The anticancer mechanisms of exopolysaccharide from Weissella cibaria D-2 on colorectal cancer via apoptosis induction.

Exopolysaccharide (EPS) from Weissella cibaria has been devoted to the study of food industry. However, the anticancer activity of W. cibaria derived EPS has not yet been investigated. In this study, we obtained the EPS from W. cibaria D-2 isolated from the feces of healthy infants and found that D-2-EPS, a homopolysaccharide with porous web like structure, could effectively inhibit the proliferation, migration, invasion and induce cell cycle arrest in G0/G1 phase of colorectal cancer (CRC) cells. In HT-29 tumor xenografts, D-2-EPS significantly retarded tumor growth without obvious cytotoxicity to normal organs. Furthermore, we revealed that D-2-EPS promoted the apoptosis of CRC cells by increasing the levels of Fas, FasL and activating Caspase-8/Caspase-3, indicating that D-2-EPS might induce apoptosis through the extrinsic Fas/FasL pathway. Taken together, the D-2-EPS has the potential to be developed as a nutraceutical or drug to prevent and treat colorectal cancer.

Exopolysaccharide from Weissella confusa J4-1 inhibits colorectal cancer via induction of cell cycle arrest.

Exopolysaccharide (EPS), a bioproduct of lactic acid bacteria (LAB), has various health-promoting biological activities that may be beneficial for cancer therapy. This in vivo and in vitro study aimed to elucidate the anti-colorectal cancer (CRC) capacity of a homopolysaccharide EPS obtained from Weissella confusa J4-1 (EPSJ4-1) isolated from the faeces of healthy infants. We confirmed that EPSJ4-1 contained glucose and effectively suppressed the proliferation, migration, and invasion of CRC cells. EPSJ4-1 treatment significantly retarded the growth of HT-29 tumour xenografts without causing cytotoxicity to normal organs. EPSJ4-1 exerts an inhibitory effect on cell proliferation by inducing G0/G1 phase cell cycle arrest in CRC cells. Furthermore, EPSJ4-1 upregulated p21 levels and downregulated mutant p53 and cyclin kinase 2 levels. This is the first study to demonstrate the antitumour effects of EPS from W. confusa on CRC via cell cycle arrest and inhibition of cell migration and invasion, suggesting that EPSJ4-1 has the potential to be developed as a nutraceutical or pharmaceutical drug to prevent and treat CRC.

Weld Seam Tracking and Detection Robot Based on Artificial Intelligence Technology.

The regular detection of weld seams in large-scale special equipment is crucial for improving safety and efficiency, and this can be achieved effectively through the use of weld seam tracking and detection robots. In this study, a wall-climbing robot with integrated seam tracking and detection was designed, and the wall climbing function was realized via a permanent magnet array and a Mecanum wheel. The function of weld seam tracking and detection was realized using a DeepLabv3+ semantic segmentation model. Several optimizations were implemented to enhance the deployment of the DeepLabv3+ semantic segmentation model on embedded devices. Mobilenetv2 was used to replace the feature extraction network of the original model, and the convolutional block attention module attention mechanism was introduced into the encoder module. All traditional 3×3 convolutions were substituted with depthwise separable dilated convolutions. Subsequently, the welding path was fitted using the least squares method based on the segmentation results. The experimental results showed that the volume of the improved model was reduced by 92.9%, only being 21.8 Mb. The average precision reached 98.5%, surpassing the original model by 1.4%. The reasoning speed was accelerated to 21 frames/s, satisfying the real-time requirements of industrial detection. The detection robot successfully realizes the autonomous identification and tracking of weld seams. This study remarkably contributes to the development of automatic and intelligent weld seam detection technologies.

Injectable Hydrogels Encapsulating Dual-Functional Au@Pt Core-Shell Nanoparticles Regulate Infarcted Microenvironments and Enhance the Therapeutic Efficacy of Stem Cells through Antioxidant and Electrical Integration.

