88, 95% CI 0.62 to 1.14, P < 0.00001, I2 = 1%). CONCLUSIONS: Open-label placebos appear to have positive clinical effects compared to no treatment. Caution is warranted when interpreting these results due to the limited number of trials identified, lack of blinding, and the fact that positive messages were included alongside open-label placebos. Larger definitive trials are now warranted to explore the potential patient benefit of open-label placebos, to investigate the relative contributions of positive suggestions, and ethical implications.

Effects of placebos without deception compared with no treatment: protocol for a systematic review and meta-analysis.

INTRODUCTION: Placebos have long provided a robust control for evaluating active pharmacological preparations, but frequently demonstrate a variable therapeutic effect when delivered in double-blinded placebo-controlled trials. Delivery of placebos as treatment alone has been considered unethical, as it has been thought that deception is essential for their effect. However, recent evidence suggests that clinical benefit can be derived from placebos delivered without deception (unblinded/open-label) manner. Here, we present a protocol for the first systematic review and meta-analysis of studies of the effects of non-deceptive placebos compared with no treatment. METHODS AND ANALYSIS: This protocol will compare the effect of placebos delivered non-deceptively to no treatment. It will also assess the methods of delivery used for non-deceptive placebos. Studies will be sought through relevant database searches and will include those within disease settings and those among healthy controls. To be included, trials must include both non-deceptive (open-label) placebo and no treatment groups. All data extraction and analysis will be conducted by two independent reviewers. The analysis will evaluate any differences in outcome measures between the non-deceptive placebo and no treatment groups. Outcome measures will be the clinically-relevant outcomes detailed in the primary papers. The delivery methods, such as verbal instructions, which may provide positive expectations and outcomes, of non-deceptive placebos will also be assessed. Each study will be comprehensively assessed for bias. Subgroup analyses will identify any discrepancies among heterogeneous data. ETHICS AND DISSEMINATION: This review does not require ethical approval. The completed review will be widely disseminated by publication and social media where appropriate. This protocol has been registered on PROSPERO (2015:CRD42015023347).

Untapped Resources: Biotechnological Potential of Peptides and Secondary Metabolites in Archaea.

Archaea are an understudied domain of life often found in "extreme" environments in terms of temperature, salinity, and a range of other factors. Archaeal proteins, such as a wide range of enzymes, have adapted to function under these extreme conditions, providing biotechnology with interesting activities to exploit. In addition to producing structural and enzymatic proteins, archaea also produce a range of small peptide molecules (such as archaeocins) and other novel secondary metabolites such as those putatively involved in cell communication (acyl homoserine lactones), which can be exploited for biotechnological purposes. Due to the wide array of metabolites produced there is a great deal of biotechnological potential from antimicrobials such as diketopiperazines and archaeocins, as well as roles in the cosmetics and food industry. In this review we will discuss the diversity of small molecules, both peptide and nonpeptide, produced by archaea and their potential biotechnological applications.

Detection and characterization of N-acyl-l-homoserine lactones using GFP-based biosensors in conjunction with thin-layer chromatography.

Many microorganisms use quorum sensing to regulate several complex phenotypes, and this is accomplished by the release of a signal molecule(s) into the environment. N-acyl-homoserine lactones (AHLs) are a common class of signalling molecule utilized by a range of microorganisms (primarily Gram negative bacteria but most recently also archaea) and are often detected through the use of bacterial biosensors. Biosensors can be limited by both their specificity and sensitivity, and the aim of this study was to modify and improve current AHL detection strategies. The biosensor employed in the present study was Escherichia coli MT102 harbouring a plasmid containing a LuxR based biosensor, which produces green fluorescent protein (GFP) as a reporting mechanism. A new method of visualizing the GFP based biosensor overlaid on silica sheets for the purpose of thin-layer chromatography (TLC) is presented. This new method vastly improves sensitivity of AHL detection by a GFP biosensor than previously reported and as such represents a powerful new tool in AHL research.

Quorum sensing in extreme environments.

Microbial communication, particularly that of quorum sensing, plays an important role in regulating gene expression in a range of organisms. Although this phenomenon has been well studied in relation to, for example, virulence gene regulation, the focus of this article is to review our understanding of the role of microbial communication in extreme environments. Cell signaling regulates many important microbial processes and may play a pivotal role in driving microbial functional diversity and ultimately ecosystem function in extreme environments. Several recent studies have characterized cell signaling in modern analogs to early Earth communities (microbial mats), and characterization of cell signaling systems in these communities may provide unique insights in understanding the microbial interactions involved in function and survival in extreme environments. Cell signaling is a fundamental process that may have co-evolved with communities and environmental conditions on the early Earth. Without cell signaling, evolutionary pressures may have even resulted in the extinction rather than evolution of certain microbial groups. One of the biggest challenges in extremophile biology is understanding how and why some microbial functional groups are located where logically they would not be expected to survive, and tightly regulated communication may be key. Finally, quorum sensing has been recently identified for the first time in archaea, and thus communication at multiple levels (potentially even inter-domain) may be fundamental in extreme environments.

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection.

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.