Human granzyme B (hGzmB), which is present in various immune cells, has attracted much attention due to its role in various pathophysiological conditions. The hGzmB activity is triggered at a catalytic triad (His59, Asp103, Ser198), cleaving its specific substrates. To date, the drug design strategy against hGzmB mainly targets the catalytic triad, which causes the non-specificity problem of inhibitors due to the highly conserved active site in serine proteases. In the present work, microsecond classical molecular dynamics simulations are devoted to exploring the structural dynamics of the hGzmB catalytic cycle in the presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and inactive forms of hGzmB in the six different stages of the hGzmB catalytic cycle, we revealed, for the very first time, an additional network of interactions involving Arg216, a residue located outside the conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge, expanding the active site to facilitate the substrate-binding. On the contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the Arg216-mediated interactions within the catalytic triad, thus preventing hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216 in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the mutated hGzmB. Importantly, as Arg216 is not conserved amongst the various granzymes, the current findings can be a major step to guide the design of hGzmB specific therapeutics.
The effective and reasonable construction of the low impact development (LID) facilities in loess area depend on the functionality of typical LID facilities and the safety of surrounding structures in areas. A full-scale field test on rainwater concentrated infiltration of bioretentions in a collapsible loess site was conducted in this study. The water content and deformation law of the site were analyzed, and the water movement law of the rainwater-concentrated infiltration at bioretention facilities in the loess site was determined. The site settlements were calculated as per the wetting deformation curve and infiltration depths were calculated on an improved infiltration depth model tailored to the loess area. The rainwater infiltration rules of different bioretention structural forms are different in the collapsible loess field. The diffusion rate of the retaining wall type in loess decreases over time, while that on a sloping type does not. Within the same infiltration time, the retaining wall has a stronger influence on the site than the sloping type. When the water is concentrated in the site, its influence on the subgrade settlement is small (generally less than 1.5 mm) enough to satisfy the relevant engineering requirements. A modified Green-Ampt model based on assumed loess saturated unsaturated stratification can be used to predict the infiltration depth of facility water at the site. The adverse effects of water infiltration related to stagnant bioretentions can be mitigated by adjusting the initial water content and saturated water content at the loess site.
The increased frequency of flood events has motivated interest in natural flood management (NFM), in particular the potential for woodlands to reduce flooding. Woodlands can reduce the risk of rainfall-generated flooding through increased interception, soil infiltration, and available storage. Despite growing evidence, there is still low confidence in woodlands as a flood mitigation method due to limited empirical data available, particularly for semi-natural woodlands. We established a correlation catchment study in Haweswater, Cumbria, UK. Nine small upland catchments, each less than 0.2 km2 in area, were established on semi-natural broadleaf woodland sites where no stock grazing occurs or pasture with varied grazing intensity. At each site soil characteristics were investigated, namely soil moisture, permeability and bulk density. In addition, a v-notch weir was installed within in each catchment to calculate flow. The specific peak discharge (SPD), peak runoff coefficient, volume runoff coefficient and time taken to flow response was determined at each site for 28 storm events, of up to 205 mm, identified over a 13-month period. We found that semi-natural woodland reduced SPD by 33-52 % compared with pasture, reducing SPD by 36 % during larger storms (> 1 mm/hr peak discharge). Woodland reduced the peak runoff coefficient by 31-52 % and the volume runoff coefficient by 13-22 % compared to pasture. Additionally, response to storm events took 1-4 hours longer in woodland. These differences in flood response can be somewhat explained by the more permeable woodland soils, 4.6 times greater than pasture soil. Our analysis strengthens the argument that woodlands can reduce rainfall-generated flooding as a land use management method of NFM. Data collected here should be used to inform the parameters in flood prediction models and contribute to the evidence base for NFM.