Injectable functional biomaterials have made significant progress in cardiac regenerative. In addition, how to adjust the abominable infarction microenvironment and introduce therapeutic stem cells to improve the healing effect has become a hotspot. Herein, injectable stem cell vector is prepared by combining natural alginate hydrogel and Au@Pt nanoparticles (Au@Pt/Alg hydrogel) to encapsulate brown adipose stem cells (BASCs). Au@Pt nanoparticles with both antioxidative and conductive properties could effectively eliminate reactive oxygen species, enhance the frequency of action potential release of cardiomyocytes, and further reduce the inflammatory factors of macrophage in vitro. The Au@Pt/Alg hydrogel enhances the antioxidant, differentiation, and paracrine capability of BASCs. The effect of BASCs loaded Au@Pt/Alg hydrogel is evaluated in a rat myocardial infarction (MI) model. The antioxidant, anti-inflammatory, and heart electrical integration are showed in the MI model. More interestingly, Au@Pt/Alg hydrogel can effectively maintain the paracrine efficiency and pro-angiogenesis effects of BASCs in the infarcted area. This study led us to recognize the great value of Au@Pt/Alg hydrogels for their ability to actively regulate the microenvironment and carry stem cells for MI treatment.

Oral nanozyme-engineered probiotics for the treatment of ulcerative colitis.

Probiotic-based therapy for ulcerative colitis (UC) is a novel and promising approach that has gained much popularity in recent years. However, probiotics may be easily captured and destroyed by the harsh in vivo environment, which limits their application prospects to a large extent. This work introduces a versatile facile approach for decorating individual probiotics with nanozyme coatings, named Pt-Lipid@EcN. A bimolecular lipid coating is deposited on the surface of probiotics used in the oral treatment of UC using simple self-assembly with a liposome under cytocompatible conditions, and it acts as armor to effectively protect the probiotics from strong digestive acids and enzymes. The obtained results indicated that the liposome-coated Escherichia coli Nissle (EcN) 1917 could significantly improve the colonization rate of the microorganisms in the intestinal tract. We also explored the specific mechanism of Pt-Lipid@EcN to improve the behavior of murine UC models, including reduction of inflammatory effects and histological injury, and regulation of epithelial barrier homeostasis in the intestine. The nanozyme-shielded probiotic exhibited enhanced anti-inflammatory ability in vivo, introducing novel strategies for the application of nanozyme biomedicine in the treatment of colonic inflammatory diseases.

Lung transcriptome analysis for the identification of genes involved in the hypoxic adaptation of plateau pika (Ochotona curzoniae).

The plateau pika, a typical hypoxia-tolerant mammal lives 3000-5000 m above sea level on the Qinghai-Tibet Plateau, has acquired many physiological and morphological characteristics and strategies in its adaptation to sustained, high-altitude hypoxia. Blunted hypoxic pulmonary vasoconstriction is one such strategy, but the genes involved in this strategy have not been elucidated. Here, we investigated the genes involved and their expression profiles in the lung transcriptome of plateau pikas subjected to different hypoxic conditions (using low-pressure oxygen cabins). A slight, right ventricular hypertrophy was observed in pikas of the control group (altitude: 3200 m) vs. those exposed to 5000 m altitude conditions for one week. Our assembly identified 67,774 genes; compared with their expression in the control animals, 866 and 8364 genes were co-upregulated and co-downregulated, respectively, in pikas subjected to 5000 m altitude conditions for 1 and 4 w. We elucidated pathways that were associated with pulmonary vascular arterial pressure, including vascular smooth muscle contraction, HIF-1 signalling, calcium signalling, cGMP-PKG signalling, and PI3K-Akt signalling based on the differentially expressed genes; the top-100 pathway enrichments were found between the control group and the group exposed to 5000 m altitude conditions for 4 w. The mRNA levels of 18 candidate gene showed that more than 83% of genes were expressed and the number of transcriptome The up-regulated genes were EPAS1, Hbα, iNOS, CX40, CD31, PPM1B, HIF-1α, MYLK, Pcdh12, Surfactant protein B, the down-regulated genes were RYR2, vWF, RASA1, CLASRP, HIF-3α. Our transcriptome data are a valuable resource for future genomic studies on plateau pika.

