Many proteins can be partially or completely disordered under physiological conditions. Structural characterization of these disordered states using experimental methods can be challenging, since ...they are composed of a structurally heterogeneous ensemble of conformations rather than a single dominant conformation. Molecular dynamics (MD) simulations should in principle provide an ideal tool for elucidating the composition and behavior of disordered states at an atomic level of detail. Unfortunately, MD simulations using current physics-based models tend to produce disordered-state ensembles that are structurally too compact relative to experiments. We find that the water models typically used in MD simulations significantly underestimate London dispersion interactions, and speculate that this may be a possible reason for these erroneous results. To test this hypothesis, we create a new water model, TIP4P-D, that approximately corrects for these deficiencies in modeling water dispersion interactions while maintaining compatibility with existing physics-based models. We show that simulations of solvated proteins using this new water model typically result in disordered states that are substantially more expanded and in better agreement with experiment. These results represent a significant step toward extending the range of applicability of MD simulations to include the study of (partially or fully) disordered protein states.
The international spread of poliovirus exposes all countries to the risk of outbreaks and is designated a Public Health Emergency of International Concern by WHO. This risk can be exacerbated in ...countries using inactivated polio vaccine, which offers excellent protection against paralysis but is less effective than oral vaccine against poliovirus shedding, potentially allowing circulation without detection of paralytic cases for long periods of time. Our study investigated the molecular properties of type 2 poliovirus isolates found in sewage with an aim to detect virus transmission in the community.
We performed environmental surveillance in London, UK, testing sewage samples using WHO recommended methods that include concentration, virus isolation in cell culture, and molecular characterisation. We additionally implemented direct molecular detection and determined whole-genome sequences of every isolate using novel nanopore protocols.
118 genetically linked poliovirus isolates related to the serotype 2 Sabin vaccine strain were detected in 21 of 52 sequential sewage samples collected in London between Feb 8 and July 4, 2022. Expansion of environmental surveillance sites in London helped localise transmission to several boroughs in north and east London. All isolates have lost two key attenuating mutations, are recombinants with a species C enterovirus, and an increasing proportion (20 of 118) meet the criterion for a vaccine-derived poliovirus, having six to ten nucleotide changes in the gene coding for VP1 capsid protein.
Environmental surveillance allowed early detection of poliovirus importation and circulation in London, permitting a rapid public health response, including enhanced surveillance and an inactivated polio vaccine campaign among children aged 1–9 years. Whole-genome sequences generated through nanopore sequencing established linkage of isolates and confirmed transmission of a unique recombinant poliovirus lineage that has now been detected in Israel and the USA.
Medicines and Healthcare products Regulatory Agency, UK Health Security Agency, Bill & Melinda Gates Foundation, and National Institute for Health Research Medical Research Council.
Late-onset bloodstream infection (LO-BSI) is a common complication of prematurity, and lack of timely diagnosis and treatment can have life-threatening consequences. We sought to identify clinical ...characteristics and microbial signatures in the gastrointestinal microbiota preceding diagnosis of LO-BSI in premature infants.
Daily faecal samples and clinical data were collected over two years from 369 premature neonates (<32 weeks gestation). We analysed samples from 22 neonates who developed LO-BSI and 44 matched control infants. Next-generation sequencing of 16S rRNA gene regions amplified by PCR from total faecal DNA was used to characterise the microbiota of faecal samples preceding diagnosis from infants with LO-BSI and controls. Culture of selected samples was undertaken, and bacterial isolates identified using MALDI-TOF. Antibiograms from bloodstream and faecal isolates were compared to explore strain similarity.
From the week prior to diagnosis, infants with LO-BSI had higher proportions of faecal aerobes/facultative anaerobes compared to controls. Risk factors for LO-BSI were identified by multivariate analysis. Enterobacteriaceal sepsis was associated with antecedent multiple lines, low birth weight and a faecal microbiota with prominent Enterobacteriaceae. Staphylococcal sepsis was associated with Staphylococcus OTU faecal over-abundance, and the number of days prior to diagnosis of mechanical ventilation and of the presence of centrally-placed lines. In 12 cases, the antibiogram of the bloodstream isolate matched that of a component of the faecal microbiota in the sample collected closest to diagnosis.
The gastrointestinal tract is an important reservoir for LO-BSI organisms, pathogens translocating across the epithelial barrier. LO-BSI is associated with an aberrant microbiota, with abundant staphylococci and Enterobacteriaceae and a failure to mature towards predominance of obligate anaerobes.
Background. Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from ...sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants. Methods. Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization–time of flight. Clostridial isolates were typed and toxin genes detected. Results. A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk. Conclusions. Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC.
Background
Amyotrophic lateral sclerosis (ALS) is a prognostically heterogeneous neurodegenerative disease. Blood creatine kinase (CK) level has been inconsistently reported as a prognostic biomarker ...and raised levels in some ALS patients have been presumed to reflect muscle wasting, which is also variable.
