Routine approaches for the efficient expression of heterogenous proteins in
Pichia pastoris
include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of ...expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the
Streptoalloteichus hindustanus
bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (P
TEF1
). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (P
Dog2p300
) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the P
Dog2p300
has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter–based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.
Key points
• P
Dog2p300
was identified as a novel glucose-regulated promoter.
•
The expression of antibiotic-resistant gene driven by P
Dog2p300
was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.
•
The transformants harboring multiple integrations were cost-effectively selected by using the P
Dog2p300
for driving antibiotic-resistant genes.
Abstract
Climate change has resulted in an increase in drought severity in the species-rich tropical and subtropical forests of southern China. Exploring the spatiotemporal relationship between ...drought-tolerance trait and tree abundance provides a means to elucidate the impact of droughts on community assembly and dynamics. In this study, we measured the leaf turgor loss point (πtlp) for 399 tree species from three tropical forest plots and three subtropical forest plots. The plot area was 1 ha and tree abundance was calculated as total basal area per hectare according to the nearest community census data. The first aim of this study was to explore πtlp abundance relationships in the six plots across a range of precipitation seasonality. Additionally, three of the six plots (two tropical forests and one subtropical forest) had consecutive community censuses data (12–22 years) and the mortality ratios and abundance year slope of tree species were analyzed. The second aim was to examine whether πtlp is a predictor of tree mortality and abundance changes. Our results showed that tree species with lower (more negative) πtlp were more abundant in the tropical forests with relative high seasonality. However, πtlp was not related to tree abundance in the subtropical forests with low seasonality. Moreover, πtlp was not a good predictor of tree mortality and abundance changes in both humid and dry forests. This study reveals the restricted role of πtlp in predicting the response of forests to increasing droughts under climate change.
B and T lymphocyte attenuator (BTLA) is an immune checkpoint molecule that mediates the escape of tumor cells from immunosurveillance. Consequently, BTLA and its ligand herpesvirus entry mediator ...(HVEM) are potentially immunotherapeutic targets. However, the potential effects of BTLA on tumor cells remain incompletely unknown. Here, we show that BTLA is expressed across a broad range of tumor cells. The depletion of BTLA or HVEM promotes cell proliferation and colony formation, which is reversed by the overexpression of BTLA in BTLA knockout cells. In contrast, overexpression of BTLA or HVEM inhibits tumor cell proliferation and colony formation. Furthermore, the proliferation of a subpopulation with high BTLA was also significantly slower than that of the low BTLA subpopulation. Mechanistically, the coordination of BTLA and HVEM inhibits its major downstream extracellular regulated protein kinase (ERK1/2) signaling pathway, thus preventing tumor cell growth. This study demonstrates that tumor cell-intrinsic BTLA/HVEM is a potential tumor suppressor and is likely to have a potential antagonist for immunotherapy, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.
A new RNA-selective fluorescent dye integrated with a thiazole orange and a p-(methylthio)styryl moiety shows better nucleolus RNA staining and imaging performance in live cells than the commercial ...stains. It also exhibits excellent photostability, cell tolerance, and counterstain compatibility with 4',6-diamidino-2-phenylindole for specific RNA-DNA colocalization in bioassays.
Rechargeable lithium–metal batteries with a cell‐level specific energy of >400 Wh kg−1 are highly desired for next‐generation storage applications, yet the research has been retarded by poor ...electrolyte–electrode compatibility and rigorous safety concerns. We demonstrate that by simply formulating the composition of conventional electrolytes, a hybrid electrolyte was constructed to ensure high (electro)chemical and thermal stability with both the Li‐metal anode and the nickel‐rich layered oxide cathodes. By employing the new electrolyte, Li∥LiNi0.6Co0.2Mn0.2O2 cells show favorable cycling and rate performance, and a 10 Ah Li∥LiNi0.8Co0.1Mn0.1O2 pouch cell demonstrates a practical specific energy of >450 Wh kg−1. Our findings shed light on reasonable design principles for electrolyte and electrode/electrolyte interfaces toward practical realization of high‐energy rechargeable batteries.
Formulation of conventional electrolyte composition yields a hybrid solid/liquid electrolyte that is electrochemically compatible with the Li‐metal anode and the nickel‐rich layered oxide cathodes, which promises stable operation of a practical 10‐Ah‐grade pouch cell with a specific energy of >450 Wh kg−1.
Photoperiod- and thermosensitive genic male sterility (P/TGMS) lines are widely used in crop breeding. The fertility conversion of Arabidopsis (
) TGMS lines including
, which is defective in callose ...wall formation, relies on slow development under low temperatures. In this study, we discovered that
also exhibits PGMS. Fertility of
was restored when pollen development was slowed under short-day photoperiods or low light intensity, suggesting that slow development restores the fertility of
2 under these conditions. We found that several other TGMS lines with defects in pollen wall formation also exhibited PGMS characteristics. This similarity indicates that slow development is a general mechanism of PGMS fertility restoration. Notably, slow development also underlies the fertility recovery of TGMS lines. Further analysis revealed the pollen wall features during the formation of functional pollens of these P/TGMS lines under permissive conditions. We conclude that slow development is a general mechanism for fertility restoration of P/TGMS lines and allows these plants to take different strategies to overcome pollen formation defects.
