Objectives/Goals: This study investigates the contribution of the stool secretome (the soluble factors secreted by microbes into extracellular space) to in vitro α-synuclein (αSyn) oligomerization using stool cultures from patients with multiple system atrophy (MSA), a rare neurodegenerative disease hallmarked by pathologic αSyn aggregates. Methods/Study Population: Stool samples from MSA patients (n = 20), household controls (n = 20), and healthy controls (n = 20) will be cultured using an adapted dilution-to-extinction approach. The goal is to reduce microbial complexity progressively to produce random secretome combinations that may affect αSyn oligomerization differentially. The original inoculant and dilutions will be cultured anaerobically to collect conditioned media (CM) enriched with microbial secretomes. CM will be used to expose a fluorescence resonance energy transfer (FRET) biosensor assay and a Gaussia luciferase protein complementation assay – both modified to quantify αSyn-αSyn interaction indicating oligomerization. Any CM-altering αSyn oligomerization will undergo multiomic characterization to identify potential causative agent(s). Results/Anticipated Results: Specific microbe-produced molecules from the literature are anticipated to modulate αSyn oligomerization, identified by targeted, reductionist studies that selected and tested separately single microbial factors on αSyn aggregation in vitro and in vivo. From these studies, lipopolysaccharide and bacterial amyloid protein are expected to increase αSyn oligomerization, while short-chain fatty acids, such as butyrate, are expected to interfere with and decrease oligomerization. As a complementary systemic approach, this study’s agnostic methods involving MSA stool culture combined with the proposed dilution-to-extinction method are expected to identify additional MSA stool secretome components modulating αSyn oligomerization that might otherwise be missed in earlier reductionist approaches. Discussion/Significance of Impact: Completion of this reverse-translational work will aid in discovering MSA stool secretome components modulating αSyn oligomerization. Identification of specific factors contributing to pathologic αSyn behavior might set the stage for patient screenings for identified stool markers and could lead to microbiome-based interventions for MSA.

OBJECTIVES/GOALS: Aim 1: We will determine whether temporal changes in the fecal microbiome signature correlate with a clinical multiple system atrophy (MSA) phenotype. Aim 2: We will evaluate whether secretomes cultured from fecal samples from MSA patients enhance intracellular and extracellular α-synuclein (αSyn) aggregation using in vitro functional assays. METHODS/STUDY POPULATION: Aim 1: Gut microbiome profiling will be performed by 16S rRNA gene sequencing, tandem mass spectrometry for expression proteomics, and targeted metabolomics in fecal samples from 30 MSA cases matched to 30 healthy controls, a Parkinson’s disease comparison group, and household controls. Aim 2: Microbial species will be isolated using dilution-to-extinction on MSA fecal samples and then will be cultured to obtain secretomes. To assess the effect of MSA fecal secretomes on αSyn aggregation, culture media from microbial isolates will be used in fluorescence resonance energy transfer (FRET) assays and luciferase reporter assays, both modified to measure αSyn aggregation. Positive tests will undergo expanded metagenomic characterization of the microbes and secretome to identify potential causative agent(s). RESULTS/ANTICIPATED RESULTS: Based on cross-sectional metagenomic studies on MSA, MSA cases are expected to have genus reductions in Blautia and Dorea (acetate production); Paraprevotella (succinic and acetic acid production); and Ruminococcus, Coprococcus, and Faecalibacterium (butyrate production). Increases in genus Bacteroides (clinical pathogen) and Akkermansia (mucin degradation) and pro-inflammatory families Clostridiaceae and Rikenellaceae are also expected. MSA is predicted to be associated with reduced levels of short chain fatty acids and increased lipopolysaccharide. These microbial proteins and metabolites are anticipated to modulate intracellular and extracellular αSyn aggregation in vitro. Microbe isolation and secretome culturing methods are expected to identify additional drivers of αSyn aggregation. DISCUSSION/SIGNIFICANCE: This study’s novel use of longitudinal sampling, household controls, and secretome culturing aim to develop a more comprehensive understanding of the complex interactions between the gut microbiome and MSA. The success of this work offers the potential for new insights into the impact of the gut microbiome and secretome on MSA and αSyn aggregation.

The multi-cell Penning–Malmberg trap concept has been proposed as a way to accumulate and confine unprecedented numbers of antiparticles, an attractive but challenging goal. We report on the commissioning and first results (using electron plasmas) of the World's second prototype of such a trap, which builds and improves on the findings of its predecessor. Reliable alignment of both ‘master’ and ‘storage’ cells with the axial magnetic field has enabled confinement of plasmas, without use of the ‘rotating wall’ (RW) compression technique, for over an hour in the master cell and tens of seconds in the storage cells. In the master cell, attachment to background neutrals is found to be the main source of charge loss, with an overall charge-confinement time of 8.6 h. Transfer to on-axis and off-axis storage cells has been demonstrated, with an off-axis transfer rate of $50\,\%$ of the initial particles, and confinement times in the storage cells in the tens of seconds (again, without RW compression). This, in turn, has enabled the first simultaneous plasma confinement in two off-axis cells, a milestone for the multi-cell trap concept.

