A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid

BACKGROUND: The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. RESULTS: A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. CONCLUSIONS: The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications

Microtomography-based numerical simulations of heat transfer and fluid flow through β-SiC open-cell foams for catalysis

β-SiC open-cell foams are promising materials for catalytic supports with improved heat and mass transfer at moderate pressure drops. In this work, 3-dimensional (3D) models of a 30 ppi (pores per inch) β-SiC open-cell foam were generated using X-ray microtomography data. The resulting foam models were then used for finite element analysis (FEA) and computational fluid dynamics (CFD) simulations of heat transfer and fluid flow on the pore-scale. The FEA results demonstrate that (i) the overall effective thermal conductivity from direct simulations is comparable to the results estimated by experimental measurement, and are in the order of 10−1 W m−1 K−1 and (ii) thermal transport through fluid-saturated β-SiC foams depends on the solid-to-fluid conductivity ratio. By employing realistic foam models, pore-scale CFD simulations of fluid flows revealed the microscopic characteristics of laminar flow through open-cell foams. The anisotropic feature of realistic foam models promotes the axial and radial mixing of fluids in and after the foam element. The diffusion coefficient of laminar flow within foams was estimated at 10−4 m2 s−1, which is much larger than the molecular diffusion coefficient in a typical laminar flow in an open channel

The Impact of Responsive Feeding Practice Training on Teacher Feeding Behaviors in Tribal Early Care and Education: The Food Resource Equity and Sustainability for Health (FRESH) Study

Background: Establishing healthy eating habits early affects lifelong dietary intake, which has implications for many health outcomes. With children spending time in early care and education (ECE) programs, teachers establish the daytime meal environment through their feeding practices. Objective: We aimed to determine the effect of a teacher-focused intervention to increase responsive feeding practices in 2 interventions, 1 focused exclusively on the teacher’s feeding practices and the other focused on both the teacher’s feeding practices and a nutrition classroom curriculum, in ECE teachers in a Native American (NA) community in Oklahoma. Methods: Nine tribally affiliated ECE programs were randomly assigned to 1 of 2 interventions: 1) a 1.5-h teacher-focused responsive feeding practice training (TEACHER; n = 4) and 2) TEACHER plus an additional 3-h training to implement a 15-wk classroom nutrition curriculum (TEACHER + CLASS; n = 5). Feeding practice observations were conducted during lunch at 1 table in 1 classroom for 2- to 5-y-olds at each program before and 1 mo after the intervention. The Mealtime Observation in Child Care (MOCC) organizes teacher behaviors into 8 subsections. Descriptive statistics and the Shapiro–Wilk test for normality were calculated. Paired t tests were calculated to determine change in each group. Results: Amean± SD of 5.2 ± 2.0 (total n = 47) children and 1.7 ± 0.5 (total n = 14) teachers/center were observed at baseline, and 5.6 ± 1.7 (total n = 50) children and 1.7 ± 0.7 teachers (total n = 14) were observed/center postintervention. Total MOCC scores (max possible = 10) improved for TEACHER (6.1 ± 0.9 compared with 7.5 ± 0.3, t = 4.12, P = 0.026) but not for TEACHER + CLASS (6.5 ± 0.8 compared with 6.4 ± 1.0, t =−0.11, P = 0.915). No other changes were observed. Conclusions: Teacher intervention–only programs demonstrated improvements in responsive feeding practices, whereas the programs receiving teacher and classroom training did not. Greater burden likely decreased capacity to make changes in multiple domains. We demonstrated the ability to implement interventions in NA ECE. Further research with larger communities is necessary. This trial was registered at clinicaltrials.gov as NCT03251950

A New Field Protocol for Monitoring Forest Degradation

Forest degradation leads to the gradual reduction of forest carbon stocks, function, and biodiversity following anthropogenic disturbance. Whilst tropical degradation is a widespread problem, it is currently very under-studied and its magnitude and extent are largely unknown. This is due, at least in part, to the lack of developed and tested methods for monitoring degradation. Due to the relatively subtle and ongoing changes associated with degradation, which can include the removal of small trees for fuelwood or understory clearance for agricultural production, it is very hard to detect using Earth Observation. Furthermore, degrading activities are normally spatially heterogeneous and stochastic, and therefore conventional forest inventory plots distributed across a landscape do not act as suitable indicators: at best only a small proportion of plots (often zero) will actually be degraded in a landscape undergoing active degradation. This problem is compounded because the metal tree tags used in permanent forest inventory plots likely deter tree clearance, biasing inventories toward under-reporting change. We have therefore developed a new forest plot protocol designed to monitor forest degradation. This involves a plot that can be set up quickly, so a large number can be established across a landscape, and easily remeasured, even though it does not use tree tags or other obvious markers. We present data from a demonstration plot network set up in Jalisco, Mexico, which were measured twice between 2017 and 2018. The protocol was successful, with one plot detecting degradation under our definition (losing greater than 10% AGB but remaining forest), and a further plot being deforested for Avocado (Persea americana) production. Live AGB ranged from 8.4 Mg ha–1 to 140.8 Mg ha–1 in Census 1, and from 0 Mg ha–1 to 144.2 Mg ha–1 Census 2, with four of ten plots losing AGB, and the remainder staying stable or showing slight increases. We suggest this protocol has great potential for underpinning appropriate forest plot networks for degradation monitoring, potentially in combination with Earth Observation analysis, but also in isolation

