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The human gut microbiome harbors substantial ecological diversity at the species level as well as at the strain level within species. In healthy hosts, species abundance fluctuations in the microbiome are thought to be stable, and these fluctuations can be described by macroecological laws. However, it is less clear how strain abundances change over time. An open question is whether individual strains behave like species themselves, exhibiting stability and following the macroecological relationships known to hold at the species level, or whether strains have different dynamics, perhaps due to the relatively close phylogenetic relatedness of cocolonizing lineages. Here, we analyze the daily dynamics of intraspecific genetic variation in the gut microbiomes of four healthy, densely longitudinally sampled hosts. First, we find that the overall genetic diversity of a large majority of species is stationary over time despite short-term fluctuations. Next, we show that fluctuations in abundances in approximately 80% of strains analyzed can be predicted with a stochastic logistic model (SLM), an ecological model of a population experiencing environmental fluctuations around a fixed carrying capacity, which has previously been shown to capture statistical properties of species abundance fluctuations. The success of this model indicates that strain abundances typically fluctuate around a fixed carrying capacity, suggesting that most strains are dynamically stable. Finally, we find that the strain abundances follow several empirical macroecological laws known to hold at the species level. Together, our results suggest that macroecological properties of the human gut microbiome, including its stability, emerge at the level of strains. IMPORTANCE To date, there has been an intense focus on the ecological dynamics of the human gut microbiome at the species level. However, there is considerable genetic diversity within species at the strain level, and these intraspecific differences can have important phenotypic effects on the host, impacting the ability to digest certain foods and metabolize drugs. Thus, to fully understand how the gut microbiome operates in times of health and sickness, its ecological dynamics may need to be quantified at the level of strains. Here, we show that a large majority of strains maintain stable abundances for periods of months to years, exhibiting fluctuations in abundance that can be well described by macroecological laws known to hold at the species level, while a smaller percentage of strains undergo rapid, directional changes in abundance. Overall, our work indicates that strains are an important unit of ecological organization in the human gut microbiome.

Surveys were conducted to assess Citrus tristeza virus (CTV) in California. Orchard surveys in central California during 2009 to 2013 estimated CTV incidence from 0.05% to 2.9%. Similar surveys in Ventura, Riverside, San Bernardino, and San Diego Counties in 2020-22 estimated CTV incidence from 6.3% to 34.9% while CTV was rarely found in the other five counties surveyed. T30 comprised over 95% of CTV detected alone or in mixtures with other strains in southern California and constituted 59% of 550 CTV accessions maintained in Tulare, California. VT, RB, and S1 genotypes were also found but T36 was rarely detected. No evidence of CTV-induced economic damage was noted except for occasional CTV quick decline on sour orange rootstock.

Elucidating the sources of a microbiome can provide insight into the ecological dynamics responsible for the formation of these communities. Source tracking approaches to date leverage species abundance information; however, single nucleotide variants (SNVs) may be more informative because of their high specificity to certain sources. To overcome the computational burden of utilizing all SNVs for a given sample, we introduce a novel method to identify signature SNVs for source tracking. Signature SNVs used as input into a previously designed source tracking algorithm, FEAST, can more accurately estimate contributions than species and provide novel insights, demonstrated in three case studies.

BackgroundMetagenomics analyses can be negatively impacted by DNA contamination. While external sources of contamination such as DNA extraction kits have been widely reported and investigated, contamination originating within the study itself remains underreported.ResultsHere, we applied high-resolution strain-resolved analyses to identify contamination in two large-scale clinical metagenomics datasets. By mapping strain sharing to DNA extraction plates, we identified well-to-well contamination in both negative controls and biological samples in one dataset. Such contamination is more likely to occur among samples that are on the same or adjacent columns or rows of the extraction plate than samples that are far apart. Our strain-resolved workflow also reveals the presence of externally derived contamination, primarily in the other dataset. Overall, in both datasets, contamination is more significant in samples with lower biomass.ConclusionOur work demonstrates that genome-resolved strain tracking, with its essentially genome-wide nucleotide-level resolution, can be used to detect contamination in sequencing-based microbiome studies. Our results underscore the value of strain-specific methods to detect contamination and the critical importance of looking for contamination beyond negative and positive controls. Video Abstract.