High levels of dissolved arsenic (As) have been reported in many rivers running though the Tibetan Plateau (TP), the “Water Tower of Asia”. However, the source, spatiotemporal variations, and ...geochemical behavior of dissolved As in these rivers remain poorly understood. In this study, hot spring, river water, and suspended particulate material samples collected from the Yarlung Tsangpo River (YTR) (upper reaches of the Brahmaputra River) system in 2017 and 2018 were analyzed. Spatial results shown that the upper reaches of YTR (Zone I) have comparatively high levels of dissolved As (Asdissolved: mean 31.7 μg/L; 4.7–81.6 μg/L; n = 16), while the tributaries of the lower reaches (Zone II) have relatively low levels (mean 0.54 μg/L; 0.11–1.3 μg/L; n = 7). Seasonal results shown that the high Asdissolved (6.1–22.4 μg/L) were found in September to June and low Asdissolved (1.4–3.7 μg/L) were observed in July to August. Geothermal water is suspected as the main source of the elevated As levels in YTR due to the extremely high Asdissolved in hot springs (1.13–9.76 mg/L) and abundance of geothermal systems throughout TP. However, the seasonal results suggested that weathering of As-containing rocks and minerals is also a key factor affecting the Asdissolved in the river water in July to August (wet-season). Natural attenuation of As in main channel is dominated by dilution process due to the lower As concentrations in tributaries, but mostly occurred by both dilution and adsorption (or co-precipitation) processes in tributaries. This work highlights that the weathering process may have an important contribution to the dissolved As in the river waters in wet-season, and the geochemical behavior of As is largely transported conservatively in the main channel and relative non-conservatively in the tributaries in YTR system.
Display omitted
•High levels of dissolved As are observed in YTR system.•Hot springs are main source of dissolved As in the upper reaches of YTR.•Natural attenuation of dissolved As in main channel is mainly due to dilution.•Lowest dissolved As is observed in July and August due to dilution process.•Weathering of As-containing minerals inputs much of As in rivers in wet-season.
Influenza virus infection can alter the composition of the gut microbiota, while its pathogenicity can, in turn, be highly influenced by the gut microbiota. However, the details underlying these ...associations remain to be determined. The H7N9 influenza virus is an emerging zoonotic pathogen which has caused the death of 616 humans and has incurred huge losses in the poultry industry. Here, we investigated the effects of infection with highly pathogenic H7N9 on gut microbiota and determined potential anti-influenza microbes. 16S rRNA sequencing results show that H7N9 infection alters the mouse gut microbiota by promoting the growth of
, and
, and reducing the abundance of
gut group and
. Although the abundance of
is positively related to H7N9 infection, the oral administration of cultures, especially of pasteurized
, can significantly reduce weight loss and mortality caused by H7N9 infection in mice. Furthermore, oral administration of live or pasteurized
significantly reduces pulmonary viral titers and the levels IL-1β and IL-6 but enhances the levels of IFN-β, IFN-γ, and IL-10 in H7N9-infected mice, suggesting that the anti-influenza role of
is due to its anti-inflammatory and immunoregulatory properties. Taken together, we showed that the changes in the gut microbiota are associated with H7N9 infection and demonstrated the anti-influenza role of
, which enriches current knowledge about how specific gut bacterial strains protect against influenza infection and suggests a potential anti-influenza probiotic.
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•GO possesses intrinsic antibacterial activity to various bacterial pathogens.•A parabolic relation exists between the size and antibacterial activity of GO.•Increasing GO size ...enhances cell entrapment effect but reduces cutting effect.•Decreasing GO size reduces cell entrapment effect but enhances cutting effect.
The antibiotics-independent antimicrobial activity of graphene oxide (GO) is of great importance since antibiotic therapy is facing great challenges from drug resistance. However, the relations of GO size with its antimicrobial activity and how the size regulates the antibacterial mechanisms are still unknown. Herein, we fabricated four GO suspensions with different sizes and demonstrated the parabolic relationship between GO size and its antibacterial activity against the Gram-positive cariogenic bacterium Streptococcus mutans. More interestingly, we found out how GO size regulated the nano-bio interaction-based physical antibacterial mechanisms. Increasing the size reduced the cutting effect but enhanced the cell entrapment effect, and vice versa. In conclusion, GO size affects its edge density and lateral dimension, further regulates its physical antibacterial mechanisms in different orientations and ultimately determines its activity. These findings provide a deep understanding of GO antibacterial property and may guide the design and development of GO for clinical use.
YIM B00359
T
, a novel bacterial strain was isolated from the saline soil of Turpan city in Xinjiang province, north–west China. The strain was Gram-stain-positive, motile, aerobic, produced oval ...subterminal endospores in swollen sporangia. The whole-cell hydrolysates contain
meso
-diaminopimelic acid as the cell-wall diamino acid, with xylose, glucose, and ribose as the major whole-cell sugars. The phospholipids are diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, unidentified glycolipids, and one unidentified glycophospholipid. The predominant menaquinone is MK-7. The major fatty acids are anteiso-C
15:0
, iso-C
14:0
, iso-C
15:0
, and iso-C
16:0
. The DNA G + C content of the type strain is 37.5 mol%. Phylogenetic analysis indicated that the isolate belongs to the genus
Oceanobacillus
. However, it differed from its closest relatives,
Oceanobacillus halophilus
DSM 23996
T
and
Oceanobacillus senegalensis
Marseille-P3587
T
in many physiological and chemotaxonomic characteristics. Based on comparative analysis of polyphasic taxonomic data, strain YIM B00359
T
represents a novel species of the genus
Oceanobacillus
, for which the name
Oceanobacillus salinisoli
sp. nov. is proposed. The type strain is YIM B00359
T
(= CGMCC 1.17509
T
= KCTC 43185
T
).
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