A critical role for transcription factor Smad4 in T cell function that is independent of transforming growth factor ß receptor signaling.

Transforming growth factor-beta (TGF-ß) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-ß signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-ßR) deletion and compromised T-cell-mediated tumor rejection. Although Smad4 was dispensable for T cell generation, homeostasis, and effector function, it was essential for T cell proliferation after activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-ßR-independent role for Smad4 in promoting T cell function, autoimmunity, and anti-tumor immunity.

The effectiveness of out-of-hours palliative care telephone advice lines: A rapid systematic review.

BACKGROUND: People with palliative care needs and their carers often rely on out-of-hours services to remain at home. Policymakers have recommended implementing telephone advice lines to ensure 24/7 access to support. However, the impact of these services on patient and carer outcomes, as well as the health care system, remains poorly understood. AIM: To evaluate the clinical- and cost-effectiveness of out-of-hours palliative care telephone advice lines, and to identify service characteristics associated with effectiveness. DESIGN: Rapid systematic review (PROSPERO ID: CRD42023400370) with narrative synthesis. DATA SOURCES: Three databases (Medline, EMBASE and CINAHL) were searched in February 2023 for studies of any design reporting on telephone advice lines with at least partial out-of-hours availability. Study quality was assessed using the Mixed Methods Appraisal Tool, and quantitative and qualitative data were synthesised narratively. RESULTS: Twenty-one studies, published 2000-2022, were included. Most studies were observational, none were experimental. While some evidence suggested that telephone advice lines offer guidance and reassurance, supporting care at home and potentially reducing avoidable emergency care use in the last months of life, variability in reporting and poor methodological quality across studies limit our understanding of patient/carer and health care system outcomes. CONCLUSION: Despite their increasing use, evidence for the clinical- and cost-effectiveness of palliative care telephone advice lines remains limited, primarily due to the lack of robust comparative studies. There is a need for more rigorous evaluations incorporating experimental or quasi-experimental methods and longer follow-up, and standardised reporting of telephone advice line models and outcomes, to guide policy and practice.

Drift dynamics in microbial communities and the effective community size.

The structure and diversity of all open microbial communities are shaped by individual births, deaths, speciation and immigration events; the precise timings of these events are unknowable and unpredictable. This randomness is manifest as ecological drift in the population dynamics, the importance of which has been a source of debate for decades. There are theoretical reasons to suppose that drift would be imperceptible in large microbial communities, but this is at odds with circumstantial evidence that effects can be seen even in huge, complex communities. To resolve this dichotomy we need to observe dynamics in simple systems where key parameters, like migration, birth and death rates can be directly measured. We monitored the dynamics in the abundance of two genetically modified strains of Escherichia coli, with tuneable growth characteristics, that were mixed and continually fed into 10 identical chemostats. We demonstrated that the effects of demographic (non-environmental) stochasticity are very apparent in the dynamics. However, they do not conform to the most parsimonious and commonly applied mathematical models, where each stochastic event is independent. For these simple models to reproduce the observed dynamics we need to invoke an 'effective community size', which is smaller than the census community size.

NUFEB: A massively parallel simulator for individual-based modelling of microbial communities.

We present NUFEB (Newcastle University Frontiers in Engineering Biology), a flexible, efficient, and open source software for simulating the 3D dynamics of microbial communities. The tool is based on the Individual-based Modelling (IbM) approach, where microbes are represented as discrete units and their behaviour changes over time due to a variety of processes. This approach allows us to study population behaviours that emerge from the interaction between individuals and their environment. NUFEB is built on top of the classical molecular dynamics simulator LAMMPS (Large-scale Atomic/Molecular Massively Parallel Simulator), which we extended with IbM features. A wide range of biological, physical and chemical processes are implemented to explicitly model microbial systems, with particular emphasis on biofilms. NUFEB is fully parallelised and allows for the simulation of large numbers of microbes (107 individuals and beyond). The parallelisation is based on a domain decomposition scheme that divides the domain into multiple sub-domains which are distributed to different processors. NUFEB also offers a collection of post-processing routines for the visualisation and analysis of simulation output. In this article, we give an overview of NUFEB's functionalities and implementation details. We provide examples that illustrate the type of microbial systems NUFEB can be used to model and simulate.

