Effective bioreactor pH control using only sparging gases

pH control is critical in bioreactor operations, typically realized through a two-sided control loop, where CO₂ sparging and base addition are used in bicarbonate-buffered media. Though a common approach, base addition could compromise culture performance due to the potential impact from pH excursions and osmolality increase in large-scale bioreactors. In this study, the feasibility of utilizing control of sparge gas composition as part of the pH control loop was assessed in Chinese hamster ovary (CHO) fed-batch cultures. Fine pH control was evaluated in multiple processes at different setpoints in small-scale ambr®250 bioreactors. Desired culture pH setpoints were successfully maintained via air sparge feedback control. As part of the pH control loop, air sparging was increased to improve CO₂ removal automatically, hence increase culture pH, and vice versa. The effectiveness of this pH control strategy was seamlessly transferred from ambr®250 to 200 L scale, demonstrating scalability of the proposed methodology. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2743, 2019     

Substrate specificity of human thymine-DNA glycosylase on exocyclic cytosine adducts

The environmental carcinogen glycidaldehyde (GDA) and therapeutic chloroethylnitrosoureas (CNUs) can form hydroxymethyl etheno and ring-saturated ethano bases, respectively. The mutagenic potential of these adducts relies on their miscoding properties and repair efficiency. In this work, the ability of human thymine-DNA glycosylase (TDG) to excise 8-(hydroxymethyl)-3,N(4)-ethenocytosine (8-hm-varepsilonC) and 3,N(4)-ethanocytosine (EC) was investigated and compared with varepsilonC, a known substrate for TDG. When tested using defined oligonucleotides containing a single adduct, TDG is able to excise 8-hm-varepsilonC but not EC. The 8-hm-varepsilonC activity mainly depends on guanine pairing with the adduct. TDG removes 8-hm-varepsilonC less efficiently than varepsilonC but its activity can be significantly enhanced by human AP endonuclease 1 (APE1), a downstream enzyme in the base excision repair. TDG did not show any detectable activity toward EC when placed in various neighboring sequences, including the 5'-CpG site. Molecular modeling revealed a possible steric clash between the non-planar EC exocyclic ring and residue Asn 191 within the TDG active site, which could account for the lack of TDG activity toward EC. TDG was not active against the bulkier exocyclic adduct 3,N(4)-benzethenocytosine, nor the two adenine derivatives with same modifications as the cytosine derivatives, 7-hm-varepsilonA and EA. These findings expand the TDG substrate range and aid in understanding the structural requirements for TDG substrate specificity.     

1H NMR investigation on interaction between ibuprofen and lipoproteins

A large number of studies indicate that oxidative modification of plasma lipoproteins, especially low-density lipoprotein (LDL), is a critical factor in initiation and progression of atherosclerosis. We have previously found that ibuprofen (IBP), a potential antioxidant drug to inhibit LDL oxidation, interacted with lipoproteins in intact human plasma. In the present study, we compare the binding affinities of IBP to LDL and HDL (high-density lipoprotein) by (1)H NMR spectroscopy. When IBP is added into the HDL and LDL samples, the - N(+)(CH(3))(3) moieties of phosphatidylcholine (PC) and sphingomyelin (SM) in lipoprotein particles experience the chemical shift up-field drift. Intermolecular cross-peaks observed in NOESY spectra imply that there are direct interactions between ibuprofen and lipoproteins at both hydrophobic and hydrophilic (ionic) regions. These interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group ( - N(+)(CH(3))(3)) and - (CH(2))(n) - group in HDL than that in LDL. This could be explained by either IBP has higher binding affinity to HDL than to LDL, or IBP induces orientation of the phospholipid head group at the surface of the lipoprotein particles.     

Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA

Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.     

ADAM-15/metargidin mediates homotypic aggregation of human T lymphocytes and heterotypic interactions of T lymphocytes with intestinal epithelial cells

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.     