Systemic immunosuppression depletes peripheral blood regulatory B cells in patients with immune thrombocytopenia

Regulatory B (Breg) cells are potentially implicated in the pathogenesis of immune thrombocytopenia (ITP). We analysed a prospective cohort of newly diagnosed steroid naïve ITP patients enrolled in the multicentre FLIGHT trial and found that the numbers of Bregs in their peripheral blood were similar to healthy controls. In contrast, Breg numbers were significantly reduced in ITP patients treated with systemic immunosuppression (glucocorticoids or mycophenolate mofetil). We also demonstrate that glucocorticoid treatment impairs Breg interleukin-10 production via an indirect T-cell-mediated mechanism

Immunomodulatory Therapy Reduces the Severity of Placental Lesions in Chronic Histiocytic Intervillositis

From Frontiers via Jisc Publications RouterHistory: collection 2021, received 2021-08-04, accepted 2021-09-23, epub 2021-10-18Publication status: PublishedChronic histiocytic intervillositis (CHI) is a rare, but highly recurrent inflammatory placental lesion wherein maternal macrophages infiltrate the intervillous space. Pregnancies with CHI are at high risk of fetal growth restriction, miscarriage or stillbirth. Presently, the diagnosis can only be made after histopathological examination of the placenta. Given its proposed immunological etiology, current treatments include aspirin, heparin, and immunomodulatory agents. However, the rationale for these medications is largely based upon small case series and reports as there is a lack of larger studies investigating treatment efficacy. Therefore, this study sought to determine whether inclusion of immunomodulatory medications was effective at reducing the severity of lesions and improving pregnancy outcomes in subsequent pregnancies. Thirty-three women with a history of CHI in at least one pregnancy (index case) were identified retrospectively through medical records. Twenty-eight participants presented with a first subsequent pregnancy and a further 11 with a second subsequent pregnancy at a specialist clinic for pregnancy after loss. Data on maternal demographics, medical history, medication, pregnancy outcome, and placental pathology was collected and compared between pregnancies. Twenty-seven (69%) subsequent pregnancies were treated with at least one or both of prednisolone and hydroxychloroquine. Inclusion of at least one immunomodulatory agent in treatment regimen resulted in an almost 25% increase in overall livebirth rate (61.5 vs. 86.2%). In women treated with immunomodulatory medication a greater proportion of placentas had reduced severity of lesions compared to those treated without (86.7 vs. 33.3%, respectively). A reduction in CHI severity was associated with a 62.3% improvement in livebirth rate compared to those where severity remained unchanged in relation to the index case. These data provide preliminary evidence that the use of immunomodulatory medication in the management of CHI improves histopathological lesions and the chance of livebirth in subsequent pregnancies. Due to CHI's rarity and ethical and feasibility issues, randomized controlled trials in affected women are challenging to conduct. As a result, collaboration between centers is required in future to increase study sample sizes and elucidate the mechanisms of hydroxychloroquine and prednisolone in reducing pathology

Interferon-γ inhibits interleukin-1β-induced matrix metalloproteinase production by synovial fibroblasts and protects articular cartilage in early arthritis

Introduction: The first few months after symptom onset represents a pathologically distinct phase in rheumatoid arthritis (RA). We used relevant experimental models to define the pathological role of interferon-γ (IFN-γ) during early inflammatory arthritis. Methods: We studied IFN-γ's capacity to modulate interleukin-1β (IL-1β) induced degenerative responses using RA fibroblast-like synoviocytes (FLS), a bovine articular cartilage explant (BACE)/RA-FLS co-culture model and an experimental inflammatory arthritis model (murine antigen-induced arthritis (AIA)). Results: IFN-γ modulated IL-1β driven matrix metalloproteinases (MMP) synthesis resulting in the down-regulation of MMP-1 and MMP-3 production in vitro. IFN-γ did not affect IL-1β induced tissue inhibitor of metalloproteinase-1 (TIMP-1) production by RA FLS but skewed the MMP/TIMP-1 balance sufficiently to attenuate glycosaminoglycan-depletion in our BACE model. IFN-γ reduced IL-1β expression in the arthritic joint and prevented cartilage degeneration on Day 3 of AIA. Conclusions: Early therapeutic intervention with IFN-γ may be critical to orchestrate tissue-protective responses during inflammatory arthritis