Developing an ultra-intensified fed-batch cell culture process with greatly improved performance and productivity.

Intensified fed-batch (IFB), a popular cell culture intensification strategy, has been widely used for productivity improvement through high density inoculation followed by fed-batch cultivation. However, such an intensification strategy may counterproductively induce rapidly progressing cell apoptosis and difficult-to-sustain productivity. To improve culture performance, we developed a novel cell culture process intermittent-perfusion fed-batch (IPFB) which incorporates one single or multiple cycles of intermittent perfusion during an IFB process for better sustained cellular and metabolic behaviors and notably improved productivity. Unlike continuous perfusion or other semi-continuous processes such as hybrid perfusion fed-batch with only early-stage perfusion, IPFB applies limited times of intermittent perfusion in the mid-to-late stage of production and still inherits bolus feedings on nonperfusion days as in a fed-batch culture. Compared to IFB, an average titer increase of ~45% was obtained in eight recombinant CHO cell lines studied. Beyond IPFB, ultra-intensified IPFB (UI-IPFB) was designed with a markedly elevated seeding density of 20-80 × 106 cell/mL, achieved through the conventional alternating tangential flow filtration (ATF) perfusion expansion followed with a cell culture concentration step using the same ATF system. With UI-IPFB, up to ~6 folds of traditional fed-batch and ~3 folds of IFB productivity were achieved. Furthermore, the application grounded in these two novel processes showed broad-based feasibility in multiple cell lines and products of interest, and was proven to be effective in cost of goods reduction and readily scalable to a larger scale in existing facilities.

Multifunctional Nanoprobe for 3D Nanoresolution Imaging of Intact Cell HER2 Protein with Hard X-ray Tomography.

Three-dimensional nondestructive, nanoresolution, and in situ visualization of protein spatial localization in a large, thick single cell remains challenging. In this study, we designed a multifunctional iron oxide (Fe@BFK) nanoprobe that possesses fluorescence and hard X-ray imaging signals. This probe can specifically target the human epidermal growth factor receptor 2 (HER2) protein and help optimize the label condition and selection of suitable samples for X-ray imaging. Combining 30 nm resolution synchrotron radiation hard X-ray nanocomputed tomography and the X-ray-sensitive Fe@BFK nanoprobe, a 3D localization of HER2 on SK-BR-3 cells was obtained for the first time. HER2 was mainly localized and cluster-distributed on the cell membrane with a heterogeneous pattern. This study provides a novel method for the in situ and nondestructive synchrotron radiation imaging of the desired protein localization in large, thick cells and evaluation of the true cellular distribution of a nanoprobe with high resolution.

Composition and evolutionary characterization of the gut microbiota in pigs.

The intestinal microbiota plays significant role in the physiology and functioning of host organisms. However, there is limited knowledge of the composition and evolution of microbiota-host relationships from wild ancestors to modern domesticated species. In this study, the 16S rRNA gene V3-V4 in the intestinal contents of different pig breeds was analyzed and was compared using high-throughput sequencing. This identified 18 323 amplicon sequence variants, of which the Firmicutes and Actinobacteria phyla and Bifidobacterium and Allobaculum genera were most prevalent in wild pigs (WP). In contrast, Proteobacteria and Firmicutes predominated in Chinese Shanxi Black pigs (CSB), while Firmicutes were the most prevalent phylum in Large White pigs (LW) and Iberian pigs (IB), followed by Bacteroidetes in IB and Proteobacteria in LW. At the genus level, Shigella and Lactobacillus were most prevalent in CSB and LW, while Actinobacillus and Sarcina predominated in IB. Differential gene expression together with phylogenetic and functional analyses indicated significant differences in the relative abundance of microbial taxa between different pig breeds. Although many microbial taxa were common to both wild and domestic pigs, significant diversification was observed in bacterial genes that potentially influence host phenotypic traits. Overall, these findings suggested that both the composition and functions of the microbiota were closely associated with domestication and the evolutionary changes in the host. The members of the microbial communities were vertically transmitted in pigs, with evidence of co-evolution of both the hosts and their intestinal microbial communities. These results enhance our understanding and appreciation of the complex interactions between intestinal microbes and hosts and highlight the importance of applying this knowledge in agricultural and microbiological research.

