Dynamic fecal microenvironment properties enable predictions and understanding of peripartum blood oxidative status and nonesterified fatty acids in dairy cows.

The transition period in dairy cows is a critical stage and peripartum oxidative status, negative energy balance (NEB), and inflammation are highly prevalent. Fecal microbial metabolism is closely associated with blood oxidative status and nonesterified fatty acids (NEFA) levels. Here, we investigated dynamic changes in total oxidative status markers and NEFA in blood, fecal microbiome, and metabolome of 30 dairy cows during transition (-21, -7, +7, +21 d relative to calving). Then the Bayesian network and 9 machine-learning algorithms were applied to dismantle their relationship. Our results show that the oxidative status indicator (OSI) of -21, -7, +7 d was higher than +21 d. The plasma concentration of NEFA peaked on +7 d. For fecal microenvironment, a decline in bacterial α diversity was observed at postpartum and in bacterial interactions at +7 d. Conversely, microbial metabolites involved in carbohydrate, lipid, and energy metabolism increased on +7 d. A correlation analysis revealed that 11 and 10 microbial metabolites contributed to OSI and NEFA variations, respectively (arc strength >0.5). The support vector machine (SVM) radial model showed the highest average predictive accuracy (100% and 88.9% in the test and external data sets) for OSI using 1 metabolite and 3 microbiota. The SVM radial model also showed the highest average diagnostic accuracy (100% and 91% in the test and external data sets) for NEFA with 2 metabolites and 3 microbiota. Our results reveal a relationship between variation in the fecal microenvironment and indicators of oxidative status, NEB, and inflammation, which provide a theoretical basis for the prevention and precise regulation of peripartum oxidative status and NEB.

Calcium-binding motif-mediated binding of redundant calcium offers a chimeolysin enhanced bactericidal activity and extended host range under physiological conditions.

OBJECTIVES: Calcium-binding motifs are shared by multiple bacteriophage lysins; however, the influence of calcium on the enzymatic activity and host range of these enzymes is still not understood. To address this, ClyF, a chimeric lysin with a putative calcium-binding motif, was used as a model for in vitro and in vivo investigations. METHODS: The concentration of calcium bound to ClyF was determined by atomic absorption spectrometry. The influence of calcium on the structure, activity and host range of ClyF was assessed by circular dichroism and time-kill assays. The bactericidal activity of ClyF was evaluated in various sera and a mouse model of Streptococcus agalactiae bacteraemia. RESULTS: ClyF has a highly negatively charged surface around the calcium-binding motif that can bind extra calcium, thereby increasing the avidity of ClyF for the negatively charged bacterial cell wall. In line with this, ClyF exhibited significantly enhanced staphylolytic and streptolytic activity in various sera containing physiological calcium, including human serum, heat-inactivated human serum, mouse serum and rabbit serum. In a mouse model of S. agalactiae bacteraemia, intraperitoneal administration of a single dose of 25 µg/mouse ClyF fully protected the mice from lethal infection. CONCLUSIONS: The present data collectively showed that physiological calcium improves the bactericidal activity and host range of ClyF, making it a promising candidate for the treatment of infections caused by multiple staphylococci and streptococci.

Genome assembly of the Chinese maize elite inbred line RP125 and its EMS mutant collection provide new resources for maize genetics research and crop improvement.

Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan-genome characterization. In this study, we generated a high-quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long-read sequencing and Hi-C scaffolding, which yield highly contiguous, chromosome-length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high-quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: "missing a small part" in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis-like cell death were detected in qk1 endosperm. Our study provides the community with a high-quality genome sequence and a large collection of mutant germplasm.