The α-synuclein protein can adopt several different conformations that cause neurodegeneration. Different α-synuclein conformers cause at least three distinct α-synucleinopathies: multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and Parkinson's disease (PD). In earlier studies, we transmitted MSA to transgenic (Tg) mice and cultured HEK cells both expressing mutant α-synuclein (A53T) but not to cells expressing α-synuclein (E46K). Now, we report that DLB is caused by a strain of α-synuclein prions that is distinct from MSA. Using cultured HEK cells expressing mutant α-synuclein (E46K), we found that DLB prions could be transmitted to these HEK cells. Our results argue that a third strain of α-synuclein prions likely causes PD, but further studies are needed to identify cells and/or Tg mice that express a mutant α-synuclein protein that is permissive for PD prion replication. Our findings suggest that other α-synuclein mutants should give further insights into α-synuclein prion replication, strain formation, and disease pathogenesis, all of which are likely required to discover effective drugs for the treatment of PD as well as the other α-synucleinopathies.

While genome databases are nearing a complete catalog of species commonly inhabiting the human gut, their representation of intraspecific diversity is lacking for all but the most abundant and frequently studied taxa. Statistical deconvolution of allele frequencies from shotgun metagenomic data into strain genotypes and relative abundances is a promising approach, but existing methods are limited by computational scalability. Here we introduce StrainFacts, a method for strain deconvolution that enables inference across tens of thousands of metagenomes. We harness a "fuzzy" genotype approximation that makes the underlying graphical model fully differentiable, unlike existing methods. This allows parameter estimates to be optimized with gradient-based methods, speeding up model fitting by two orders of magnitude. A GPU implementation provides additional scalability. Extensive simulations show that StrainFacts can perform strain inference on thousands of metagenomes and has comparable accuracy to more computationally intensive tools. We further validate our strain inferences using single-cell genomic sequencing from a human stool sample. Applying StrainFacts to a collection of more than 10,000 publicly available human stool metagenomes, we quantify patterns of strain diversity, biogeography, and linkage-disequilibrium that agree with and expand on what is known based on existing reference genomes. StrainFacts paves the way for large-scale biogeography and population genetic studies of microbiomes using metagenomic data.

In many types of familial amyotrophic lateral sclerosis (fALS), mutations cause proteins to gain toxic properties that mediate neurodegenerative processes. It is becoming increasingly clear that the proteins involved in ALS, and those responsible for a host of other neurodegenerative diseases, share many characteristics with a growing number of prion diseases. ALS is a heterogenous disease in which the majority of cases are sporadic in their etiology. Studies investigating the inherited forms of the disease are now beginning to provide evidence that some of this heterogeneity may be due to the existence of distinct conformations that ALS-linked proteins can adopt to produce the equivalent of prion strains. In this review, we discuss the in vitro and in vivo evidence that has been generated to better understand the characteristics of these proteins and how their tertiary structure may impact the disease phenotype.

The value of bioassays as analytical methods for assessing the potency of particular stressors on live animal models depends on the precision of their results, which are greatly influenced by the choice of test subjects. The genetic makeup of experimental subjects varies, and, as such, so will their responses to the test environment. Genetic diversity of test populations may contribute to statistical variability; therefore, the use of genetically similar subjects may enhance the utility of bioassays. This study addresses the efficacy of using isogenic homozygous zebrafish (Danio rerio) as subjects for bioassays. Stress responses (acidic conditions) were compared during early development for gynogenetically produced isogenic homozygous line of zebrafish (C32) and wildtype (WT) zebrafish. Experiments evaluated early life stage milestones after exposure to low pH in water of a different electrolyte composition. Because the isogenic homozygous clonal (IHC) fish possessed far less genetic variability than the WT fish tested, it was predicted that the IHC fish would exhibit less variability in their response to stress. Although we found no significant differences in the variability between the responses of the IHC and WT fish, pH and water hardness level had a differential effect on the two groups. Simple strain differences may be the probable cause of the response differences to environmental stress. Factors that may affect stress response, such as heterogeneity, co-adapted gene complexes, and domestication, are discussed. Our findings and review of recent zebrafish literature stress the need for researchers to carefully consider breeding histories and trait characteristics for each potential test subject to maximize the sensitivity of the assay.