Enrichment of Nitrogen-Fixing Bacteria in a Nitrogen-Deficient Wastewater Treatment System.

Anthropogenic nitrogen fixation is essential to sustain a global population of 7.7 billion. However, there has been a long-standing desire to find cheaper and more environmentally friendly alternatives to the Haber-Bosch process. In this study, we developed a new strategy of nitrogen fixation by enriching free-living N2-fixing bacteria (NFB) in reactors fed with low nitrogen wastewater, analogous to those usually found in certain industrial effluents such as paper mills. Our reactors fixed appreciable quantities of nitrogen with a rate of 11.8 mg N L-1 day-1. This rate is comparable to recent "breakthrough" nitrogen-fixing technologies and far higher than observed in low C/N reactors (fed with organic matter and nitrogen). NFB were quantified using quantitative polymerase chain reaction (qPCR) of the nifH (marker gene used to identify biological nitrogen fixation) and 16S rRNA genes. The nifH gene was enriched by a factor of 10 in the nitrogen-fixing reactors (compared to controls) attaining 13% of the bacterial population (1:4.2 copies of nifH to 16S rRNA). The Illumina MiSeq 16S rRNA gene amplicon sequencing of reactors showed that the microbial community was dominated (19%) by Clostridium pasteurianum. We envisage that nitrogen-enriched biomass could potentially be used as a biofertilizer and that the treated wastewater could be released to the environment with very little post-treatment.

The effect of anode potential on current production from complex substrates in bioelectrochemical systems: a case study with glucose.

Anode potential can affect the degradation pathway of complex substrates in bioelectrochemical systems (BESs), thereby influencing current production and coulombic efficiency. However, the intricacies behind this interplay are poorly understood. This study used glucose as a model substrate to comprehensively investigate the effect of different anode potentials (- 150 mV, 0 mV and + 200 mV) on the relationship between current production, the electrogenic pathway and the abundance of the electrogenic microorganisms involved in batch mode fed BESs. Current production in glucose-acclimatized reactors was a function of the abundance of Geobacteraceae and of the availability of acetate and formate produced by glucose degradation. Current production was increased by high anode potentials during acclimation (0 mV and + 200 mV), likely due to more Geobacteraceae developing. However, this effect was much weaker than a stimulus from an artificial high acetate supply: acetate was the rate-limiting intermediate in these systems. The supply of acetate could not be influenced by anode potential; altering the flow regime, batch time and management of the upstream fermentation processes may be a greater engineering tool in BES. However, these findings suggest that if high current production is the focus, it will be extremely difficult to achieve success with complex waste streams such as domestic wastewater.

EBI Metagenomics in 2017: enriching the analysis of microbial communities, from sequence reads to assemblies.

EBI metagenomics (http://www.ebi.ac.uk/metagenomics) provides a free to use platform for the analysis and archiving of sequence data derived from the microbial populations found in a particular environment. Over the past two years, EBI metagenomics has increased the number of datasets analysed 10-fold. In addition to increased throughput, the underlying analysis pipeline has been overhauled to include both new or updated tools and reference databases. Of particular note is a new workflow for taxonomic assignments that has been extended to include assignments based on both the large and small subunit RNA marker genes and to encompass all cellular micro-organisms. We also describe the addition of metagenomic assembly as a new analysis service. Our pilot studies have produced over 2400 assemblies from datasets in the public domain. From these assemblies, we have produced a searchable, non-redundant protein database of over 50 million sequences. To provide improved access to the data stored within the resource, we have developed a programmatic interface that provides access to the analysis results and associated sample metadata. Finally, we have integrated the results of a series of statistical analyses that provide estimations of diversity and sample comparisons.