Large-scale preparative isolation of bergenin standard substance from Saxifraga atrata using polyamide coupled with MCI GEL® CHP20P as stationary phases in medium pressure chromatography.

In this study, polyamide and MCI GEL® CHP20P were employed as stationary phases in medium pressure chromatography (MPC) for the efficient preparative separation of bergenin from Saxifraga atrata. Ethanol-water, methanol-water, and acetonitrile-water mobile phases all showed good enrichment capacity for bergenin fraction when polyamide was used as a stationary phase. After 5 cycles of polyamide MPC using acetonitrile/water, 1.2 g of bergenin fraction was isolated from 180 g Saxifraga atrata herb. Further purification of this fraction was conducted using MCI GEL® CHP20P styrene-divinylbenzene beads. The bergenin fraction was separated into two fractions, and after three runs of MPC, 714.2 mg of bergenin with purity above 99% was obtained. The results demonstrate that the combination of polyamide and styrene-divinylbenzene MPC can be utilized for preparative isolation of compounds from natural products with high yield and purity.

Targeted isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata using medium- and high- pressure liquid chromatography combined with online high performance liquid chromatography-1,1-diphenyl-2- picrylhydrazyl detection.

Traditional Tibetan medicine (TTM) is a valuable source of novel therapeutic lead molecules inspired by natural products (NPs). The health benefits of Saxifraga atrata are well documented in TTM, but reports on its chemical composition are limited, most likely due to the complicated purification process. Herein, target separation and identification of 4 main radical scavenging compounds from the methanolic extract of S. atrata was were performed using medium- and high-pressure liquid chromatography coupled with online HPLC-DPPH detection. The sample was pretreated using medium pressure liquid chromatography with MCI GELⓇ CHP20P styrene-divinylbenzene beads as a stationary phase, yielding 1.4 g of the target DPPH inhibitors (Fr4, 11.9% recovery). The compounds were further purified and isolated using HPLC on RP-C18 (ReproSil-Pur C18 AQ) followed by HILIC (Click XIon) column separation, resulting in 2.8 mg of fraction Fr4-1-1, 6.8 mg of fraction Fr4-2, 244.9 mg of the Fr4-3-1 sample, and 38.3 mg of Fr4-4-1. The structure and purity of the target compounds were determined, and four compounds (ethyl gallate, 11-O-galloylbergenin, rutin and isoquercitrin) were isolated with >95% purity. The developed methodology is efficient for targeted isolation of high-purity radical scavengers from NP extracts and could be used for rapid identification and isolation of DPPH inhibitors from various NPs.

Weissella cibaria Attenuated LPS-Induced Dysfunction of Intestinal Epithelial Barrier in a Caco-2 Cell Monolayer Model.