A Choline-Recognizing Monomeric Lysin, ClyJ-3m, Shows Elevated Activity against Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading pathogen for bacterial pneumonia, which can be treated with bacteriophage lysins harboring a conserved choline binding module (CBM). Such lysins regularly function as choline-recognizing dimers. Previously, we reported a pneumococcus-specific lysin ClyJ comprising the binding domain from the putative endolysin gp20 from the Streptococcus phage SPSL1 and the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain from the PlyC lysin. A variant of ClyJ with a shortened linker, i.e., ClyJ-3, shows improved activity and reduced cytotoxicity. Resembling typical CBM-containing lysins, ClyJ-3 dimerized upon binding with choline. Herein, we further report a choline-recognizing variant of ClyJ-3, i.e., ClyJ-3m, constructed by deleting its C-terminal tail. Biochemical characterization showed that ClyJ-3m remains a monomer after it binds to choline yet exhibits improved bactericidal activity against multiple pneumococcal strains with different serotypes. In an S. pneumoniae-infected bacteremia model, a single intraperitoneal administration of 2.32 µg/mouse of ClyJ-3m showed 70% protection, while only 20% of mice survived in the group receiving an equal dose of ClyJ-3 (P < 0.05). A pharmacokinetic analysis following single intravenously doses of 0.29 and 1.16 mg/kg of ClyJ-3 or ClyJ-3m in BALB/c mice revealed that ClyJ-3m shows a similar half-life but less clearance and a greater area under curve than ClyJ-3. Taken together, the choline-recognizing monomer ClyJ-3m exhibited enhanced bactericidal activity and improved pharmacokinetic proprieties compared to those of its parental ClyJ-3 lysin. Our study also provides a new way for rational design and programmed engineering of lysins targeting S. pneumoniae.

Construction and characterization of a chimeric lysin ClyV with improved bactericidal activity against Streptococcus agalactiae in vitro and in vivo.

The emergence of antibiotic-resistant beta-hemolytic Streptococcus agalactiae strains poses increasing threat to human beings globally. As an attempt to create a novel lysin with improved activity against S. agalactiae, a chimeric lysin, ClyV, was constructed by fusing the enzymatically active domain (EAD) from PlyGBS lysin (GBS180) and the cell wall binding domain (CBD) from PlyV12 lysin (V12CBD). Plate lysis assay combined with lytic kinetic analysis demonstrated that ClyV has improved activity than its parental enzymatic domain GBS180 against multiple streptococci. Biochemical characterization showed that ClyV is active from pH 7 to 10, with the optimum pH of 9, and is stable under NaCl concentration of < 500 mM. In a S. agalactiae infection model, a single intraperitoneally administration of 0.1 mg/mouse of ClyV protected 100% mice, while it was observed that ~ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180. Moreover, a high dose of 0.8 mg/mouse ClyV did not show any adverse effects to the health or survival rate of the mice. Considering the robust bactericidal activity and good safety profile of ClyV, it represents a potential candidate for the treatment of S. agalactiae infections.

High-power passively Q-switched Yb:YCa4O(BO3)3 laser with a GaAs crystal plate as saturable absorber.

We report on efficient high-power passively Q-switched operation of a Yb:YCa4O(BO3)3 laser with a GaAs crystal plate acting as the saturable absorber. An average output power of 5.7 W at 1032 nm is generated at a pulse repetition rate of 166.7 kHz when the incident pump power is 26.8 W, with a slope efficiency determined to be 24.5%. The averaged pulse energy achieved is roughly 30 µJ and is increased to about 40 µJ when the output coupling used changes from 30% to 50%, while the shortest pulse width is measured to be 153 ns.

New insights into the enteric methane production based on the archaeal genome map of ruminant gastrointestinal tract.

INTRODUCTION: As one of the important components of ruminant gastrointestinal tract (GIT) microbiome, archaea are involved in many biological processes, especially methanogenesis. However, the diversity and individual metabolic characteristics of archaea in this habitat remain largely understudied, partly due to the lack of a unified reference genome catalog. OBJECTIVES: This study aimed to construct a unified genome map for taxonomic and functional exploration of ruminant GIT archaea in the future. METHODS: We collected archaeal genomes from public sources and new data of this study. We performed phylogenetic analysis, functional genomics analyses, prophages identification based on the genomes. Using collected genomes as a reference, we conducted metagenomic and metatranscriptomic analyses on rumen fluid samples from 18 dairy cows with different methane (CH4) production. RESULTS: We constructed the ruminant GIT archaeal genomes (RGAG) by compiling 405 strain-level (160 species) non-redundant archaeal genomes from more than 10 ruminant species. The functional heterogeneity and methanogenic structure within RGAG was investigated. RGAG possessed 1,124 (99.5 %) unknown microbial biosynthetic gene clusters. A survey of RGAG-borne prophages identified 63 prophages with 122 host-beneficial genes and 18 auxiliary metabolic genes (AMGs). The pipeline for both metagenomics and metatranscriptomics generated in the study revealed the roles of archaeal genomes under-assessed in general analyses of muti-omics. The highly expressed genus Methanosphaera was negatively correlated with CH4 production. CONCLUSION: A unified genome map of ruminant GIT archaea is constructed in the study. Functional genomics indicates that the multifaceted functions of RGAG remains undiscovered. Multi-omics analyses reveals the advantages of metatranscriptomics over metagenomics in studying rumen archaeal communities. Differences in rumen archaeal community structure among cattle with different CH4 production may reflect the balance between rumen hydrogen production and methanogenesis. Our work provides new understanding of archaeal functions in the ruminant GIT and potential targets for future CH4 reduction.