Characterization of slowly-biodegradable organic compounds and hydrolysis kinetics in tropical wastewater for biological nitrogen removal.

Many developing countries, mostly situated in the tropical region, have incorporated a biological nitrogen removal process into their wastewater treatment plants (WWTPs). Existing wastewater characteristic data suggested that the soluble chemical oxygen demand (COD) in tropical wastewater is not sufficient for denitrification. Warm wastewater temperature (30 °C) in the tropical region may accelerate the hydrolysis of particulate settleable solids (PSS) to provide slowly-biodegradable COD (sbCOD) for denitrification. This study aimed to characterize the different fractions of COD in several sources of low COD-to-nitrogen (COD/N) tropical wastewater. We characterized the wastewater samples from six WWTPs in Malaysia for 22 months. We determined the fractions of COD in the wastewater by nitrate uptake rate experiments. The PSS hydrolysis kinetic coefficients were determined at tropical temperature using an oxygen uptake rate experiment. The wastewater samples were low in readily-biodegradable COD (rbCOD), which made up 3-40% of total COD (TCOD). Most of the biodegradable organics were in the form of sbCOD (15-60% of TCOD), which was sufficient for complete denitrification. The PSS hydrolysis rate was two times higher than that at 20 °C. The high PSS hydrolysis rate may provide sufficient sbCOD to achieve effective biological nitrogen removal at WWTPs in the tropical region.

Regulating, Measuring, and Modeling the Viscoelasticity of Bacterial Biofilms

Biofilms occur in a broad range of environments under heterogeneous physicochemical conditions, such as in bioremediation plants, on surfaces of biomedical implants, and in the lungs of cystic fibrosis patients. In these scenarios, biofilms are subjected to shear forces, but the mechanical integrity of these aggregates often prevents their disruption or dispersal. Biofilms' physical robustness is the result of the multiple biopolymers secreted by constituent microbial cells which are also responsible for numerous biological functions. A better understanding of the role of these biopolymers and their response to dynamic forces is therefore crucial for understanding the interplay between biofilm structure and function. In this paper, we review experimental techniques in rheology, which help quantify the viscoelasticity of biofilms, and modeling approaches from soft matter physics that can assist our understanding of the rheological properties. We describe how these methods could be combined with synthetic biology approaches to control and investigate the effects of secreted polymers on the physical properties of biofilms. We argue that without an integrated approach of the three disciplines, the links between genetics, composition, and interaction of matrix biopolymers and the viscoelastic properties of biofilms will be much harder to uncover.

Metabolites of an Oil Field Sulfide-Oxidizing, Nitrate-Reducing Sulfurimonas sp. Cause Severe Corrosion.

Oil reservoir souring and associated material integrity challenges are of great concern to the petroleum industry. The bioengineering strategy of nitrate injection has proven successful for controlling souring in some cases, but recent reports indicate increased corrosion in nitrate-treated produced water reinjection facilities. Sulfide-oxidizing, nitrate-reducing bacteria (soNRB) have been suggested to be the cause of such corrosion. Using the model soNRB Sulfurimonas sp. strain CVO obtained from an oil field, we conducted a detailed analysis of soNRB-induced corrosion at initial nitrate-to-sulfide (N/S) ratios relevant to oil field operations. The activity of strain CVO caused severe corrosion rates of up to 0.27 millimeters per year (mm y-1) and up to 60-µm-deep pitting within only 9 days. The highest corrosion during the growth of strain CVO was associated with the production of zero-valent sulfur during sulfide oxidation and the accumulation of nitrite, when initial N/S ratios were high. Abiotic corrosion tests with individual metabolites confirmed biogenic zero-valent sulfur and nitrite as the main causes of corrosion under the experimental conditions. Mackinawite (FeS) deposited on carbon steel surfaces accelerated abiotic reduction of both sulfur and nitrite, exacerbating corrosion. Based on these results, a conceptual model for nitrate-mediated corrosion by soNRB is proposed.IMPORTANCE Ambiguous reports of corrosion problems associated with the injection of nitrate for souring control necessitate a deeper understanding of this frequently applied bioengineering strategy. Sulfide-oxidizing, nitrate-reducing bacteria have been proposed as key culprits, despite the underlying microbial corrosion mechanisms remaining insufficiently understood. This study provides a comprehensive characterization of how individual metabolic intermediates of the microbial nitrogen and sulfur cycles can impact the integrity of carbon steel infrastructure. The results help explain the dramatic increases seen at times in corrosion rates observed during nitrate injection in field and laboratory trials and point to strategies for reducing adverse integrity-related side effects of nitrate-based souring mitigation.

