A novel excretory–secretory (ES) protein of Trichinella pseudospiralis was produced. A cDNA library was constructed from mRNA of muscle larvae at 30 days post infection (p.i.) and immunoscreened with the antibody against ES products. A clone, designated Tp22-3, contained a cDNA transcript of 815 bp in length with a single open reading frame which encoded 244-amino acids (28407 Da in the estimated molecular mass). A database search revealed that no sequences had a homology to this predicted protein. The recombinant protein was produced in an Escherichia coli expression system. Stage specific expression of this protein was suggested from the following experiments. An antibody against the recombinant protein could stain proteins migrating at about 28 kDa (which is the expected size from the sequence) on Western blotting of crude extracts or ES products from 30 days p.i. muscle larvae, but failed to stain any proteins in crude extracts from newborn larvae or 15 days p.i. muscle larvae. The antibody reacted to the stichocytes of larvae at 30 days p.i., but did not react to 15 days p.i. muscle larvae. The production of an mRNA transcript for Tp22-3 gene was restricted largely to the 30 days p.i. muscle larvae and adult worms.

Recombinant protein was produced from the cDNA library of Trichinella pseudospiralis, which seemed to form part of the excretory–secretory (ES) products. The library was constructed from cDNA of muscle larvae at 1 month post-infection, and immunoscreened with antibody against T. pseudospiralis ES products. A clone, designated Tp21-3, contained a cDNA transcript of 657 bp in length with a single open reading frame, which encoded 172 amino acids (19617 Da in the estimated molecular mass). The predicted amino acid sequence of clone Tp21-3 had a similarity of 76% to that of clone ORF 17.20 (GenBank under accession number U88239) from T. spiralis. The recombinant fusion proteins encoded by clone Tp21-3 were produced in an Escherichia coli expression system and affinity purified. On Western blotting analysis, Tp21-3 recombinant proteins migrated at 40 kDa and reacted to antibody against T. pseudospiralis ES products and T. pseudospiralis-infected sera. Sera were developed against Tp 21-3 recombinant proteins, which reacted to a single band migrating at 21 kDa in crude worm extract and ES products from T. pseudospiralis on Western blotting analysis, and reacted with stichocytes of T. pseudospiralis on immunohistochemical staining.

The nurse cell in the cyst of Trichinella spiralis comprises at least two kinds of cytoplasm, derived from muscle or satellite cells, as indicated by the pattern of staining using regular dye (haematoxylin and eosin, or toluidine blue), alkaline phosphatase (ALP) expression, acid phosphatase (ACP) expression and immunostaining with an anti-intermediate filament protein (desmin or keratin). Muscle cells undergo basophilic changes following a T. spiralis infection and transform to the nurse cells, accompanied by an increase in ACP activity and the disappearance of desmin. Satellite cells are activated, transformed and joined to the nurse cells but remain eosinophilic. The eosinophilic cytoplasm is accompanied by an increase in desmin and ALP expression but not an increase in ACP activity. Differences in the staining results for ALP or ACP suggest that the two kinds of cytoplasm have different functions. Trichinella pseudospiralis infection results in an increase of ACP activity at a later stage than T. spiralis. There is also a difference in the location pattern of ACP in the cyst of T. spiralis compared with T. pseudospiralis. In T. spiralis, ACP is diffused within the cell, but in T. pseudospiralis, ACP distribution is spotty corresponding to the location of the nucleus. Trichinella pseudospiralis infection is accompanied by a slight increase in ALP activity. Activated satellite cells following a T. pseudospiralis infection exhibit an increase in desmin expression. The present study therefore reveals that nurse cell cytoplasm differs between the two Trichinella species and between the two origins of cytoplasm in the cyst of T. spiralis.