Prion diseases are rapidly progressive, incurable neurodegenerative disorders caused by misfolded, aggregated proteins known as prions, which are uniquely infectious. Remarkably, these infectious proteins have been responsible for widespread disease epidemics, including kuru in humans, bovine spongiform encephalopathy in cattle, and chronic wasting disease in cervids, the latter of which has spread across North America and recently appeared in Norway and Finland. The hallmark histopathological features include widespread spongiform encephalopathy, neuronal loss, gliosis, and deposits of variably sized aggregated prion protein, ranging from small, soluble oligomers to long, thin, unbranched fibrils, depending on the disease. Here, we explore recent advances in prion disease research, from the function of the cellular prion protein to the dysfunction triggering neurotoxicity, as well as mechanisms underlying prion spread between cells. We also highlight key findings that have revealed new therapeutic targets and consider unanswered questions for future research.

Prions are proteins capable of adopting misfolded conformations and transmitting these conformations to other normally folded proteins. Prions are most commonly known for causing fatal neurodegenerative diseases in mammals but are also associated with several harmless phenotypes in yeast. A distinct feature of prion propagation is the existence of different phenotypical variants, called strains. It is widely accepted that these strains correspond to different conformational states of the protein, but the mechanisms driving their interactions remain poorly understood. This study uses mathematical modeling to provide insight into this problem. We show that the classical model of prion dynamics allows at most one conformational strain to stably propagate. In order to conform to biological observations of strain coexistence and co-stability, we develop an extension of the classical model by introducing a novel prion species consistent with biological studies. Qualitative analysis of this model reveals a new variety of behavior. Indeed, it allows for stable coexistence of different strains in a wide parameter range, and it also introduces intricate initial condition dependency. These new behaviors are consistent with experimental observations of prions in both mammals and yeast. As such, our model provides a valuable tool for investigating the underlying mechanisms of prion propagation and the link between prion strains and strain specific phenotypes. The consideration of a novel prion species brings a change in perspective on prion biology and we use our model to generate hypotheses about prion infectivity.

Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole-genome sequencing (WGS) analysis. We analyzed M. circinelloides isolates from patients in our burn unit (BU1, Hôpital Saint-Louis, Paris, France) together with nonoutbreak isolates from Burn Unit 2 (BU2, Paris area) and from France over a 2-year period (2013 to 2015). A total of 21 isolates, including 14 isolates from six BU1 patients, were analyzed by whole-genome sequencing (WGS). Phylogenetic classification based on de novo assembly and assembly free approaches showed that the clinical isolates clustered in four highly divergent clades. Clade 1 contained at least one of the strains from the six epidemiologically linked BU1 patients. The clinical isolates were specific to each patient. Two patients were infected with more than two strains from different clades, suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients from BU1 shared one strain, which could correspond to direct transmission or contamination with the same environmental source. In conclusion, WGS of several isolates per patients coupled with precise epidemiological data revealed a complex situation combining potential cross-transmission between patients and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.IMPORTANCE Invasive wound mucormycosis (IWM) is a severe infection due to environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used whole-genome sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission among them. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on WGS combined with careful sampling.

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson's disease (PD), we developed nine α-synuclein-YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.