The dysfunction of the intestinal epithelial barrier contributes to local or systemic infection and inflammation. Some lactic acid bacteria (LAB) strains had been shown to improve the conditions of barrier function and, for this reason, are recognized as probiotics. Weissella cibaria, a species belonging to the LAB group, is known to promote several health benefits. However, the role of W. cibaria in regulating the integrity of the intestinal epithelial barrier has not yet been investigated. In this study, W. cibaria MW01 was isolated from Chinese sauerkraut and was selected based on its functional features, such as gastric juice and bile salt tolerance, besides antagonistic activity against pathogenic bacteria. In a cellular model of the intestinal barrier, it was observed that W. cibaria was able to adhere more efficiently than Lactobacillus rhamnosus GG in Caco-2 cells. Moreover, the LPS-induced inflammation in Caco-2 cells was attenuated by the treatment with W. cibaria MW01, which reduced the synthesis of TNF-α, IL-6, and IL-8. In addition, it was noted that the treatment with W. cibaria MW01 recovered the integrity of the Caco-2 cell monolayer exposed to LPS. Furthermore, W. cibaria MW01 significantly alleviated LPS-induced downregulation of tight junction proteins (TJP) (claudin, occludin, and tight junction protein-1). Mechanistically, W. cibaria MW01 inhibited the translocation of NF-κB to the nucleus and deactivated the MLCK-pMLC pathway during LPS exposure. Thus, W. cibaria MW01, as a potential probiotic, can protect intestinal epithelial barrier function by regulating inflammation and expression of TJP via the NF-κB-mediated MLCK-pMLC pathway.

An efficient strategy based on liquid-liquid extraction and pH-zone-refining counter-current chromatography for selective enrichment, separation, and purification of alkaloids and organic Acids from natural products.

This study presents an efficient strategy based on liquid-liquid extraction and pH-zone-refining counter-current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two-phase solvent system with maximum or minimum partition coefficient was developed for the liquid-liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH-zone-refining counter-current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin-3-adipoyl-l-arginine, 42 mg of telocinobufagin-3-pimeloyl-l-arginine, 51 mg of telocinobufagin-3-suberoyl-l-arginine, 132 mg of marinobufagin-3-suberoyl-l-arginine, and 57 mg of bufalin-3-suberoyl-l-arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.

Preparative isolation of arylbutanoid-type phenol [(-)-rhododendrin] with peak tailing on conventional C18 column using middle chromatogram isolated gel column coupled with reversed-phase liquid chromatography.

Reversed-phase liquid chromatography coupled with middle chromatogram isolated gel column was employed for the efficient preparative separation of the arylbutanoid-type phenol [(-)-rhododendrin] from Saxifraga tangutica. Universal C18 (XTerra C18) and XCharge C18 columns were compared for (-)-rhododendrin fraction analysis and preparation. Although tailing and overloading occurred on the XTerra C18 column, the positively charged reversed-phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading at a on a preparative scale (3.69 mg per injection). The general separation process was as follows. First, 365.0 mg of crude (-)-rhododendrin was enriched from 165 g Saxifraga tangutica extract via a middle chromatogram isolated gel column. Second, separation was performed on an XTerra C18 preparative column, from which 73.8 mg of the target fraction was easily obtained. Finally, the 24.0 mg tailing peak of (-)-rhododendrin on XTerra C18 column was selectively purified on the XCharge C18 analytical column. These results demonstrate that the tailing nonalkaloid peaks can be effectively used for preparative isolation on XCharge C18 columns.

DNA barcodes and their characteristic diagnostic sites analysis of Schizothoracinae fishes in Qinghai province.