A clone, designated as TsTM, was selected from the cDNA library of newborn larvae (NBL) of Trichinella spiralis through immunoscreening against infected sera. The clone contained a cDNA transcript of 855 bp in length with a single open reading frame, which encoded 285-amino acids (33 kDa in the estimated molecular weight). A sequence analysis revealed that the clone TsTM encoded the full-length of tropomyosin gene. The phylogenetic analysis of the tropomyosin gene was in good agreement with the classical taxonomical position of T. spiralis. The fusion proteins encoded by the clone TsTM were produced in an Escherichia coli expression system and affinity purified, and the antibody was raised against the protein for the following studies. The antibody against the fusion protein positively bound to the hypodermal muscle layer in immunolocalization analysis, and the 35 kDa band in crude extracts of muscle larvae but not in excretory and secretory (ES) products on Western blots. The antigenicity of the clone TsTM was recognized by host mice but exhibited little species specificity.

OBJECTIVES/GOALS: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks effective targeted treatment options. TNBC's greater degree of immunogenicity than other breast tumors makes immunotherapy a viable strategy. Strategies to improve the immunotherapy response includes targeting the cGAS-STING innate immune pathway with STING agonists. METHODS/STUDY POPULATION: We have previously shown in vitro that eribulin, a microtubule destabilizer currently used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. Separately, eribulin also significantly enhances type I interferon expression induced by STING agonists measured by qRT-PCR through a second TBK1-dependent mechanism downstream of STING activation through detecting higher amounts of phosphorylated IRF-3 by western blot protein analysis. Mechanisms of eribulin-mediated interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizing agent paclitaxel. RESULTS/ANTICIPATED RESULTS: We determine that the enhancement of type I interferon expression by eribulin is pharmacologically synergistic with multiple STING agonists. The significant enhancement by eribulin led us to evaluate the antitumor efficacy of eribulin in combination the STING agonist ADU-S100 in a challenging spontaneous mammary tumor model MMTV-PyVT. We show that the combination treatment significantly decreased tumor growth which allowed for longer survival compared to other groups. This is particularly interesting because of our previous studies showing that eribulin alone, but not paclitaxel, promotes the activation of CD4+ T-cells in the spleen and draining lymph nodes of BALB/c mice with 4T1 tumors through flow cytometric analysis. DISCUSSION/SIGNIFICANCE: These data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC and suggest that eribulin can act as an immune adjuvant in addition to its anti-mitotic effect.

Mixing rates, relaxation rates, and decay of correlations for dynamics defined by potentials with summable variations are well understood, but little is known for non-summable variations. This paper exhibits upper bounds for these quantities for dynamics defined by potentials with square-summable variations. We obtain these bounds as corollaries of a new block coupling inequality between pairs of dynamics starting with different histories. As applications of our results, we prove a new weak invariance principle and a Hoeffding-type inequality.

Studies on community-acquired pneumonia (CAP) and pneumococcal pneumonia (PP) related to the 13-valent pneumococcal conjugate vaccine (PCV13) introduction in Asia are scarce. This study aimed to investigate the epidemiological and microbiological determinants of hospitalised CAP and PP after PCV13 was introduced in Japan. This observational hospital-based surveillance study included children aged ⩽15 years, admitted to hospitals in and around Chiba City, Japan. Participants had bacterial pneumonia based on a positive blood or sputum culture for bacterial pathogens. Serotype and antibiotic-susceptibility testing of Streptococcus pneumoniae and Haemophilus influenzae isolates from patients with bacterial pneumonia were assessed. The CAP hospitalisation rate per 1000 child-years was 17.7, 14.3 and 9.7 in children aged <5 years and 1.18, 2.64 and 0.69 in children aged 5–15 years in 2008, 2012 and 2018, respectively. There was a 45% and 41% reduction in CAP hospitalisation rates, between the pre-PCV7 and PCV13 periods, respectively. Significant reductions occurred in the proportion of CAP due to PP and PCV13 serotypes. Conversely, no change occurred in the proportion of CAP caused by H. influenzae. The incidence of hospitalised CAP in children aged ⩽15 years was significantly reduced after the introduction of PCV13 in Japan. Continuous surveillance is necessary to detect emerging PP serotypes.