Origin and differentiation of endemism in the flora of China

The present paper analyzed 239 endemic genera in 67 families in the flora of seed plants in China.The results showed that there are five families containing more than ten endemic genera,namely,Gesneriaceae (27),which hereafter refers to the number of endemic genera in China,Composite (20),Labiatae (12),Cruciferae (11),and Umbelliferae (10),15 families with two endemic genera,and another 30 families with only one endemic genus.Four monotypic families (Ginkgoaceae,Davidiaceae,Eucommiaceae and Acanthochlamydaceae)are the most ancient,relict and characteristic in the flora of seed plants in China.Based on integrative data of systematics,fossil history,and morphological and molecular evidence of these genera,their origin,evolution and relationships were discussed.In gymnosperms,all endemic genera are relicts of the Arctic-Tertiary flora,having earlier evolutionary history,and can be traced back to the Cretaceous or to the Jurassic and even earlier.In angiosperms,the endemic genera are mostly relicts,and are represented in all lineages in the"Eight-Class System ofClassification of Angiosperms",and endemism can be found in almost every evolutionary stage of extant angiosperms.The relict genera once occupied huge areas in the northern hemisphere in the Tertiary or the late Cretaceous,while neo-endemism mostly originated in the late Tertiary.They came from Arctic-Tertiary,Paleo-tropical-Tertiary and Tethys-Tertiary florisitic elements,and the blend of the three elements with many genera of autochthonous origin.The endemism was formed when some dispersal routes such as the North Atlantic Land Bridge,and the Bering Bridge became discontinuous during the Tertiary,as well as the climate change and glaciations in the late Tertiary and the Quaternary.Therefore,the late Tertiary is the starting point of extant endemism of the flora in China.     

Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis

We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea.     

Damming river shapes distinct patterns and processes of planktonic bacterial and microeukaryotic communities

Planktonic bacterial and microeukaryotic communities play important roles in biogeochemical cycles, but their biogeographic patterns and community assembly processes in large damming rivers still remain unclear. In this study, 16S rRNA and 18S rRNA coding genes were used for sample sequencing analysis of planktonic bacterial and microeukaryotic communities in the upper Yangtze River. The upper Yangtze River was divided into dam-affected zones and river zones based on the influence of dams. The results showed that there were significant differences in the bacterial and microeukaryotic communities between the two zones and that dams significantly reduced the α-diversity of the bacterial communities. Co-occurrence network analysis indicated that networks in the river zone were denser than those in the dam-affected zone. The relationships among species in bacterial networks were more complex than those in microeukaryotic networks. Dispersal limitation and ecological drift were the main processes influencing planktonic bacterial and microeukaryotic communities in the dam-affected zone respectively, whereas the role of deterministic processes increased in the river zone. Anthropogenic activities and hydraulic conditions affected suspended sediment and controlled microbial diversity in the river zone. These results suggest that dams impact planktonic bacteria more strongly than planktonic microeukaryotes, indicating that the distribution patterns and processes of the bacterial and microeukaryotic communities in large rivers are significantly different.     

Infection and pathogenesis of the Delta variant of SARS-CoV-2 in Rhesus macaque

Highlights
1. Delta variant of SARS-CoV-2 can effectively infect the Rhesus macaque.
2. The Delta variant grows faster than the early strain isolated from Wuhan in late 2019.
3. The shedding pattern, viral load and disease severity of Delta variant are similar with the early strain isolated from Wuhan in late 2019.
4. This study supports the attributed rapid disease spread of the Delta variant.     

叉角厉蝽对亚洲玉米螟幼虫的捕食功能反应

叉角厉蝽Eocanthecona furcellata是一种在生物防治方面有重要作用和潜力的捕食性天敌昆虫,为探明叉角厉蝽对亚洲玉米螟Ostrinia furnacalis幼虫的控害潜力,在室内温度26℃±1℃、相对湿度65%±10%、光周期16 L∶8 D条件下,观察了叉角厉蝽4龄、5龄若虫和成虫分别对亚洲玉米螟3龄、4龄幼虫的捕食行为,测定了叉角厉蝽对不同亚洲玉米螟密度下的捕食量、搜寻效应以及自身密度和种间干扰对捕食作用的影响。结果表明,叉角厉蝽对亚洲玉米螟的捕食功能反应符合Holling-Ⅱ功能反应模型,叉角厉蝽3个虫龄对亚洲玉米螟3龄与4龄幼虫的理论最大取食量顺序均为：成虫（30.21头）>5龄若虫（24.69头）>4龄若虫（11.96头）,相同虫龄的叉角厉蝽对亚洲玉米螟3龄幼虫的最大取食量均大于对亚洲玉米螟4龄幼虫的最大取食量。在亚洲玉米螟相同虫龄内,搜寻效应与猎物密度的增加呈负相关。在生存环境和亚洲玉米螟幼虫密度固定的条件下,其平均捕食量和搜寻效应与叉角厉蝽成虫密度的增加呈负相关。叉角厉蝽成虫与亚洲玉米螟幼虫以相同比例增加时,叉角厉蝽成虫种间会产生干扰作用。结果表明了叉角厉蝽对亚洲玉米螟具有较好的控害潜力,为今后高效利用叉角厉蝽田间防治亚洲玉米螟提供了理论依据。