- Invited Review - Understanding the functionality of the rumen microbiota: searching for better opportunities for rumen microbial manipulation.

Rumen microbiota play a central role in the digestive process of ruminants. Their remarkable ability to break down complex plant fibers and proteins, converting them into essential organic compounds that provide animals with energy and nutrition. Research on rumen microbiota not only contributes to improving animal production performance and enhancing feed utilization efficiency but also holds the potential to reduce methane emissions and environmental impact. Nevertheless, studies on rumen microbiota face numerous challenges, including complexity, difficulties in cultivation, and obstacles in functional analysis. This review provides an overview of microbial species involved in the degradation of macromolecules, the fermentation processes, and methane production in the rumen, all based on cultivation methods. Additionally, the review introduces the applications, advantages, and limitations of emerging omics technologies such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics, in investigating the functionality of rumen microbiota. Finally, the article offers a forward-looking perspective on the new horizons and technologies in the field of rumen microbiota functional research. These emerging technologies, with continuous refinement and mutual complementation, have deepened our understanding of rumen microbiota functionality, thereby enabling effective manipulation of the rumen microbial community.

Advances in single-cell transcriptomics in animal research.

Understanding biological mechanisms is fundamental for improving animal production and health to meet the growing demand for high-quality protein. As an emerging biotechnology, single-cell transcriptomics has been gradually applied in diverse aspects of animal research, offering an effective method to study the gene expression of high-throughput single cells of different tissues/organs in animals. In an unprecedented manner, researchers have identified cell types/subtypes and their marker genes, inferred cellular fate trajectories, and revealed cell‒cell interactions in animals using single-cell transcriptomics. In this paper, we introduce the development of single-cell technology and review the processes, advancements, and applications of single-cell transcriptomics in animal research. We summarize recent efforts using single-cell transcriptomics to obtain a more profound understanding of animal nutrition and health, reproductive performance, genetics, and disease models in different livestock species. Moreover, the practical experience accumulated based on a large number of cases is highlighted to provide a reference for determining key factors (e.g., sample size, cell clustering, and cell type annotation) in single-cell transcriptomics analysis. We also discuss the limitations and outlook of single-cell transcriptomics in the current stage. This paper describes the comprehensive progress of single-cell transcriptomics in animal research, offering novel insights and sustainable advancements in agricultural productivity and animal health.

Single-cell transcriptomics across 2,534 microbial species reveals functional heterogeneity in the rumen microbiome.

Deciphering the activity of individual microbes within complex communities and environments remains a challenge. Here we describe the development of microbiome single-cell transcriptomics using droplet-based single-cell RNA sequencing and pangenome-based computational analysis to characterize the functional heterogeneity of the rumen microbiome. We generated a microbial genome database (the Bovine Gastro Microbial Genome Map) as a functional reference map for the construction of a single-cell transcriptomic atlas of the rumen microbiome. The atlas includes 174,531 microbial cells and 2,534 species, of which 172 are core active species grouped into 12 functional clusters. We detected single-cell-level functional roles, including a key role for Basfia succiniciproducens in the carbohydrate metabolic niche of the rumen microbiome. Furthermore, we explored functional heterogeneity and reveal metabolic niche trajectories driven by biofilm formation pathway genes within B. succiniciproducens. Our results provide a resource for studying the rumen microbiome and illustrate the diverse functions of individual microbial cells that drive their ecological niche stability or adaptation within the ecosystem.

Green synthesis of silk fibroin-silver nanoparticle composites with effective antibacterial and biofilm-disrupting properties.