The Qinghai-Tibetan Plateau (QTP), the source and upper reaches of many Asian rivers, are crisscrossed by rivers and dotted with lakes. Schizothoracinae fishes, species native to the QTP, are distributed widely through these rivers and lakes. Over the past decades, ecological protection has become increasingly intense. The rapid acquisition of the genetic information and accurate gene sequence database are assumed to play an important role in the conservation of species diversity and biodiversity. In this study, 153 COI sequences (648bp in length) covering 13 species in 8 genera of Schizothoracinae fishes in Qinghai Province were used to determine whether barcode could identify Schizothoracinae species accurately. The average Kimura two parameter (K2P) genetic distances within and among species were 0.35% and 8.83%, respectively. The maximum K2P distance within species was observed in Gymnocypris eckloni (1.36%) while minimum K2P distance among species was observed between Chuanchia labiosa and Schizopygopsis pylzovi (0.23%). Overlaps existed in K2P distance intra- and inter- species based on both the genes. Eleven groups with 9 single-species groups and 2 multi-species groups were identified through Automatic Barcode Gap Discovery System, which were consistent with the overlaps of K2P distance. 96.7% as the accurate ratio for COI barcode was calculated and high solution was observed in the phylogenetic trees based on COI gene and Cyt b gene. Except for the similar results based on two genes above, COI barcode was more economical than Cyt b gene. The SOM model successfully predicted characteristic-diagnostic sites at species level: 36 characteristic-diagnostic sites from eight species, in which 12 from Gmnodiptychus pachycgeilus, 2 from Platypharodon extremus, 7 from Ptychobarbus kaznakovi, 2 from Schizopygopsis anteroventris, 2 from Schizopygopsis malacanthus, 3 from Schizopygopsis malacanthus chengi, 3 from Schizothorax dolichonema and 5 from Schizothorax lantsangensis. Our results show that Schizothoracinae fishes can be identified validly by using COI DNA barcode. Thirty-six characteristic-diagnostic sites were proposed to be applied into works of species identification for the Schizothoracinae fishes in Qinghai Province.

Testosterone-retinoic acid signaling directs spermatogonial differentiation and seasonal spermatogenesis in the Plateau pika (Ochotona curzoniae).

During evolution, animals optimize their reproductive strategies to increase offspring survival. Seasonal breeders reproduce only during certain times of the year. In mammals, reproduction is tightly controlled by hypothalamus-pituitary-gonad axis. Although pathways regulating gametogenesis in non-seasonal model species have been well established, molecular insights into seasonal reproduction are severely limited. Using the Plateau pika (Ochotona curzoniae), a small rodent animal species native to the Qinghai-Tibetan plateau, as a model, here we report that seasonal spermatogenesis is governed at the level of spermatogonial differentiation. In testis of the reproductively dormant animals, undifferentiated spermatogonia failed to differentiate and accumulated in the seminiferous tubules. RNA-seq analyses of the active and dormant testes revealed that genes modulating retinoic acid biogenesis and steriodogenesis were differentially regulated. A single injection of all-trans retinoic acid (ATRA) reinitiated spermatogenesis and inhibition the function of RA-degrading enzyme CYP26B1 for 10 days induced spermatogonial differentiation. Strikingly, testosterone injection reinitiated spermatogenesis in short day adapted animals. Testosterone provides a permissive environment of RA biogenesis and actions in testis, therefore, indirectly controls spermatogonial differentiation. Collectively, these findings provide a key mechanistic insight regarding the molecular regulation of seasonal reproduction in mammals.

PH-zone-refining counter-current chromatography with a hydrophilic organic/salt-containing two-phase solvent system for preparative separation of polar alkaloids from natural products.

This study presents an efficient strategy based on pH-zone-refining counter-current chromatography with a hydrophilic organic/salt-containing two-phase system composed of acetonitrile, sodium chloride and water for preparative separation of polar alkaloids from natural products. Acetonitrile-sodium chloride-water system provides a wider range of polarity for polar alkaloids than classical aqueous two-phase systems. It gets rid of the effect of free hydrogen ion, strong ionic strength, hold low viscosity and the sharp retainer border could be formed easily. So acetonitrile-sodium chloride-water system showed great advantages to pH-zone-refining counter-current chromatography for polar alkaloids. The separation of polar indole alkaloids from toad venom was selected as an example to show the advantage and practicability of this strategy. An optimized acetonitrile-sodium chloride-water (54%:5%:41%, w%) system was applied in this study, where 10 mM triethylamine (TEA) as the retainer and 15 mM hydrochloric acid (HCl) as the eluter were added. As a result, three polar indole alkaloids, including 19 mg of serotonin, 45 mg of 5-Hydroxy-N'-methyl tryptamine, 33 mg of bufotenine were simultaneously separated from 500 mg of 5% ethanol elution fraction of toad venom on macroporous resin chromatography, with the purity of 91.3%, 97.5% and 89.4%, respectively. Their structures were identified by spectroscopic analysis.