A coupled atmospheric-hydrologic system models the complex interactions between the land surface and the atmospheric boundary layer, and the water-energy cycle from groundwater across the land surface to the top of the atmosphere. A regional climate model called WRF (Weather Research Forecasting) was coupled with a land surface scheme (Noah) to simulate intensive storms in central Alberta, Canada. Accounting for the land-atmosphere feedback enhances the predictability of the fine-tuned WRF-Noah system. Soil moisture, vegetation, and land surface temperature influence latent and sensible heat fluxes, and modulate both thermal and dynamical characteristics of land and lower atmosphere. WRF was set up in a two-way, three-domain nested framework so that the output of the outermost domain (D1) was used to run the second domain (D2) and the output of D2 was used to run the innermost domain (D3). In two-way nesting, D3 and D2 provide the feedback to their outer domains (D2 and D1), respectively. D3 was set at a 3-km resolution adequate to simulate convective storms. WRF-Noah was forced with climate outputs from Global Climate Models (GCMs) for the baseline period 1980–2005. A quantile-quantile bias correction method and a regional frequency analysis were applied to develop intensity-duration-frequency (IDF) curves from precipitation simulated by WRF-Noah. The simulated baseline precipitation of central Alberta agreed well with observed rain gauge data of Edmonton. The 5th‐generation NCAR mesoscale atmospheric model (MM5) was also set up in a 3-domain, but one-way nesting configuration. As expected, after bias correction, precipitation simulated by MM5 was less accurate than that simulated by WRF-Noah. For storms of short durations and return periods of more than 25 years, both MM5 driven by SRES climate scenarios of CMIP3 and WRF-Noah driven by RCP climate scenarios of CMIP5 projected storm intensities in central Alberta to increase from the base period to the 2050s, and to the 2080s.
Protein structure networks (PSNs) have long been used to provide a coarse yet meaningful representation of protein structure, dynamics, and internal communication pathways. An important question is what criteria should be applied to construct the network so that to include relevant interresidue contacts while avoiding unnecessary connections. To address this issue we systematically considered varying residue distance cutoff length and the probability threshold for contact formation to construct PSNs based on atomistic molecular dynamics in order to assess the amount of mutual information within the resulting representations. We found that the minimum in mutual information is universally achieved at the cutoff length of 5 Å, irrespective of the applied contact formation probability threshold in all considered, distinct proteins. Assuming that the optimal PSNs should be characterised by the least amount of redundancy, which corresponds to the minimum in mutual information, this finding suggests an objective criterion for cutoff distance and supports the existing preference towards its customary selection around 5 Å length, typically based to date on heuristic criteria.
The hyper-diverse order Coleoptera comprises a staggering ~25% of known species on Earth. Despite recent breakthroughs in next generation sequencing, there remains a limited representation of beetle diversity in assembled genomes. Most notably, the ground beetle family Carabidae, comprising more than 40,000 described species, has not been studied in a comparative genomics framework using whole genome data. Here we generate a high-quality genome assembly for Nebria riversi, to examine sources of novelty in the genome evolution of beetles, as well as genetic changes associated with specialization to high elevation alpine habitats. In particular, this genome resource provides a foundation for expanding comparative molecular research into mechanisms of insect cold adaptation. Comparison to other beetles shows a strong signature of genome compaction, with N. riversi possessing a relatively small genome (~147 Mb) compared to other beetles, with associated reductions in repeat element content and intron length. Small genome size is not, however, associated with fewer protein-coding genes, and an analysis of gene family diversity shows significant expansions of genes associated with cellular membranes and membrane transport, as well as protein phosphorylation and muscle filament structure. Finally, our genomic analyses show that these high elevation beetles have endosymbiotic Spiroplasma, with several metabolic pathways (e.g. propanoate biosynthesis) that might complement N. riversi, although its role as a beneficial symbiont or as a reproductive parasite remains equivocal.