Natural polymer Bombyx mori silk fibroin is used as a biotemplate to produce silver nanoparticles in situ under light (both incandescent light and sunlight) at room temperature. Silk fibroin provides multiple functions in the whole reaction system, serving as the reducing agent of silver, and the dispersing and stabilizing agent of the resulted silver nanoparticles. As the reaction needs not any other chemicals and only uses light as power source, the synthetic route of silver nanoparticles reported here is rather environment-friendly and energy-saving. The silk fibroin-silver nanoparticle composite prepared by this method can be stably stored in a usual environment (room temperature, exposure to light, and so forth) for at least one month. Such a silk fibroin-silver nanoparticle composite shows an effective antibacterial activity against the methicillin-resistant Staphylococcus aureus (S. aureus) and subsequently inhibits the biofilm formation caused by the same bacterium. Moreover, a maturely formed biofilm created by methicillin-resistant S. aureus can be destroyed by the silk fibroin-silver nanoparticle composite, which meets the demand of clinical application. Therefore, the silk fibroin-silver nanoparticle composite prepared by this clean and facile method is expected to be an effective and economical antimicrobial material in biomedical fields.

Effects of bamboo leaf extract on energy metabolism, antioxidant capacity, and biogenesis of small intestine mitochondria in broilers.

The present study was carried out to investigate the effects of bamboo leaf extract (BLE) on energy metabolism, antioxidant capacity, and biogenesis of broilers' small intestine mitochondria. A total of 384 one-day-old male Arbor Acres broiler chicks were randomly divided into four groups with six replicates each for 42 d. The control group was fed a basal diet, whereas the BLE1, BLE2, and BLE3 groups consumed basal diets with 1.0, 2.0, and 4.0 g/kg of BLE, respectively. Some markers of mitochondrial energy metabolism including isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase and some markers of redox system including total superoxide dismutase, malondialdehyde, and glutathione were measured by commercial colorimetric kits. Mitochondrial and cellular antioxidant genes, mitochondrial biogenesis-related genes, and mitochondrial DNA copy number were measured by quantitative real-time-polymerase chain reaction (qRT-PCR). Data were analyzed using the SPSS 19.0, and differences were considered as significant at P < 0.05. BLE supplementation linearly increased jejunal mitochondrial isocitrate dehydrogenase (P < 0.05) and total superoxide dismutase (P < 0.05) activity. The ileal manganese superoxide dismutase mRNA expression was linearly affected by increased dietary BLE supplementation (P < 0.05). Increasing BLE supplementation linearly increased jejunal sirtuin 1 (P < 0.05) and nuclear respiratory factor 1 (P < 0.05) mRNA expression. Linear (P < 0.05) and quadratic (P < 0.05) responses of the ileal nuclear respiratory factor 2 mRNA expression occurred with increased dietary BLE levels. In conclusion, BLE supplementation was beneficial to the energy metabolism, antioxidant capacity, and biogenesis of small intestine mitochondria in broilers. The dose of 4.0 g/kg BLE demonstrated the best effects.

The intensive breeding model of broilers exposes broilers directly to oxidative stress, which is associated with mitochondrial dysfunction. Some researches have shown that bamboo leaf extract (BLE) exhibited antioxidant capacity both in vitro and vivo. However, few researches have been conducted to explore the effects of BLE supplementation on small intestine mitochondrial functions in broilers. The study aimed to evaluate whether BLE can improve energy metabolism, antioxidant capacity, and biogenesis of broilers' small intestine mitochondria. All broilers were randomly divided into four groups. The control (CTR) group was fed a basal diet, and the three experimental groups of BLE1, BLE2, and BLE3 were fed the basal diet supplemented with 1.0, 2.0, and 4.0 g of BLE per kg of feed between 1 d and 42 d of age, respectively. Based on our results, we obtained interesting evidence that BLE supplementation enhanced metabolic efficiency of small intestine mitochondria in broilers.

Gut microbiome is linked to functions of peripheral immune cells in transition cows during excessive lipolysis.