We describe here efforts to create and study magnetized electron–positron pair plasmas, the existence of which in astrophysical environments is well-established. Laboratory incarnations of such systems are becoming ever more possible due to novel approaches and techniques in plasma, beam and laser physics. Traditional magnetized plasmas studied to date, both in nature and in the laboratory, exhibit a host of different wave types, many of which are generically unstable and evolve into turbulence or violent instabilities. This complexity and the instability of these waves stem to a large degree from the difference in mass between the positively and the negatively charged species: the ions and the electrons. The mass symmetry of pair plasmas, on the other hand, results in unique behaviour, a topic that has been intensively studied theoretically and numerically for decades, but experimental studies are still in the early stages of development. A levitated dipole device is now under construction to study magnetized low-energy, short-Debye-length electron–positron plasmas; this experiment, as well as a stellarator device that is in the planning stage, will be fuelled by a reactor-based positron source and make use of state-of-the-art positron cooling and storage techniques. Relativistic pair plasmas with very different parameters will be created using pair production resulting from intense laser–matter interactions and will be confined in a high-field mirror configuration. We highlight the differences between and similarities among these approaches, and discuss the unique physics insights that can be gained by these studies.

Background: In RRMS patients with inadequate response to prior therapy, 2 alemtuzumab courses (12 mg/day; baseline: 5 days; 12 months later: 3 days) significantly improved outcomes over 2 years (y) versus SC IFNB-1a (CARE-MS II [NCT00548405]), with durable efficacy over a 4-y extension (NCT00930553). We present 6-y efficacy (2-y core study plus 4-y extension) in patients with relapse (relapsers) between Courses (C) 1 and 2. Methods: Annualized relapse rate (ARR); 6-month confirmed disability worsening (CDW); MRI disease activity (Gd-enhancing lesions; new/enlarging T2 hyperintense lesions); brain volume loss (BVL; derived by relative change in brain parenchymal fraction). Results: 105/435 (24%) patients relapsed between C1 and C2; 33% (relapsers) versus 55% without relapse (non-relapsers) received neither alemtuzumab retreatment nor another disease-modifying therapy through Y6. ARR (Y1: 1.2) declined post-C2 (0.5), remaining low through Y6 (0.2 [0.1, non-relapsers]; 10/105 [10%] relapsed). Through Y6, patients remained CDW-free (60% [relapsers]; 75% [non-relapsers]), Gd-enhancing lesion-free (94% [relapsers]; 90% [non-relapsers]), new/enlarging T2 hyperintense lesion-free (68% [relapsers]; 69% [non-relapsers]), and MRI disease activity-free (68% [relapsers]; 69% [non-relapsers]). Alemtuzumab slowed median percent yearly BVL (Y6: -0.13% [relapsers]; -0.10% [non-relapsers]). Conclusions: Patients relapsing between C1 and C2 improved post-C2 through Y6. These findings support administering 2 alemtuzumab courses to achieve optimal and durable benefit.

Shigellosis causes significant morbidity and mortality in developing and developed countries, mostly among infants and young children. The World Health Organization estimates that more than one million people die from Shigellosis every year. In order to evaluate trends in Shigellosis in Israel in the years 2002–2015, we analysed national notifiable disease reporting data. Shigella sonnei was the most commonly identified Shigella species in Israel. Hospitalisation rates due to Shigella flexenri were higher in comparison with other Shigella species. Shigella morbidity was higher among infants and young children (age 0–5 years old). Incidence of Shigella species differed among various ethnic groups, with significantly high rates of S. flexenri among Muslims, in comparison with Jews, Druze and Christians. In order to improve the current Shigellosis clinical diagnosis, we developed machine learning algorithms to predict the Shigella species and whether a patient will be hospitalised or not, based on available demographic and clinical data. The algorithms’ performances yielded an accuracy of 93.2% (Shigella species) and 94.9% (hospitalisation) and may consequently improve the diagnosis and treatment of the disease.

There is a growing concern about the role of the environment in the dissemination of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG). In this systematic review, we summarize evidence for increases of ARG in the natural environment associated with potential sources of ARB and ARG such as agricultural facilities and wastewater treatment plants. A total of 5247 citations were identified, including studies that ascertained both ARG and ARB outcomes. All studies were screened for relevance to the question and methodology. This paper summarizes the evidence only for those studies with ARG outcomes (n = 24). Sixteen studies were at high (n = 3) or at unclear (n = 13) risk of bias in the estimation of source effects due to lack of information or failure to control for confounders. Statistical methods were used in nine studies; three studies assessed the effect of multiple sources using modeling approaches, and none reported effect measures. Most studies reported higher ARG concentration downstream/near the source, but heterogeneous findings hindered making any sound conclusions. To quantify increases of ARG in the environment due to specific point sources, there is a need for studies that emphasize analytic or design control of confounding, and that provide effect measure estimates.