Background: Iron deficiency is the most common micronutrient deficiency affecting nearly one-third of the population and is the leading cause of anemia worldwide. n this study, we evaluated the effectiveness of ferrous gluconate and ferrous sulfate supplements to identify the best iron supplement with the most effective and the least side effects in toddlers 6-24 months old. Methods: A randomized, single-blind clinical trial was performed. A total of 120 healthy toddlers aged 6 to 24 months old (two groups of 60) entered the study. Toddlers receiving ferrous sulfate (FS group) and ferrous gluconate (FG group) supplements. Blood indices such as hemoglobin & ferritin levels were evaluated at baseline and 6 months post- supplementation. Results: The FG group that received ferrous gluconate chelate iron showed approximately 2.4 g/dl higher Hb level in comparison to the FS group with ferrous sulfate supplementation 6 months post-supplementation (12.51±0.58 g/dL vs. 10.10±0.83, p = 0.045). Side effects were significantly more common in the FS group than the FG group (43.3 % vs. 16.7 %, P ≤ 0.001. Conclusion: The present study shows that educating mothers to feed toddlers with breast milk and iron supplements, including ferrous sulfate and ferrous gluconate, can be helpful in the prophylaxis of iron deficiency. Our results show that ferrous gluconate can be used in cases where ferrous sulfate causes unacceptable side effects.
Aim Although the fludarabine (F-araA)-treosulfan based toxicity reduced conditioning regimen has improved hematopoietic cell transplantation (HCT) outcome in patients with high-risk beta-thalassemia major (TM), rejection and regimen related toxicities (RRT) are still of major concern. This study aims to assess the role of F-araA pharmacokinetics (PK) and pharmacogenetics (PG) in a uniform cohort of patients with TM. Methods All patients with TM who receiving F-araA based regimen prior to HCT between September 2010 and 2019 were enrolled in this study. F-araA plasma levels were analyzed using LC-MS/MS. Selected polymorphisms in genes encoding for the enzymes (NT5E (Ecto-5’-nucleotidase) and DCK (Deoxycytidine kinase) involved in the metabolism of F-araA were screened. The influence of F-araA PK and PG on clinical outcomes were evaluated. Results F-araA PK showed wide inter-individual variation (27 and 19 fold in F-araA AUC and CL) which was explained by a promoter polymorphism (rs2295890) in the NT5E gene. Patients carrying the NT5E promoter variant showed no graft rejection (0% vs 7.7%, p=0.07) or Sinusoidal Obstruction Syndrome (0% Vs 19%, p=0.0007) and a trend to better EFS (87.5% vs 75.7%, p=0.1). F-araA systemic exposure was not associated with HCT outcome. Conclusion Our results suggest that the NT5E promoter polymorphism could be a predictive biomarker in F-araA based HCT setting in TM, however extensive functional studies are warranted to validate the clinical utility of this finding.
Conservation research is dominated by vertebrate examples but the shorter generation times and high local population sizes of invertebrates may lead to different management strategies. Here we investigate the genetic structure of an endangered flightless grasshopper, Keyacris scurra, used in classical evolutionary studies in the 60s. It had a wide distribution across New South Wales (NSW) and Victoria in pre-European times but has now become threatened because of land clearing for agriculture. We revisited remnant sites of K. scurra, with populations now restricted to only one area in Victoria and a few small patches in NSW and the Australian Capital Territory (ACT). Using DArTseq to generate SNP markers as well as mtDNA sequence data, we show that the remaining Victorian populations in an isolated valley are genetically distinct from the NSW populations and that all populations are genetically unique, with large FST values up to 0.8. We also find that, with one exception, the NSW/ACT populations separate genetically into chromosomal races (2n = 15 vs. 2n = 17). Isolation by distance was detected across both the SNP and mtDNA data sets, and there was substantial differentiation within chromosomal races. Genetic diversity as measured by heterozygosity was not correlated with the size of remaining habitat patches, with high variation present in some remnant cemetery sites. However, inbreeding correlated negatively with estimated habitat size at 25-500 m patch radius. These findings emphasize the importance of small fragments in conserving genetic variation across the species, and they highlight populations suitable for future translocations.