BACKGROUND: Postpartum dairy cows experiencing excessive lipolysis are prone to severe immunosuppression. Despite the extensive understanding of the gut microbial regulation of host immunity and metabolism, its role during excessive lipolysis in cows is largely unknown. Herein, we investigated the potential links between the gut microbiome and postpartum immunosuppression in periparturient dairy cows with excessive lipolysis using single immune cell transcriptome, 16S amplicon sequencing, metagenomics, and targeted metabolomics. RESULTS: The use of single-cell RNA sequencing identified 26 clusters that were annotated to 10 different immune cell types. Enrichment of functions of these clusters revealed a downregulation of functions in immune cells isolated from a cow with excessive lipolysis compared to a cow with low/normal lipolysis. The results of metagenomic sequencing and targeted metabolome analysis together revealed that secondary bile acid (SBA) biosynthesis was significantly activated in the cows with excessive lipolysis. Moreover, the relative abundance of gut Bacteroides sp. OF04 - 15BH, Paraprevotella clara, Paraprevotella xylaniphila, and Treponema sp. JC4 was mainly associated with SBA synthesis. The use of an integrated analysis showed that the reduction of plasma glycolithocholic acid and taurolithocholic acid could contribute to the immunosuppression of monocytes (CD14+MON) during excessive lipolysis by decreasing the expression of GPBAR1. CONCLUSIONS: Our results suggest that alterations in the gut microbiota and their functions related to SBA synthesis suppressed the functions of monocytes during excessive lipolysis in transition dairy cows. Therefore, we concluded that altered microbial SBA synthesis during excessive lipolysis could lead to postpartum immunosuppression in transition cows. Video Abstract.

[Comparison of postoperative deglutition disorder in patients with laryngeal carcinoma after different types of supracricoid partial laryngectomy].

OBJECTIVE: To Evaluate the incidence rates and extents of deglutition disorder in patients with laryngeal carcinoma after different types of supracricoid partial laryngectomy. METHODS: Retrospective analysis of postoperative deglutition disorder in patients with laryngeal carcinoma after different types of supracricoid partial laryngectomy treated in our department from 2005 to 2009. The extents of postoperative deglutition disorder were evaluated using a homemade quantitative score table at 5-20 days postoperation. RESULTS: The score of deglutition disorder was 2.71 ± 0.31 in the supracricoid partial laryngectomy-cricohyoidoepiglottopexy (SCPL-CHEP) operation group and 3.43 ± 0.64 in the supracricoid partial laryngectomy-cricohyoidopexy (SCPL-CHP) group, respectively. The deference was statistically significant between the two groups (P < 0.001). The coefficient between age and score of postoperative deglutition disorder was assessed by Pearson correlation analysis. The coefficient of correlation was 0.947 (P < 0.0001) in the SCPL-CHEP group and 0.907 (P < 0.0001) in the SCPL-CHP group. The incidence rate of deglutition disorder was 1/37 in the SCPL-CHEP group and 7/30 in the SCPL-CHP group, evaluated at 8 weeks postoperation (P = 0.012). The deference between the two groups was significant. CONCLUSIONS: The type of operation procedure is an important factor affecting the occurrence of postoperative deglutition disorder in the patients after supracricoid partial laryngectomy, more serious in the SCPL-CHP group. The severity of postoperative deglutition disorder is more serious along with the increase of patient's age. For the elderly (> 70 years of age) patients with laryngeal carcinoma, the choice of surgical procedure should be more cautious, especially with the SCPL-CHP operation.

Effect of Bamboo Leaf Extract on Antioxidant Status and Cholesterol Metabolism in Broiler Chickens.

The objective of this study was to investigate the effects of dietary bamboo leaf extract (BLE) on antioxidant status and cholesterol metabolism in broilers. One-day-old male Arbor Acres (576) broilers were randomly divided into six groups. A control group was fed a basal diet, while five experimental groups were supplemented with 1.0, 2.0, 3.0, 4.0, and 5.0g BLE per kg feed in their basal diets. The result indicated that BLE supplementation linearly improved eviscerated yield and decreased abdominal fat (p < 0.05). A significant decrease of serum triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c) content was observed with BLE supplementation (p < 0.05). BLE supplementation linearly improved the total antioxidant capacity and catalase activity in both serum and liver (p < 0.05). Glutathione peroxidase was quadratically increased in serum and linearly increased in the liver with BLE supplementation (p < 0.05). The malonaldehyde content in liver showed a linear and quadratic decrease with BLE supplementation (p < 0.05). BLE supplementation up-regulated the mRNA expression of cholesterol 7- alpha hydroxylase and low-density lipoprotein receptor and downregulated 3-hydroxy3-methyl glutamates coenzyme A reductase mRNA expression in the liver. The antioxidant enzyme mRNA expressions were all up-regulated by BLE supplementation in the liver. In conclusion, supplemental BLE improved antioxidant status and cholesterol metabolism in broilers, which eventually led to a decrease of serum TG, LDL-c content, and abdominal fat deposition.