Low-temperature limitation of bioreactor sludge in anaerobic treatment of domestic wastewater.

Two strategies exist for seeding low-temperature anaerobic reactors: the use of specialist psychrophilic biomass or mesophilic bioreactor sludge acclimated to low temperature. We sought to determine the low-temperature limitation of anaerobic sludge from a bioreactor acclimated to UK temperatures (<15 °C). Anaerobic incubation tests using low-strength real domestic wastewater (DWW) and various alternative soluble COD sources were conducted at 4, 8 and 15 °C; methanogenesis and acidogenesis were monitored separately. Production of methane and acetate was observed; decreasing temperature resulted in decreased yields and increased 'start-up' times. At 4 °C methanogenesis not hydrolysis/acidogenesis was rate-limiting. The final methane yields at 4 °C were less than 35% of the theoretical potential whilst at 8 and 15 °C more than 75 and 100% of the theoretical yield was achieved respectively. We propose that the lower temperature limit for DWW treatment with anaerobic bioreactor sludge lies between 8 and 4 °C and that 8 °C is the threshold for reliable operation.

A national survey of UK practice for day-case vaginal prolapse surgery: Unrealistic expectations?

OBJECTIVE: The UK NHS Getting It Right First Time report (2021) recommended that a significant proportion of native tissue vaginal prolapse operations should be undertaken as day-case procedures (target: 80% anterior compartment, 70% posterior compartment, 60% combined anterior/posterior compartment). The evidence for perioperative care, options for anaesthesia and outcomes of day-case vaginal prolapse surgery is limited. This study aimed to establish current practice amongst UK gynaecologists and explore perceived barriers to implementing day-case surgery for pelvic organ prolapse. Study design A pre-tested 16-item survey was emailed to British Society of Urogynaecology members in July 2022. This survey recorded rates of day-case prolapse surgeries, barriers to implementation and practices for managing urethral catheters, vaginal packs, intraoperative anaesthetics and perioperative care. Responses to free-text questions were independently analysed by two of the authors and underwent thematic analysis. RESULTS: 121 eligible responses were received (28 % response-rate): 41 % never undertook day-case prolapse repair, 16 % undertook < 5 per year and 26 % undertook > 20 cases per year. There was no significant difference in training level or hospital setting between those groups. Reasons cited for not undertaking day-case prolapse surgery included concerns about vaginal packs and urinary catheters (92 %) postoperative complications (67 %), early discharge of elderly patients (60 %) and a lack of published evidence (39 %) or national guidance (35 %). For those currently undertaking day-case prolapse surgery; 67 % used general anaesthesia, 15 % spinal with short-acting local anaesthetic, 14 % spinal with long-acting local anaesthetic and 3 % local anaesthetic alone. Vaginal packs and self-retaining urethral catheters were used by 68 % and 70 % respectively. Concerns regarding the management of vaginal packs and urinary catheters were the most frequently cited barrier to implementing day-case surgery for pelvic organ prolapse amongst free-text responses. There were wide variations in managing catheters and packs, and in managing readmissions. CONCLUSIONS: There is significant variation in uptake and practice for day-case prolapse surgery in the UK, with legitimate clinical concerns a barrier to its implementation. Further evaluation and development of robust, evidence-based management pathways are required to make day-case prolapse surgery consistent, feasible and defensible in clinical practice.