We studied a suitable procedure for preparing of water samples used in radiocarbon intercomparisons involving dissolved inorganic carbon (DIC). The water samples must have inter-batch consistency and stable 14C concentrations and no sterilizing agent (e.g., HgCl2) should be added, in order to avoid the production of hazardous material. Six water samples, containing widely different amounts and types of salts, DIC, and 14C concentrations (1–100 pMC), were prepared in order to assess the procedure. Sample consistency was investigated through δ13C and chemical compositions; their low variabilities indicate that our procedure can be applied to radiocarbon intercomparison. A specific sample preparation protocol was developed for this kind of applications.

In November 2016, a woman in her 30s who stayed at an insecure, temporary housing facility, a manga café in Tokyo, Japan, for a year was diagnosed with sputum smear-positive tuberculosis (TB). Since the café had 31 staff members and provided with accommodation to many people, the local health office initiated a contact investigation. This study aims to characterise the cases found in the outbreak. A TB case was defined as a person tested bacteriologically positive for TB, or was determined to have TB by a physician. A latent TB infection case was defined as a person tested positive by interferon-γ release assay. From January 2016 through November 2017, there were 31 staff members at the manga café, of which, six developed TB disease (one smear-negative, culture-positive and five smear- and culture-negative) in addition to seven LTBI. Another long-term customer was found having sputum smear-positive TB. Variable numbers tandem repeat (VNTR) test revealed that the index patient and the long-term customer had the identical type of VNTR; however, one staff member had a different VNTR. Local health authorities should intensify screening long-term customers of such facilities for TB regularly as well as once a TB outbreak occurs.

In this study, direct numerical simulation of the flow around a rotating sphere at high Mach and low Reynolds numbers is conducted to investigate the effects of rotation rate and Mach number upon aerodynamic force coefficients and wake structures. The simulation is carried out by solving the three-dimensional compressible Navier–Stokes equations. A free-stream Reynolds number (based on the free-stream velocity, density and viscosity coefficient and the diameter of the sphere) is set to be between 100 and 300, the free-stream Mach number is set to be between 0.2 and 2.0, and the dimensionless rotation rate defined by the ratio of the free-stream and surface velocities above the equator is set between 0.0 and 1.0. Thus, we have clarified the following points: (1) as free-stream Mach number increased, the increment of the lift coefficient due to rotation was reduced; (2) under subsonic conditions, the drag coefficient increased with increase of the rotation rate, whereas under supersonic conditions, the increment of the drag coefficient was reduced with increasing Mach number; and (3) the mode of the wake structure becomes low-Reynolds-number-like as the Mach number is increased.

Europium LIII-edge X-ray absorption near-edge structure (XANES) was employed to determine the Eu(II)/Eu(III) ratios in minerals. This ratio can be determined based on the peak-area ratio of white lines, the resonance peak, in normalized XANES spectra for Eu(II) and Eu(III) species. For precise determination of the Eu(II)/Eu(III) ratios, however, it was revealed that the transition probabilities for each individual Eu(II) and Eu(III) species in the system must be quantified, because we found that the peak area in normalized XANES spectra is different in each Eu(II) and Eu(III) species. Despite this ambiguity, the method was applied to Eu in natural hydrothermal apatites (Eu = 39 and 64 ppm) and fluocerite (Eu = 282 ppm). The relationship between the Eu(II)/Eu(III) ratio in these hydrothermal minerals, and the distribution coefficients of Eu(II) and Eu(III) were discussed, taking into account Eu anomalies in their REE patterns. It is considered that by combining the Eu(II)/Eu(III) ratios determined by XANES and the degree of Eu anomaly in REE patterns, we can provide new information on the distribution of Eu(II) and Eu(III) in various geochemical studies.