Non-human primate (NHP) models of spinal cord injury better reflect human injury and provide a better foundation to evaluate potential treatments and functional outcomes. We combined finite element (FE) and surrogate models with impact data derived from in vivo experiments to define the impact mechanics needed to generate a moderate severity unilateral cervical contusion injury in NHPs (Macaca mulatta). Three independent variables (impactor displacement, alignment, and pre-load) were examined to determine their effects on tissue level stresses and strains. Mechanical measures of peak force, peak displacement, peak energy, and tissue stiffness were analyzed as potential determinants of injury severity. Data generated from FE simulations predicted a lateral shift of the spinal cord at high levels of compression (>64%) during impact. Submillimeter changes in mediolateral impactor position over the midline increased peak impact forces (>50%). Surrogate cords established a 0.5 N pre-load protocol for positioning the impactor tip onto the dural surface to define a consistent dorsoventral baseline position before impact, which corresponded with cerebrospinal fluid displacement and entrapment of the spinal cord against the vertebral canal. Based on our simulations, impactor alignment and pre-load were strong contributors to the variable mechanical and functional outcomes observed in in vivo experiments. Peak displacement of 4 mm after a 0.5N pre-load aligned 0.5-1.0 mm over the midline should result in a moderate severity injury; however, the observed peak force and calculated peak energy and tissue stiffness are required to properly characterize the severity and variability of in vivo NHP contusion injuries.

Prions are proteins that adopt alternative conformations that become self-propagating; the PrP(Sc) prion causes the rare human disorder Creutzfeldt-Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T-YFP) and TgM83(+/-) mice expressing α-synuclein (A53T). The TgM83(+/-) mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83(+/+) mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83(+/-) mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T-YFP in cultured cells, whereas none of six Parkinson's disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83(+/+) mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

Dopamine agonists disrupt prepulse inhibition (PPI) of startle in male rodents. In humans, this is observed only in some studies. We reported that PPI was disrupted by d-amphetamine in men, but only among those with high basal PPI levels. Here, amphetamine effects on PPI were tested in normal women and female rats.Acoustic startle and PPI were tested in normal women after placebo or 20 mg amphetamine, in a double-blind, crossover design, and in female rats after vehicle or 4.5 mg/kg amphetamine. Rats were from Sprague–Dawley (SD) and Long Evans (LE) strains that differ significantly in gene expression in PPI-regulatory circuitry, including levels of nucleus accumbens (NAC) catechol-O-methyl transferase (COMT) mRNA.Amphetamine was bioactive in humans based on quantitative autonomic and self-rating measures, but did not significantly change startle magnitude or PPI across all subjects. Amphetamine’s effects on PPI in women correlated significantly (p

Crow deaths were observed after West Nile virus (WNV) was introduced into North America, and this phenomenon has subsequently been used to monitor the spread of the virus. To investigate potential differences in the crow virulence of different WNV strains, American Crows were inoculated with Old World strains of WNV from Kenya and Australia (Kunjin) and a North American (NY99) WNV genotype. Infection of crows with NY99 genotype resulted in high serum viremia levels and death; the Kenyan and Kunjin genotypes elicited low viremia levels and minimal deaths but resulted in the generation of neutralizing antibodies capable of providing 100% protection from infection with the NY99 strain. These results suggest that genetic alterations in NY99 WNV are responsible for the crow-virulent phenotype and that increased replication of this strain in crows could spread WNV in North America.

Behavioral and pharmacological testing in mice has been revamped following the development of new tools for the manipulation of genetic information. We present the results from the peak procedure, an operant test that assesses the capacity to perceive, remember, and act upon temporal information. We studied the basic timing abilities in two different strains of mice, the C57Bl/6J and C3H/HeJ, and their response to psychoactive substances. Scopolamine and high doses of d-amphetamine disrupted performance by increasing response variability. The effect of d-amphetamine was particularly clear in C3H mice. Whereas scopolamine did not seem to affect the location of the response, the effect of a low dose of d-amphetamine, a leftward shift, was consistent with the hypothesis that it accelerates the internal time keeping mechanism. Physostigmine alone improved performance by reducing variability between trials without affecting the response location. Pretreatment with physostigmine partially blocked the deleterious effects of scopolamine. Methylphenidate did not have major effects on timing behavior in C57 but in the highest dose shifted the response of C3H mice to the left. The higher sensitivity of the C3H strain to the effects of d-amphetamine and methylphenidate support its value as an animal model of attention deficit disorder. The performance of mice in this temporal task was comparable to that observed in rats and pigeons, and seemed exquisitely sensitive to pharmacological manipulation.