Methods
MEDLINE was systematically searched for papers related to CK in ALS and the relevant studies were reviewed. Using data from 222 ALS patients in a multi-centre, prospective, longitudinal cohort, survival analyses using Kaplan–Meier and Cox proportional hazards models were undertaken in relation to CK and other prognostic factors.
Results
Twenty-five studies investigating CK in ALS were identified, of which 10 specifically studied the link between CK and survival. Five studies observed no association, four found that higher CK levels were associated with longer survival and one, the opposite. In our cohort (
n
= 222), 39% of patients had a CK level above the laboratory reference range. Levels were higher in males compared to females (
p
< 0.001), in patients with limb versus bulbar onset of symptoms (
p
< 0.001) and in patients with higher lower motor neuron burden (
p
< 0.001). There was no significant trend in longitudinal CK values. Although a higher standardised log (CK) at first visit was associated with longer survival in univariate analysis (hazard ratio 0.75,
p
= 0.003), there was no significant association after adjusting for other prognostic covariates.
Conclusion
While raised CK levels in ALS do reflect lower motor neuron denervation to a large extent, they are not independently associated with survival when measured in the symptomatic phase of the disease.
The clinical syndromes caused by frontotemporal lobar degeneration are heterogeneous, including the behavioural variant frontotemporal dementia (bvFTD) and progressive supranuclear palsy. Although ...pathologically distinct, they share many behavioural, cognitive and physiological features, which may in part arise from common deficits of major neurotransmitters such as γ-aminobutyric acid (GABA). Here, we quantify the GABAergic impairment and its restoration with dynamic causal modelling of a double-blind placebo-controlled crossover pharmaco-magnetoencephalography study. We analysed 17 patients with bvFTD, 15 patients with progressive supranuclear palsy, and 20 healthy age- and gender-matched controls. In addition to neuropsychological assessment and structural MRI, participants undertook two magnetoencephalography sessions using a roving auditory oddball paradigm: once on placebo and once on 10 mg of the oral GABA reuptake inhibitor tiagabine. A subgroup underwent ultrahigh-field magnetic resonance spectroscopy measurement of GABA concentration, which was reduced among patients. We identified deficits in frontotemporal processing using conductance-based biophysical models of local and global neuronal networks. The clinical relevance of this physiological deficit is indicated by the correlation between top-down connectivity from frontal to temporal cortex and clinical measures of cognitive and behavioural change. A critical validation of the biophysical modelling approach was evidence from parametric empirical Bayes analysis that GABA levels in patients, measured by spectroscopy, were related to posterior estimates of patients' GABAergic synaptic connectivity. Further evidence for the role of GABA in frontotemporal lobar degeneration came from confirmation that the effects of tiagabine on local circuits depended not only on participant group, but also on individual baseline GABA levels. Specifically, the phasic inhibition of deep cortico-cortical pyramidal neurons following tiagabine, but not placebo, was a function of GABA concentration. The study provides proof-of-concept for the potential of dynamic causal modelling to elucidate mechanisms of human neurodegenerative disease, and explains the variation in response to candidate therapies among patients. The laminar- and neurotransmitter-specific features of the modelling framework, can be used to study other treatment approaches and disorders. In the context of frontotemporal lobar degeneration, we suggest that neurophysiological restoration in selected patients, by targeting neurotransmitter deficits, could be used to bridge between clinical and preclinical models of disease, and inform the personalized selection of drugs and stratification of patients for future clinical trials.
Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases ...with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data.
We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation.
The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.
Advances in computational chemistry create an ongoing need for larger and higher-quality datasets that characterize noncovalent molecular interactions. We present three benchmark collections of ...quantum mechanical data, covering approximately 3,700 distinct types of interacting molecule pairs. The first collection, which we refer to as DES370K, contains interaction energies for more than 370,000 dimer geometries. These were computed using the coupled-cluster method with single, double, and perturbative triple excitations CCSD(T), which is widely regarded as the gold-standard method in electronic structure theory. Our second benchmark collection, a core representative subset of DES370K called DES15K, is intended for more computationally demanding applications of the data. Finally, DES5M, our third collection, comprises interaction energies for nearly 5,000,000 dimer geometries; these were calculated using SNS-MP2, a machine learning approach that provides results with accuracy comparable to that of our coupled-cluster training data. These datasets may prove useful in the development of density functionals, empirically corrected wavefunction-based approaches, semi-empirical methods, force fields, and models trained using machine learning methods.
Using metabolic screening, Allen et al. identify an adenosine to inosine deamination defect in astrocytes from ALS patients. This defect is the result of reduced expression of adenosine deaminase, ...leading to increased susceptibility to adenosine-mediated toxicity. Astrocyte inosine supplementation reverses the motor neuron toxicity observed with patient astrocytes in co-culture.
Abstract
As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.
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