Comparison of type I tympanoplasty with acellular dermal allograft and cartilage perichondrium.

Background: Acellular dermal allograft (AlloDerm) and cartilage perichondrium are two common materials used for repair of tympanic membrane perforations (TMPs). To date, comparative evaluations of their efficacy have rarely been reported. Aim/objectives: To compare anatomical and audiological outcomes between AlloDerm and cartilage perichondrium in type I tympanoplasty. Methods: A total of 61 patients of TMP were studied. In total, 27 patients (Group 1) underwent AlloDerm myringoplasty, and the remaining 34 patients (Group 2) underwent perichondrium myringoplasty. Operating time, closure rate and hearing gain were compared between Groups 1 and 2. Results: Successful closure rates at 6-month follow-up were 88.9% (Group 1) and 82.4% (group 2). The average improvement of air-bone gap (ABG) was 13.5 ± 11.8 dB for Group 1 and 13.1 ± 13.1 dB for Group 2. The difference in between preoperative and 6 months postoperative ABG values was statistically significant (p < .001). Conclusions and significance: Success rates and improvement of hearing level were similar for the AlloDerm (Group 1) and the cartilage perichondrium (Group 2) groups. However, AlloDerm requires shorter operative time and avoids the incisions in the harvest of allografts. Our results suggest that AlloDerm can be recommended as an attractive alternative to cartilage grafts.

PTEN promotes apoptosis of H2O2­injured rat nasal epithelial cells through PI3K/Akt and other pathways.

Chronic rhinosinusitis (CRS) is a form of chronic inflammation of the nasal cavity and paranasal sinus with multi­causal pathogenesis, including oxidative stress. Several lines of evidence have demonstrated that the phosphatase and tensin homolog gene (PTEN) can inhibit the activation of phosphoinositide 3­kinase (PI3K) to affect phosphorylation of Akt. Importantly, the PI3K/PTEN/Akt signaling pathway is associated with various types of tumors, chronic inflammatory diseases, and autoimmune disease through its regulation of cell growth, apoptosis, proliferation, and metabolism. This in vitro study aimed to investigate the role of PTEN and the relationship between PTEN and the PI3K/Akt pathway in nasal epithelial cells under oxidative stress. H2O2 treatment was applied to induce a cell injury model of oxidative stress in rat nasal epithelial cells. Cells were divided into control, H2O2, H2O2+PTEN, and H2O2+siPTEN groups. Cell viability was measured using the CCK­8 assay, and reactive oxygen species (ROS) levels and apoptosis rates were analyzed by flow cytometry (FCM). Oxidative parameters, including ROS, catalase (CAT), and malondialdehyde (MDA), were tested by enzyme­linked immunosorbent assay (ELISA). The expression of apoptosis­related genes and PI3K/Akt pathway was assayed by quantitative PCR (qPCR) and western blot. In H2O2­injured cells, oxidative stress, due to increased ROS levels and apoptosis rates, was induced, and PTEN aggravated the injury. The levels of both p­Akt and PTEN in H2O2­injured cells were positively correlated and higher than in control cells. Unknown regulatory protein(s) may exist in the PI3K/PTEN/Akt pathway or the PTEN and PI3K/Akt pathways may be two independent signaling pathways that have cross interactions.

Inflammatory immune response in rabbits with Staphylococcus aureus biofilm-associated sinusitis.