Effects of glucose and lactate on Streptococcus mutans abundance in a novel multispecies oral biofilm model.

The oral microbiome plays an important role in protecting oral health. Here, we established a controlled mixed-species in vitro biofilm model and used it to assess the impact of glucose and lactate on the ability of Streptococcus mutans, an acidogenic and aciduric species, to compete with commensal oral bacteria. A chemically defined medium was developed that supported the growth of S. mutans and four common early colonizers of dental plaque: Streptococcus gordonii, Actinomyces oris, Neisseria subflava, and Veillonella parvula. Biofilms containing the early colonizers were developed in a continuous flow bioreactor, exposed to S. mutans, and incubated for up to 7 days. The abundance of bacteria was estimated by quantitative polymerase chain reaction (qPCR). At high glucose and high lactate, the pH in bulk fluid rapidly decreased to approximately 5.2, and S. mutans outgrew other species in biofilms. In low glucose and high lactate, the pH remained above 5.5, and V. parvula was the most abundant species in biofilms. By contrast, in low glucose and low lactate, the pH remained above 6.0 throughout the experiment, and the microbial community in biofilms was relatively balanced. Fluorescence in situ hybridization confirmed that all species were present in the biofilm and the majority of cells were viable using live/dead staining. These data demonstrate that carbon source concentration is critical for microbial homeostasis in model oral biofilms. Furthermore, we established an experimental system that can support the development of computational models to predict transitions to microbial dysbiosis based on metabolic interactions.IMPORTANCEWe developed a controlled (by removing host factor) dynamic system metabolically representative of early colonization of Streptococcus mutans not measurable in vivo. Hypotheses on factors influencing S. mutans colonization, such as community composition and inoculation sequence and the effect of metabolite concentrations, can be tested and used to predict the effect of interventions such as dietary modifications or the use of toothpaste or mouthwash on S. mutans colonization. The defined in vitro model (species and medium) can be simulated in an in silico model to explore more of the parameter space.

Cooperation between autotrophic and heterotrophic denitrifiers under low C/N ratios revealed by individual-based modelling.

Denitrifying biofilms, in which autotrophic denitrifiers (AD) and heterotrophic denitrifiers (HD) coexist, play a crucial role in removing nitrate from water or wastewater. However, it is difficult to elucidate the interactions between HD and AD through sequencing-based experimental methods. Here, we developed an individual-based model to describe the interspecies dynamics and priority effects between sulfur-based AD (Thiobacillus denitrificans) and HD (Thauera phenylcarboxya) under different C/N ratios. In test I (coexistence simulation), AD and HD were initially inoculated at a ratio of 1:1. The simulation results showed excellent denitrification performance and a coaggregation pattern of denitrifiers, indicating that cooperation was the predominant interaction at a C/N ratio of 0.25 to 1.5. In test II (invasion simulation), in which only one type of denitrifier was initially inoculated and the other was added at the invasion time, denitrifiers exhibited a stratification pattern in biofilms. When HD invaded AD, the final HD abundance decreased with increasing invasion time, indicating an enhanced priority effect. When AD invaded HD, insufficient organic carbon sources weakened the priority effect by limiting the growth of HD populations. This study reveals the interaction between autotrophic and heterotrophic denitrifiers, providing guidance for optimizing wastewater treatment process.

Modelling the effects of dispersal mechanisms and hydrodynamic regimes upon the structure of microbial communities within fluvial biofilms.