Prevalence and genetic characterization of Theileria equi and Babesia caballi in grazing horses in Xinjiang, northwestern China.

Piroplasmosis is a disease that negatively affects equine health worldwide. Hence, 324 blood samples were collected from grazing horses in ten sites in Xinjiang and testing them for the presence of Theileria equi and Babesia caballi by PCR of the EMA-1 gene and BC48 gene, respectively. Of the 324 blood samples, 161 (49.7%) were positive for equine piroplasms. The prevalence of T. equi was 38.9% (126/324), while that of B. caballi was 30.2% (98/324). The T. equi and B. caballi co-infection rate was 19.4% (63/324). From the 126 EMA-1 gene sequences and 98 BC48 gene sequences we obtained, 21 and 27 genotypes were identified, respectively. The EMA-1 sequences together with the GenBank reference sequences grouped into four clusters, with those from the present study forming two distinct clusters. In contrast, the BC48 sequences formed eight clusters with the GenBank reference sequences, while those obtained in the present study formed five distinct clusters. Our results highlight the widespread distribution and abundant gene polymorphism of T. equi and B. caballi in grazing horses from Xinjiang.

A Comprehensive Review of Inorganic Sonosensitizers for Sonodynamic Therapy.

Sonodynamic therapy (SDT) is an emerging non-invasive cancer treatment method in the field of nanomedicine, which has the advantages of deep penetration, good therapeutic efficacy, and minimal damage to normal tissues. Sonosensitizers play a crucial role in the process of SDT, as their structure and properties directly determine the treatment outcome. Inorganic sonosensitizers, with their high stability and longer circulation time in the human body, have great potential in SDT. In this review, the possible mechanisms of SDT including the ultrasonic cavitation, reactive oxygen species generation, and activation of immunity are briefly discussed. Then, the latest research progress on inorganic sonosensitizers is systematically summarized. Subsequently, strategies for optimizing treatment efficacy are introduced, including combination therapy and image-guided therapy. The challenges and future prospects of sonodynamic therapy are discussed. It is hoped that this review will provide some guidance for the screening of inorganic sonosensitizers.

Strong two-photon absorption induced by energy funneling in chiral quasi-2D perovskites.

Quasi-2D Ruddlesden-Popper-type perovskites (RPPs) exhibit excellent nonlinear optical properties due to their multiple quantum well structures with large exciton binding energy. Herein, we introduce chiral organic molecules into RPPs and investigate their optical properties. It is found that the chiral RPPs possess effective circular dichroism in the ultraviolet to visible wavelengths. Two-photon absorption (TPA)-induced efficient energy funneling from small- to large-n domains is observed in the chiral RPP films, which induces strong TPA with a coefficient up to 4.98 cm MW-1. This work will broaden the application of quasi-2D RPPs in chirality-related nonlinear photonic devices.

Urea Synthesis from Isocyanides and O-Benzoyl Hydroxylamines Catalyzed by a Copper Salt.

In the presence of CuOAc, a series of unsymmetric ureas can be generated in moderate to good yields under mild reaction conditions (10 mol% of CuOAc, 2 equiv t-BuONa or PhONa, 30 °C), using aryl isocyanides and O-benzoyl hydroxylamines as the readily accessible starting materials. The reactions might undergo a cascade process involving isocyanide insertion into the N-O bond and Mumm-type rearrangement. This work represents a rare example of isocyanide insertion into N-O bonds, which would extend isocyanide insertion chemistry.

Development of a duplex PCR assay for detecting Theileria luwenshuni and Anaplasma phagocytophilum in sheep and goats.