An Efficient Strategy Based on Liquid-Liquid Extraction with Three-Phase Solvent System and High Speed Counter-Current Chromatography for Rapid Enrichment and Separation of Epimers of Minor Bufadienolide from Toad Meat.

This study presents an efficient strategy based on liquid-liquid extraction with three-phase solvent system and high speed counter-current chromatography for rapid enrichment and separation of epimers of minor bufadienolide from toad meat. The reflux extraction conditions were optimized by response surface methodology first, and a novel three-phase solvent system composed of n-hexane/methyl acetate/acetonitrile/water (3:6:5:5, v/v) was developed for liquid-liquid extraction of the crude extract. This integrative extraction process could enrich minor bufadienolide from complex matrix efficiently and minimize the loss of minor targets induced by repeated extraction with different kinds of organic solvents occurring in the classical liquid two-phase extraction. As a result, four epimers of minor bufadienolide were greatly enriched in the middle phase and total content of these epimers of minor bufadienolide was increased from 3.25% to 46.23%. Then, the enriched four epimers were separated by HSCCC with a two-phase solvent system composed of chloroform/methanol/water (4:2:2, v/v) successfully. Furthermore, we tested Na+,K+-ATPase (NKA) inhibitory effect of the four epimers. 3ß-Isomers of bufadienolide showed stronger (>8-fold) inhibitory activity than 3α-isomers. The characterization of minor bufadienolide in toad meat and their significant difference of inhibitory effect on NKA would promote the further quantitative analysis and safety evaluation of toad meat as a food source.

Microsatellite Loci Analysis Reveals Post-bottleneck Recovery of Genetic Diversity in the Tibetan Antelope.

The Tibetan antelope (chiru, Pantholops hodgsoni) is one of the most endangered mammals native to the Qinghai-Tibetan Plateau. The population size has rapidly declined over the last century due to illegal hunting and habitat damage. In the past 10 years, the population has reportedly been expanding due to conservation efforts. Several lines of evidence suggest that the Tibetan antelope has undergone a demographic bottleneck. However, the consequences of the bottleneck on genetic diversity and the post-bottleneck genetic recovery remain unknown. In this study, we investigate the genetic variation of 15 microsatellite loci from two Tibetan antelope populations sampled in 2003 (Pop2003) and 2013 (Pop2013). A higher level of genetic diversity (NA, 13.286; He, 0.840; PIC, 0.813; I, 2.114) was detected in Pop2013, compared to Pop2003 (NA, 12.929; He, 0.818; PIC, 0.789; I, 2.033). We observe that despite passing through the bottleneck, the Tibetan antelope retains high levels of genetic diversity. Furthermore, our results show significant or near significant increases in genetic diversity (He, PIC and I) in Pop2013 compared with Pop2003, which suggests that protection efforts did not arrive too late for the Tibetan antelope.

The complete mitochondrial genome sequence of Platypharodon extremus (Cypriniformes: Cyprinidae).

Platypharodon extemus is a monotypic species of Schizothoracine fishes and it was listed as Endangered species in the "China Red Data Book (Pisces)", Vulnerable (V) by the National Environmental Protection Agency and Endangered Species Scientific Commission. So far, little mitochondrial genome information of this genus has been described. In this study, we obtained the complete mitochondrial DNA genome sequences of this species. The mitogenome was 16,668 bp in length, which consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 noncoding regions. The base composition of this mitochondrial genome was 28.6% A, 27.3% T, 18.2% G, 25.9% C, with a high A + T content (55.9%). The complete mitochondrial genome of P. extremus would be of great utility in the phylogenetic analysis of the schizothoracine fishes and also provide meritorious insights into the deeper problems of the phylogenic analysis.

Inhibition of inducible nitric oxide synthase expression and nitric oxide production in plateau pika (Ochotona curzoniae) at high altitude on Qinghai-Tibet Plateau.