At Lago Buenos Aires, Argentina, 10Be, 26Al, and 40Ar/39Ar ages range from 190,000 to 109,000 yr for two moraines deposited prior to the last glaciation, 23,000�16,000 yr ago. Two approaches, maximum boulder ages assuming no erosion, and the average age of all boulders and an erosion rate of 1.4 mm/103 yr, both yield a common estimate age of 150,000�140,000 yr for the two moraines. The erosion rate estimate derives from 10Be and 26Al concentrations in old erratics, deposited on moraines that are >760,000 yr old on the basis of interbedded 40Ar/39Ar dated lavas. The new cosmogenic ages indicate that a major glaciation during marine oxygen isotope stage 6 occurred in the mid-latitude Andes. The next five youngest moraines correspond to stage 2. There is no preserved record of a glacial advance during stage 4. The distribution of dated boulders and their ages suggest that at least one major glaciation occurred between 760,000 and >200,000 yr ago. The mid-latitude Patagonian glacial record, which is well preserved because of low erosion rates, indicates that during the last two glacial cycles major glaciations in the southern Andes have been in phase with growth and decay of Northern Hemisphere ice sheets, especially at the 100,000 yr periodicity. Thus, glacial maxima are global in nature and are ultimately paced by small changes in Northern Hemisphere insolation.

Understanding the timing of mountain glacier and paleolake expansion and retraction in the Great Basin region of the western United States has important implications for regional-scale climate change during the last Pleistocene glaciation. The relative timing of mountain glacier maxima and the well-studied Lake Bonneville highstand has been unclear, however, owing to poor chronological limits on glacial deposits. Here, this problem is addressed by applying terrestrial cosmogenic 10Be exposure dating to a classic set of terminal moraines in Little Cottonwood and American Fork Canyons in the western Wasatch Mountains. The exposure ages indicate that the main phase of deglaciation began at 15.7 ± 1.3 ka in both canyons. This update to the glacial chronology of the western Wasatch Mountains can be reconciled with previous stratigraphic observations of glacial and paleolake deposits in this area, and indicates that the start of deglaciation occurred during or at the end of the Lake Bonneville hydrologic maximum. The glacial chronology reported here is consistent with the growing body of data suggesting that mountain glaciers in the western U.S. began retreating as many as 4 ka after the start of northern hemisphere deglaciation (at ca. 19 ka).

During the last glacial maximum (LGM), the western Uinta Mountains of northeastern Utah were occupied by the Western Uinta Ice Field. Cosmogenic10Be surface-exposure ages from the terminal moraine in the North Fork Provo Valley and paired26Al and10Be ages from striated bedrock at Bald Mountain Pass set limits on the timing of the local LGM. Moraine boulder ages suggest that ice reached its maximum extent by 17.4±0.5 ka (± 2σ).10Be and26Al measurements on striated bedrock from Bald Mountain Pass, situated near the former center of the ice field, yield a mean26Al/10Be ratio of 5.7±0.8 and a mean exposure age of 14.0±0.5 ka, which places a minimum-limiting age on when the ice field melted completely. We also applied a mass/energy-balance and ice-flow model to investigate the LGM climate of the western Uinta Mountains. Results suggest that temperatures were likely 5 to 7°C cooler than present and precipitation was 2 to 3.5 times greater than modern, and the western-most glaciers in the range generally received more precipitation when expanding to their maximum extent than glaciers farther east. This scenario is consistent with the hypothesis that precipitation in the western Uintas was enhanced by pluvial Lake Bonneville during the last glaciation.

Herein we describe a protocol for a systematic review of the evidence on whether point sources of anthropogenic effluent are associated with an increase in antibiotic resistance in the adjacent environment. The review question was based on the Population, Exposure, Comparator, Outcome, Study Design (PECOS) framework as follows: Is the prevalence or concentration of antibiotic resistant bacteria or resistance genes (O) in soil, water, air or free-living wildlife (P) higher in close proximity to, or downstream from, known or suspected sources of anthropogenic effluent (E) compared to areas more distant from or upstream from these sources (C)? A comprehensive search strategy was created to capture all relevant, published literature. Criteria for two stages of eligibility screening were developed to exclude publications that were not relevant to the question, and determine if the study used a design that permitted estimation of an association between a source and levels of resistance. A decision matrix was created for assessment of risk of bias to internal validity due to sample selection bias, information bias, and confounding. The goal of this protocol is to provide a method for determining the state of knowledge about the effect of point sources on antibiotic resistance in the environment.

Between 1950 and 1964, as a result of slight federal policy shifts, Cold War civil defence went from a pro-urban policy dedicated to the preservation of communities to an anti-urban policy focused on social control in the wake of an attack. Civil defence volunteers in Baltimore along with some of the city's civil defence paid staff, who had bought the federal message that they could protect themselves and their communities for nuclear war, allied with anti-nuclear activists against an increasingly militarized programme – one that by 1961 prioritized post-attack policing and de-emphasized the imperative to preserve urban neighbourhoods.