Coinfections with the tick-borne pathogens Theileria luwenshuni and Anaplasma phagocytophilum can cause significant economic losses in sheep and goat farming. The difficulty in detecting these two pathogens by microscopic examination warrants the development of a rapid detection test to discriminate them. In this study, a duplex polymerase chain reaction (PCR) assay was developed to simultaneously detect T. luwenshuni and A. phagocytophilum. Alignment of the sequences from related pathogens allowed us to design a primer pair targeting the 18S ribosomal RNA gene in T. luwenshuni and generate a target product of 962 bp, whereas a previously reported species-specific primer (SSAP2f/SSAP2r) for A. phagocytophilum was used in the same reaction to generate a product of 641 bp. Genomic DNA from T. luwenshuni and A. phagocytophilum was 10-fold serially diluted for testing PCR sensitivity. Under the optimal PCR conditions we established, the lower limit of detection of the assay was 29.13 fg/µL for T. luwenshuni and 1.53 fg/µL for A. phagocytophilum, and PCR primers used in this study were confirmed to be 100% species-specific using other hemoparasites previously identified by other methods. No significant difference was found between conventional and duplex PCR protocols used to detect the two species. Our study provides an effective, sensitive, specific, and accurate tool for the diagnosis and epidemiological surveillance of mixed infections of the two pathogens in sheep and goats.

Leakage-Proof Electrolyte Chemistry for a High-Performance Lithium-Sulfur Battery.

Electrolyte leakage is a severe safety concern in lithium batteries. With highly volatile 1,2-dimethoxyethane as solvent, the leakage related hazards are more pronounced in lithium-sulfur (Li-S) batteries. To address this concern, a leakage-proof electrolyte is delicately designed through functionalizing the commercial electrolyte by Li6 PS5 Cl-grafted poly(ethyl cyanoacrylate), which can interact readily with the aluminum-plastic packing through hydrogen bond to immobilize the electrolyte. The moisture from ambient can also catalyze a further polymerization of the macromolecules to seal the leaking points and thereby to solve the leakage issue, endowing Li-S batteries superior safety even in an artificial cut pouch cell. With a bare S loading of 4.9 mg cm-2 , the battery can deliver good endurance owing to the suppressed polysulfide shuttle by its polar groups. This work enlightens the design of leakage-proof electrolyte and makes a milestone for high performance Li-S batteries.

Nicotinamide N-methyltransferase decreases 5-fluorouracil sensitivity in human esophageal squamous cell carcinoma through metabolic reprogramming and promoting the Warburg effect.

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor with poor prognosis. And different individuals respond to the same drug differently. Increasing evidence has confirmed that metabolism reprogramming was involved in the drug sensitivity of tumor cells. However, the potential molecular mechanism of 5-fluorouracil (5-FU) sensitivity remains to be elucidated in ESCC cells. In this study, we found that the 5-FU sensitivity of TE1 cells was lower than that of EC1 and Eca109 cells. Gas chromatography-mass spectrometry analysis results showed that nicotinate and nicotinamide metabolism and tricarboxylic acid cycle were significantly different in these three cell lines. Nicotinamide N-methyltransferase (NNMT), a key enzyme of nicotinate and nicotinamide metabolism, was significantly higher expressed in TE1 cells than that in EC1 and Eca109 cells. Therefore, the function of NNMT on 5-FU sensitivity was analyzed in vitro and in vivo. NNMT downregulation significantly increased 5-FU sensitivity in TE1 cells. Meanwhile, the glucose consumption and lactate production were decreased, and the expression of glycolysis-related enzymes hexokinase 2, lactate dehydrogenase A, and phosphoglycerate mutase 1 were downregulated in NNMT knockdown TE1 cells. Besides, overexpression of NNMT in EC1 and Eca109 cells caused the opposite effects. Moreover, when glycolysis was inhibited by 2-deoxyglucose, the roles of NNMT on 5-FU sensitivity was weakened. In vivo experiments showed that NNMT knockdown significantly increased the sensitivity of xenografts to 5-FU and suppressed the Warburg effect. Overall, these results demonstrated that NNMT decreases 5-FU sensitivity in human ESCC cells through promoting the Warburg effect, suggesting that NNMT may contribute to predict the treatment effects of the clinical chemotherapy in ESCC.