The spatial distribution of microbial taxa is determined primarily by physical and chemical environments and by dispersal. In a homogeneous landscape with limited dispersal, the similarity in abundance of taxa in samples declines with separation distance. We present a one-dimensional model for the spatial autocorrelation in abundances arising from immigration from some remote community and dispersal between environmentally similar landscape patches. Spatial correlation in taxa abundances were calculated from biofilms from the beds of two flumes which differed only in their bedform profiles; one flat and the other a periodic sawtooth shape. The hydraulic regime is approximately uniform over the flat bed, whereas the sawtooth induces fast flow over the peaks and recirculation in the troughs. On the flat bed, the correlation decline between samples was reproduced by a model using one biologically reasonable parameter. A decline was apparent in the other flume; however, a better fit was achieved when dispersal was not assumed constant everywhere. However, analysis of finer-resolution data for the heterogeneous flume suggested even this model did not adequately capture the community's complexity. We conclude that hydrodynamics are a strong driver of taxa-abundance patterns in stream biofilms. However, local adaptability must also be considered to build up a complete mechanistic model.

Combined niche and neutral effects in a microbial wastewater treatment community.

It has long been assumed that differences in the relative abundance of taxa in microbial communities reflect differences in environmental conditions. Here we show that in the economically and environmentally important microbial communities in a wastewater treatment plant, the population dynamics are consistent with neutral community assembly, where chance and random immigration play an important and predictable role in shaping the communities. Using dynamic observations, we demonstrate a straightforward calibration of a purely neutral model and a parsimonious method to incorporate environmental influence on the reproduction (or birth) rate of individual taxa. The calibrated model parameters are biologically plausible, with the population turnover and diversity in the heterotrophic community being higher than for the ammonia oxidizing bacteria (AOB) and immigration into AOB community being relatively higher. When environmental factors were incorporated more of the variance in the observations could be explained but immigration and random reproduction and deaths remained the dominant driver in determining the relative abundance of the common taxa. Consequently we suggest that neutral community models should be the foundation of any description of an open biological system.

Microstructural and Rheological Transitions in Bacterial Biofilms.

Biofilms are aggregated bacterial communities structured within an extracellular matrix (ECM). ECM controls biofilm architecture and confers mechanical resistance against shear forces. From a physical perspective, biofilms can be described as colloidal gels, where bacterial cells are analogous to colloidal particles distributed in the polymeric ECM. However, the influence of the ECM in altering the cellular packing fraction (ϕ) and the resulting viscoelastic behavior of biofilm remains unexplored. Using biofilms of Pantoea sp. (WT) and its mutant (ΔUDP), the correlation between biofilm structure and its viscoelastic response is investigated. Experiments show that the reduction of exopolysaccharide production in ΔUDP biofilms corresponds with a seven-fold increase in ϕ, resulting in a colloidal glass-like structure. Consequently, the rheological signatures become altered, with the WT behaving like a weak gel, whilst the ΔUDP displayed a glass-like rheological signature. By co-culturing the two strains, biofilm ϕ is modulated which allows us to explore the structural changes and capture a change in viscoelastic response from a weak to a strong gel, and to a colloidal glass-like state. The results reveal the role of exopolysaccharide in mediating a structural transition in biofilms and demonstrate a correlation between biofilm structure and viscoelastic response.

Diversity and metabolic energy in bacteria.

Why are some groups of bacteria more diverse than others? We hypothesize that the metabolic energy available to a bacterial functional group (a biogeochemical group or 'guild') has a role in such a group's taxonomic diversity. We tested this hypothesis by looking at the metacommunity diversity of functional groups in multiple biomes. We observed a positive correlation between estimates of a functional group's diversity and their metabolic energy yield. Moreover, the slope of that relationship was similar in all biomes. These findings could imply the existence of a universal mechanism controlling the diversity of all functional groups in all biomes in the same way. We consider a variety of possible explanations from the classical (environmental variation) to the 'non-Darwinian' (a drift barrier effect). Unfortunately, these explanations are not mutually exclusive, and a deeper understanding of the ultimate cause(s) of bacterial diversity will require us to determine if and how the key parameters in population genetics (effective population size, mutation rate, and selective gradients) vary between functional groups and with environmental conditions: this is a difficult task.