BACKGROUND: Staphylococcus aureus is the most commonly isolated bacterium from patients with surgically recalcitrant chronic rhinosinusitis (CRS). Understanding the immune responses to S aureus biofilms will provide insights into how the host response may be manipulated by therapeutic agents to improve the chances of successfully preventing and treating these infections. In this study, we investigated the inflammatory immune response in a rabbit model of S aureus biofilm-related sinusitis by analyzing the levels of some major inflammatory cytokines. METHODS: Eighteen New Zealand white rabbits were randomly divided into 3 groups: a blank-control group; a negative-control group; and a model group. Four weeks after the biofilm-associated sinusitis models were established, the sinus mucosa was harvested and examined using hematoxylin-eosin (H&E) staining, scanning electron microscopy (SEM), reverse transcription polymerase chain reaction (RT-PCR), and western blotting. The expression levels of inflammatory cytokines were analyzed statistically. RESULTS: Interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α expression levels were significantly higher in the model group than in the blank-control group (p < 0.05); mRNA levels were increased by 1600%, 230%, and 130%, respectively, and the protein levels were increased by 180%, 100%, and 100%, respectively. In contrast, IL-4 and IL-5 mRNA levels were reduced by 44% and 70%, respectively, compared with the blank-control group (p < 0.05). CONCLUSION: S aureus biofilms in the rabbit maxillary sinus mucosa were associated with increased IL-1ß, IL-8, and TNF-α expression, and decreased IL-4 and IL-5 expression.

Porous Three-Dimensional Silk Fibroin Scaffolds for Tracheal Epithelial Regeneration in Vitro and in Vivo.

The regeneration of functional epithelial lining is critical for artificial grafts to repair tracheal defects. Although silk fibroin (SF) scaffolds have been widely studied for biomedical application (e.g., artificial skin), its potential for tracheal substitute and epithelial regeneration is still unknown. In this study, we fabricated porous three-dimensional (3D) silk fibroin scaffolds and cocultured them with primary human tracheobronchial epithelial cells (HBECs) for 21 days in vitro. Examined by scanning electronic microscopy (SEM) and calcein-AM staining with inverted phase contrast microscopy, the SF scaffolds showed excellent properties of promoting cell growth and proliferation for at least 21 days with good viability. In vivo, the porous 3D SF scaffolds (n = 18) were applied to repair a rabbit anterior tracheal defect. In the control group (n = 18), rabbit autologous pedicled trachea wall without epithelium, an ideal tracheal substitute, was implanted in situ. Observing by endoscopy and computed tomography (CT) scan, the repaired airway segment showed no wall collapse, granuloma formation, or stenosis during an 8-week interval in both groups. SEM and histological examination confirmed the airway epithelial growth on the surface of porous SF scaffolds. Both the epithelium repair speed and the epithelial cell differentiation degree in the SF scaffold group were comparable to those in the control group. Neither severe inflammation nor excessive fibrosis occurred in both groups. In summary, the porous 3D SF scaffold is a promising biomaterial for tracheal repair by successfully supporting tracheal wall contour and promoting tracheal epithelial regeneration.

AMPK promotes survival and adipogenesis of ischemia-challenged ADSCs in an autophagy-dependent manner.

Some studies have shown that transplanted fat tissues usually cannot survive for long if adipose-derived stem cells (ADSCs) are removed from the tissues in advance. It is more meaningful to explore the mechanism mediating survival and differentiation of ADSCs in the transplanted microenvironment. AMP-activated protein kinase (AMPK) has been shown to be one of the energy receptors that regulate many aspects of cellular metabolism. AMPK activation has been implicated in models of adult ischemic injury, but the mechanism and the regulating effects of AMPK on survival and adipogenesis of transplanted ADSCs are still little known. In this study, we simulated the transplanted microenvironment using oxygen-glucose deprivation (OGD) to test the survival and adipogenesis of ADSCs. We found that OGD treatment triggered significant apoptosis and promoted autophagy. Simultaneously, OGD hindered the differentiation of ADSCs into mature adipocytes. After inhibiting AMPK, the OGD-induced apoptosis rate increased but autophagy was inhibited. The adipogenesis level also decreased. To show that the effects of AMPK on apoptosis and adipogenesis were autophagy-dependent, we pre-inhibited or pre-promoted autophagy with siATG7 or rapamycin while blocking AMPK. We found that inhibiting or improving autophagy exacerbated or alleviated the role of AMPK prohibition in apoptosis and adipogenesis. Furthermore, we showed that AMPK inhibition significantly lowered ULK1 activity but promoted mTOR activity, so that to inhibit autophagy. Our study shows that AMPK plays a protective role in maintaining survival and adipogenesis of OGD-challenged ADSCs partly by positively regulating autophagy. AMPK positively regulates autophagy by inhibiting mTOR but promoting ULK1 activity in OGD condition.