Correction to: Downregulation of nicotinamide N-methyltransferase inhibits migration and epithelial-mesenchymal transition of esophageal squamous cell carcinoma via Wnt/ß-catenin pathway.

Fig. 2C has been published incorrectly in the original article. The correct version of the Fig. 6 is provided in this erratum.

Effects of different expression systems on characterization of adenylate deaminase from Aspergillus oryzae.

Adenylate deaminase (AMPD) is an amino hydrolase that catalyzes the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, the effect of different hosts on the enzymatic properties of AMPD from Aspergillus oryzae GX-08 was investigated and showed that Bacillus subtilis WB600 was more suitable for producing AMPD with a higher activity of 2540 U/mL. After purification, the optimal temperature and pH of recombinant AMPD were 55 °C and pH 6.0, respectively, and its activity was significantly enhanced by 10 mM Fe3+ with an increase of 236%. More importantly, the recombinant AMPD specifically and effectively catalyzed the conversion between AMP and IMP, in which 10 mL of crude AMPD achieved a conversion ratio of 76.4% after 40 min. Therefore, B. subtilis WB600 provides a potential platform for producing AMPD with excellent catalytic ability and catalytic specificity.

Downregulation of nicotinamide N-methyltransferase inhibits migration and epithelial-mesenchymal transition of esophageal squamous cell carcinoma via Wnt/ß-catenin pathway.

Nicotinamide N-methyltransferase (NNMT) is an important methyltransferase involved in the biotransformation of many drugs and exogenous compounds. Abnormal expression of NNMT protein is closely associated with the onset and progression of many malignancies, but little is known about its role in esophageal squamous cell carcinoma (ESCC). Therefore, we aimed to explore whether NNMT plays any roles in carcinogenesis and metastasis in ESCC. NNMT expression was determined by immunohistochemistry in ESCC and corresponding adjacent normal tissues. Functional experiments were performed to elucidate the effects of NNMT knockdown on the proliferation, apoptosis, cell cycle, migration, and epithelial-mesenchymal transition (EMT) in EC9706 and TE1 cells. NNMT expression was significantly elevated in ESCC tissues compared with corresponding adjacent normal tissues. Moreover, a significant association emerged between NNMT expression and lymph node metastasis. SiRNA-mediated knockdown of NNMT in ESCC cells can significantly suppress cell viability and migration, induce cell cycle arrest, and promote cell apoptosis. In addition, NNMT downregulation led to the reversal of EMT, as reflected by upregulation of the intercellular adhesion molecule E-cadherin and downregulation of the mesenchymal markers N-cadherin and Vimentin. Further study found that NNMT knockdown suppressed the Wnt/ß-catenin signaling pathway. Taken together, these findings indicate that NNMT is a critical regulator of EMT in ESCC and may be a potential therapeutic target for ESCC metastasis.

Rapid and sensitive detection of Anaplasma phagocytophilum using a newly developed recombinase polymerase amplification assay.

Anaplasma phagocytophilum, the bacterial pathogen responsible for tick-borne fever and human granulocytic anaplasmosis, can seriously affect the health of humans and a wide range of other mammals. In this study, we developed a recombinase polymerase amplification (RPA) assay to detect A. phagocytophilum in clinical samples. Following alignment of the relevant DNA sequences, a pair of specific primers based on the 16S rRNA gene was designed to specifically detect A. phagocytophilum. The assay was performed at a constant temperature of 38 °C for 30 min, with a final primer concentration of 0.4 µM. The specificity of the primers was confirmed when DNA from A. phagocytophilum was used as the positive control, and DNA from other related pathogens were used as the negative controls, with ddH2O acting as the blank control. The results showed that the primers did not cross-react with DNA from the other related pathogens. The assay's detection limit was 1.77 × 10-5 ng/µl, a 10 × higher sensitivity level than that determined for nested PCR. The RPA assay's performance was evaluated using 44 clinical samples, and the prevalence results for A. phagocytophilum were found to not differ significantly between the RPA assay and the nested PCR. Thus, we have developed a specific, sensitive, rapid and cost-effective RPA method, requiring only a water bath, for the detection of A. phagocytophilum. The assay should be especially useful in resource-limited areas where access to laboratory equipment is limited.