CHO cells have been recently shown to produce amino acid catabolism derived byproducts, which accumulate in fed-batch cultures to growth-inhibitory levels. Residual amino acid limitation or genetic engineering strategies have been successfully employed to suppress production of these novel growth inhibitory metabolic byproducts. However, the growth advantage attained due to suppression of these metabolic byproducts in fed-batch cultures is more pronounced when lactate accumulation is also controlled. BCAT1 knock-out (KO) CHO cells, which produce negligible levels of the metabolic byproducts isovalerate, isobutyrate and 2-methylbutyrate, grow to significantly higher peak cell densities in fed-batch cultures with lactate control (HiPDOG) as compared to cultures without lactate control. Henceforth, strategies involving novel metabolic byproduct control should preferably include lactate control to more easily assess the enhanced cell growth and productivities attainable.
Research hypotheses have been a cornerstone of science since before Galileo. Many have argued that inclusion of multiple hypotheses (1) encourage discovery of mechanisms, and (2) reduce bias – both features that should increase transferability and reproducibility. However, we are entering a new era of big data and highly predictive models where some argue the hypothesis is outmoded. Indeed, using a detailed literature analysis, we found prevalence of hypotheses in eco-evo research is very low (6.7-26%) and static from 1990-2015, a pattern mirrored in an extensive literature search (N=302,558 articles). Our literature review also indicates that neither grant success or citation rates were related to the inclusion of hypotheses, which may provide disincentive for hypothesis formulation. Here we confront common justifications for avoiding hypotheses and present new arguments based on benefits to the individual. Although hypotheses are not always necessary, we expect their continued and increased use will help our fields move toward greater understanding, reproducibility, prediction, and effective conservation of nature.
In this study, we collected and analyzed PSG data to investigate that value of polysomnography (PSG) in diagnosing sleep problems in children. The results of PSG studies of children (< 18 years old) with sleep problems conducted from April 2015 to May 2017 at a children’s hospital in Taiwan were collected and analyzed retrospectively. Data for 310 patients (209 males and 101 females) who underwent PSG were collected. The final diagnoses were as follows: obstructive sleep apnea in 159 (51.3%), snoring in 81 (26.4%), limb movement sleep disorder in 25 (8.1%), hypersomnias in 12 (3.9%), central apnea in 8 (2.9%), enuresis in 7 (2.3%), bruxism in 5 (1.6%), sleep terrors in 5 (1.6%), narcolepsy in 3 (1.0%), sleep seizures in 3 (1.0%), sleep walking in 1 (0.3%), and insomnia in 1 (0.3%). PSG may help detect significant sleep-related problems in children and is useful for making therapeutic decisions regarding children. Obstructive sleep apnea syndrome (OSAS) was the primarily sleep problem for most of the children (51.3%); however, only 7.4% of them underwent surgery for OSAS, even though those with OSAS underwent surgery without undergoing PSG. We therefore suggest that children with sleep problems should undergo PSG.