Nitric oxide (NO), a potent vasodilator, plays an important role in preventing hypoxia induced pulmonary hypertension. Endogenous NO is synthesized by nitric oxide synthases (NOSs) from l-arginine. In mammals, three different NOSs have been identified, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Plateau pika (Ochotona curzoniae) is a typical hypoxia tolerant mammal that lives at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. The aim of this study was to investigate whether NOS expression and NO production are regulated by chronic hypoxia in plateau pika. Quantitative real-time PCR and western blot analyses were conducted to quantify relative abundances of iNOS and eNOS transcripts and proteins in the lung tissues of plateau pikas at different altitudes (4550, 3950 and 3200 m). Plasma NO metabolites, nitrite/nitrate (NO(x)⁻) levels were also examined by Ion chromatography to determine the correlation between NO production and altitude level. The results revealed that iNOS transcript levels were significantly lower in animals at high altitudes (decreased by 53% and 57% at altitude of 3950 and 4550 m compared with that at 3200 m). Similar trends in iNOS protein abundances were observed (26% and 41% at 3950 and 4550 m comparing with at 3200 m). There were no significant differences in eNOS mRNA and protein levels in the pika lungs among different altitudes. The plasma NO(x)⁻ levels of the plateau pikas at high altitudes significantly decreased (1.65±0.19 µg/mL at 3200 m to 0.44±0.03 µg/mL at 3950 m and 0.24±0.01 µg/mL at 4550 m). This is the first evidence describing the effects of chronic hypoxia on NOS expression and NO levels in the plateau pika in high altitude adaptation. We conclude that iNOS expression and NO production are suppressed at high altitudes, and the lower NO concentration at high altitudes may serve crucial roles for helping the plateau pika to survive at hypoxic environment.

Aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition/amino acid consensus site for membrane association.

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its ß2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

Resistin-like molecule α promotes pathogenic Th17 cell responses and bacterial-induced intestinal inflammation.

Resistin-like molecule (RELM)α belongs to a family of secreted mammalian proteins that have putative immunomodulatory functions. Recent studies have identified a pathogenic role for RELMα in chemically induced colitis through effects on innate cell populations. However, whether RELMα regulates intestinal adaptive immunity to enteric pathogens is unknown. In this study, we employed Citrobacter rodentium as a physiologic model of pathogenic Escherichia coli-induced diarrheal disease, colitis, and Th17 cell responses. In response to Citrobacter, RELMα expression was induced in intestinal epithelial cells, infiltrating macrophages, and eosinophils of the infected colons. Citrobacter-infected RELMα(-/-) mice exhibited reduced infection-induced intestinal inflammation, characterized by decreased leukocyte recruitment to the colons and reduced immune cell activation compared with wild-type (WT) mice. Interestingly, Citrobacter colonization and clearance were unaffected in RELMα(-/-) mice, suggesting that the immune stimulatory effects of RELMα following Citrobacter infection were pathologic rather than host-protective. Furthermore, infected RELMα(-/-) mice exhibited decreased CD4(+) T cell expression of the proinflammatory cytokine IL-17A. To directly test whether RELMα promoted Citrobacter-induced intestinal inflammation via IL-17A, infected WT and IL-17A(-/-) mice were treated with rRELMα. RELMα treatment of Citrobacter-infected WT mice exacerbated intestinal inflammation and IL-17A expression whereas IL-17A(-/-) mice were protected from RELMα-induced intestinal inflammation. Finally, infected RELMα(-/-) mice exhibited reduced levels of serum IL-23p19 compared with WT mice, and RELMα(-/-) peritoneal macrophages showed deficient IL-23p19 induction. Taken together, these data identify a proinflammatory role for RELMα in bacterial-induced colitis and suggest that the IL-23/Th17 axis is a critical mediator of RELMα-induced inflammation.