Preparation and Photochromic Behavior of Spiropyran-Containing Fluorinated Polyacrylate Hydrophobic Coatings.

In this study, spiropyran (SP)-containing fluorinated polyacrylate (F-PA-SP) latex was prepared by emulsion polymerization using fluorinated and SP-based acrylic monomers as raw materials. Fourier transform infrared and 1H NMR demonstrate that the F-PA-SP copolymer has been successfully synthesized, and dynamic light scattering and transmission electron microscopy analyses indicate that the synthesized latex has presented a uniform particle size of approximately 200 nm. X-ray photoelectron spectroscopy, atomic force microscopy, and water contact angle (WCA) analyses were used to investigate the surface properties of the F-PA-SP coating and demonstrate that its hydrophobicity is enhanced by the addition of a fluorinated acrylic monomer. The photochromic properties of the coating were investigated by UV/vis spectroscopy, and the results reveal that the F-PA-SP coating possesses better photoresponsiveness, fatigue resistance, and photoreversibility under UV/vis irradiation than the coating prepared using fluorinated polyacrylate/SP blended latex. Moreover, the WCA of the F-PA-SP coating subjected to UV/vis irradiation shows minimal changes and retains its excellent hydrophobicity. Finally, the F-PA-SP latex was applied to cellulosic paper, and the resulting photochromic paper exhibits outstanding reversible color changes and hydrophobicity.

Facile Synthesis of Near-Infrared Emissive CdS Quantum Dots for Live Cells Imaging.

CdS quantum dots (QDs) have attracted extensive attention owing to their great potential in optoelectronic nanodevices and biosensors. But their poor water solubility and high cytotoxicity restrict their practical application in live cell imaging. In addition, CdS QDs usually emit blue or green fluorescence, which also have some limitations for cell imaging due to the "water window" effect. In this study, we report a novel strategy to directly synthesize water-soluble and low-cytotoxic CdS QDs with near infrared (NIR) fluorescence through confinement into BSA templates with the mediation of L-cysteine (Cys). The obtained CdS QDs emit NIR fluorescence at 730 nm when exited by 468 nm excitation light, and show good water solubility and low cell cytotoxicity, which can be directly used for live RAW cell imaging. In addition, the effects of the type of amino acids for mediations, and the ratio of both Cys/Cd and Cd/BSA on the fluorescence properties of CdS QDs have also been investigated comprehensively. The mediation of L-cysteine plays a critical role on the formation of CdS QDs emitting NIR fluorescence.

Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and 'Candidatus Mycoplasma haemovis') in sheep and goats in China.