Background: Selective IgA deficiency (SIgAD) is the most prevalent primary immunodeficiency with almost unknown etiology. This study aimed to investigate the clinical diagnostic and prognostic values of lymphocytes subsets and function in symptomatic SIgAD patients. Methods: A total of 30 available SIgAD patients from the Iranian registry and 30 age-sex-matched healthy controls were included in the present study. We analyzed B and T cell peripheral subsets and T cell proliferation assay by flow cytometry in SIgAD patients with mild and severe clinical phenotypes. Results: Our results indicated a significant increase in naïve and transitional B cells and a strong decrease in marginal zone-like and switched memory B-cells in SIgAD patients. We found that naïve and central memory CD4+ T cell subsets, as well as Th1, Th2 and regulatory T cells have significantly decreased. On the other hand, there was a significant reduction in central and effector memory CD8+ T cell subsets, whereas proportions of both (CD4+ and CD8+) terminally differentiated effector memory T cells (TEMRA) were significantly elevated in our patients. Although some of T cell subsets in severe SIgAD were similar, decrease in marginal-zone and switched memory B cells and increase in CD21low B cell of severe SIgAD patients were slightly prominent. Moreover, the proliferation activity of CD4+ T cells was strongly impaired in SIgAD patients with a severe phenotype. Conclusion: SIgAD patients have varied cellular and humoral deficiencies. Therefore, T cell and B cell assessment might help in better understanding the heterogeneous pathogenesis and prognosis estimation of the disease. Keywords: Primary immunodeficiency, Selective IgA deficiency, B cell subsets, T cell subsets, flow cytometry, proliferation assay
COVID-19 is a complex disease and many difficulties are faced today especially in the proper choice of pharmacological treatments. The role of antiviral agents for COVID-19 is still being investigated. The evidence for immunomodulatory and anti-inflammatory drugs is quite conflicting, while the use of corticosteroids is supported by robust evidence. The use of heparins in hospitalized critically ill patients is preferred over other anticoagulants. Lastly, conflicting data were found regarding to the use of convalescent plasma and vitamin D. According to data shared by the WHO, many vaccines are under phase 3 clinical trials and some of them already received the marketing approval in EU countries and in the US. In conclusion, drugs repurposing has represented the main approach recently used in the treatment of patients with COVID-19. At this moment, the analysis of efficacy and safety data of drugs and vaccines used in real life context is strongly needed.
Background The maturation of innate immune responses in health and atopy is still incompletely understood. Methods We aimed to evaluate age-related trajectories of the TLR3 and TLR7/8 pathways across the lifespan and whether these differ between healthy and atopic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from 39 otherwise healthy atopic and 39 non-atopic subjects, aged 0-45 years. Selected cytokines involved in antiviral responses were measured by Luminex in culture supernatants of poly(I:C)- and R848-stimulated PBMCs. The non-parametric correlation between age and cytokine expression and differences in developmental trajectories between healthy and atopic were estimated. Patterns of cytokine development were identified with principal component analysis. Results Normal innate immune maturation entails significant and progressive age-related changes in the production of IL-1β, TNF-α, MIP-1β, MCP-3, IP-10, IL-10, IL-12p70 and IFN-γ upon TLR3 and/or TLR7/8 stimulation. Individual cytokines made small contributions to the observed variability; chemokines MCP-3 and IP-10 were key contributors. The development of these pathways deviated in atopic subjects with significant differences observed in the trajectories of IL-1β, MIP-1β and IL-10 synthesis. Conclusion TLR3 and TLR7/8 pathways mature during childhood, while atopy is associated with an abnormal maturation pattern. Suboptimal responses in Th1, inflammatory cytokine and chemokine production may be implicated in poor antiviral immunity in atopics, while deficient maturation of IL-10 producing capacity in the breaking of tolerance.
T cell receptor excision circles (TRECs) are small circularized DNA elements produced during rearrangement of T cell receptor (TCR) genes. Because TRECS are fairly stable, do not replicate during mitosis, and are not diluted during division of naïve T cells1, they are suitable for assessing the number of newly formed T cells 2. In this study, we detected TRECs in 475 healthy Chinese children aged 0–18 years in different clinical settings. We found a strong correlation between TRECs levels and peripheral CD4 naïve T cell numbers, but not between TRECs levels and effector or memory CD4 and CD8 T cell numbers. TRECs levels fell significantly compared with normal controls in patients with severe combined immunodeficiencies (SCID) (n=7), wiskott-aldrich syndrome (WAS) (n=22), or activated PI3Kδ syndrome (APDS) (n=5). TRECs levels in those with signal transducer and activator of transcription 1 (STAT1) deficiency (n=8) decreased or did not change significantly, a finding consistent with that for CD4 naïve T cells. We also measured TRECs levels in seven PIDs after hematopoietic stem cell transplantation (HSCT) (WAS=5; chronic granulomatous disease (CGD)=2), and found the complications after HSCT may reduce TRECs levels by interfering with production of naïve T cells. In conclusion, we established reference values for TRECs, which can be used to screen for primary immunodeficiency diseases (PIDs) during early life and track immune reconstitution after HSCT.