BACKGROUND: Hemotropic mycoplasmas (hemoplasmas) are emerging zoonotic pathogens with a worldwide distribution that can cause mild to severe hemolytic anemia, icterus, ill-thrift, infertility, and poor weight gain. However, understanding of the molecular epidemiology of hemoplasmas (Mycoplasma ovis and 'Candidatus Mycoplasma haemovis') is limited in sheep and goats, and the hemoplasma strain/species/variant 'Candidatus M. haemovis' was poorly studied throughout the world and had never been detected in China until now. Thus, the aim of the present study was to determine the molecular prevalence of hemoplasmas, including M. ovis and 'Candidatus M. haemovis' in sheep and goats from seven provinces and one autonomous region of China. METHODS: A total of 1364 blood samples were collected from sheep and goats in seven provinces and one autonomous region of China. All blood samples were tested for hemoplasmas (M. ovis and 'Candidatus M. haemovis') by nested PCR amplification based on 16S rRNA gene. Positive specimens underwent nucleotide sequencing and phylogenetic analysis. RESULTS: Overall, 610 specimens (44.7%, 610/1364) were shown to be hemoplasmas (M. ovis and 'Candidatus M. haemovis') -positive by nested PCR amplification based on 16S rRNA gene. The prevalence in goats was 44.1% (379/860), and 45.8% (231/504) in sheep, while that in grazing small ruminants was 54.4% (396/728) and 33.6% (214/636) in house feeding small ruminants. Sequencing of the nearly complete 16S rRNA gene was successful for the 103 randomly selected positive specimens from different farms in different sampling sites of China. Among them, analysis of the 16S rRNA gene sequences identified M. ovis (n = 56) and 'Candidatus M. haemovis' (n = 47). Two (KU983740 and KU983746) of the four novel genotypes obtained in this study were closely related to M. ovis, while the other two genotypes (KU983748 and KU983749) had high identity with 'Candidatus M. haemovis'. Remarkably, the genotype (KU983740) of M. ovis in sheep and goats in this study fell in a clade with two human hemoplasmas from USA (KF313922 and GU230144) and shared 99.8%-99.9% with them. CONCLUSIONS: In this study, 'Candidatus M. haemovis' was first detected in Chinese sheep and goats and hemoplasmas (M. ovis and 'Candidatus M. haemovis') are highly prevalent, and widely distributed in China.

Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats.

Theileria spp. and Anaplasma spp., which are important tick-borne pathogens (TBPs), impact the health of humans and animals in tropical and subtropical areas. Theileria and Anaplasma co-infections are common in sheep and goats. Following alignment of the relevant DNA sequences, two primer sets were designed to specifically target the Theileria spp. 18S rRNA and Anaplasma spp. 16S rRNA gene sequences. Genomic DNA from the two genera was serially diluted tenfold for testing the sensitivities of detection of the primer sets. The specificities of the primer sets were confirmed when DNA from Anaplasma and Theileria (positive controls), other related hematoparasites (negative controls) and ddH2O were used as templates. Fifty field samples were also used to evaluate the utility of single PCR and duplex PCR assays, and the detection results were compared with those of the PCR methods previously published. An optimized duplex PCR assay was established from the two primer sets based on the relevant genes from the two TBPs, and this assay generated products of 298-bp (Theileria spp.) and 139-bp (Anaplasma spp.). The detection limit of the assay was 29.4 × 10-3 ng per µl, and there was no cross-reaction with the DNA from other hematoparasites. The results showed that the newly developed duplex PCR assay had an efficiency of detection (P > 0.05) similar to other published PCR methods. In this study, a duplex PCR assay was developed that can simultaneously identify Theileria spp. and Anaplasma spp. in sheep and goats. This duplex PCR is a potentially valuable assay for epidemiological studies of TBPs in that it can detect cases of mixed infections of the pathogens.

Affective bias in visual working memory is associated with capacity.

How does the affective nature of task stimuli modulate working memory (WM)? This study investigates whether WM maintains emotional information in a biased manner to meet the motivational principle of approaching positivity and avoiding negativity by retaining more approach-related positive content over avoidance-related negative content. This bias may exist regardless of individual differences in WM functionality, as indexed by WM capacity (overall bias hypothesis). Alternatively, this bias may be contingent on WM capacity (capacity-based hypothesis), in which a better WM system may be more likely to reveal an adaptive bias. In two experiments, participants performed change localisation tasks with emotional and non-emotional stimuli to estimate the number of items that they could retain for each of those stimuli. Although participants did not seem to remember one type of emotional content (e.g. happy faces) better than the other type of emotional content (e.g. sad faces), there was a significant correlation between WM capacity and affective bias. Specifically, participants with higher WM capacity for non-emotional stimuli (colours or line-drawing symbols) tended to maintain more happy faces over sad faces. These findings demonstrated the presence of a "built-in" affective bias in WM as a function of its systematic limitations, favouring the capacity-based hypothesis.