Protocol to evaluate rat and mouse cardiomyocyte proliferation in vitro and in vivo.

Here, we present a protocol for the quantitative assessment of rat and mouse cardiomyocyte proliferation both in vitro and in vivo. For the in vivo approach, we describe steps for the isolation of neonatal rat cardiomyocytes and the employment of various indicators to quantify cell proliferation. We then detail in vivo procedures that incorporate comprehensive assays and a genetic lineage tracing strategy to evaluate endogenous cardiomyocyte proliferation. This protocol can be modified to investigate other mammalian cardiomyocyte proliferation. For complete details on the use and execution of this protocol, please refer to Ji et al.1.

Oral pathobiont Klebsiella chaperon usher pili provide site-specific adaptation for the inflamed gut mucosa.

The ectopic gut colonization by orally derived pathobionts has been implicated in the pathogenesis of various gastrointestinal diseases, including inflammatory bowel disease (IBD). For example, gut colonization by orally derived Klebsiella spp. has been linked to IBD in mice and humans. However, the mechanisms whereby oral pathobionts colonize extra-oral niches, such as the gut mucosa, remain largely unknown. Here, we performed a high-density transposon (Tn) screening to identify genes required for the adaptation of an oral Klebsiella strain to different mucosal sites - the oral and gut mucosae - at the steady state and during inflammation. We find that K. aerogenes, an oral pathobiont associated with both oral and gut inflammation in mice, harbors a newly identified genomic locus named "locus of colonization in the inflamed gut (LIG)" that encodes genes related to iron acquisition (Sit and Chu) and host adhesion (chaperon usher pili [CUP] system). The LIG locus is highly conserved among K. aerogenes strains, and these genes are also present in several other Klebsiella species. The Tn screening revealed that the LIG locus is required for the adaptation of K. aerogenes in its ectopic niche. In particular, we determined K. aerogenes employs a CUP system (CUP1) present in the LIG locus for colonization in the inflamed gut, but not in the oral mucosa. Thus, oral pathobionts likely exploit distinct adaptation mechanisms in their ectopically colonized intestinal niche compared to their native niche.

Sphingolipid metabolism controls mammalian heart regeneration.

Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration. SphK2 is downregulated during heart development and determines CM proliferation via nuclear S1P-dependent modulation of histone acetylation. Reactivation of SphK2 induces adult CM cell-cycle re-entry and cytokinesis, thereby enhancing regeneration. Conversely, SphK1 is upregulated during development and promotes fibrosis through an S1P autocrine mechanism in cardiac fibroblasts. By fine-tuning the activity of each SphK isoform, we develop a therapy that simultaneously promotes myocardial repair and restricts fibrotic scarring to regenerate the infarcted adult hearts.

Association Between Dietary Antioxidant Quality Score (DAQS) and All-Cause Mortality in Hypertensive Adults: A Retrospective Cohort Study from the NHANES Database.

This study aimed to explore the association between the dietary antioxidant quality scores (DAQS) and all-cause mortality in hypertensive adults. In this retrospective cohort study, participants aged ≥ 18 years with hypertension were extracted from the National Health and Nutrition Examination Survey (NAHNES) 2007-2018. Outcome was all-cause mortality of hypertensive participants. DAQS was the exposure variable calculated based on the intake of vitamin A, C, E, zinc, selenium, and magnesium. The weighted univariable and multivariable COX proportional hazards regression models were utilized to explore the association between the DAQS and the all-cause mortality in hypertensive patients and were described as hazard ratios (HRs) and 95% confidence intervals (CIs). Subgroup analyses based on different age, gender, diabetes, and cardiovascular disease (CVD) history were further assessed this association. A total of 16,240 participants were finally included in this study. Until 12 December 2019, 2710 (16.69%) all-cause deaths were documented. After adjustment for confounding variables, high DAQS was associated with the lower all-cause mortality (HR = 0.83, 95%CI: 0.72-0.96) in hypertensive patients. Subgroup analyses suggested that the association between DAQS and the all-cause mortality in hypertensive patients remain robust, especially in patients with female (HR = 0.77, 95%CI: 0.63-0.95), aged ≥ 60 years (HR = 0.81, 95%CI: 0.69-0.96). High DAQS was associated with the lower odds of all-cause mortality in adults with hypertension and are a promising intervention to be further explored in hypertensive patients.

The role of vitamin D through SphK1/S1P in the regulation of MS progression.

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-ß, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-ß, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

The Effect of Resveratrol in Sirt1/CST Pathway to Inhibit TNF-α Induced Inflammatory Response in Rat Primary Fibroblast-Like Synoviocytes.

Rheumatoid arthritis has a significant impact on the life quality, but current pharmacological therapies have limitations. As a result, there is growing interest in exploring the potential of natural plant components to intervene in the development of rheumatoid arthritis. Resveratrol, a natural polyphenol and one of the main active components of the Chinese herbal medicine Polygonum cuspidatum, has emerged as a promising candidate for this purpose. In the present study, we investigated the role and mechanism of resveratrol in inhibiting inflammatory response in rat primary fibroblast-like synoviocytes. Tumor necrosis factor (TNF)-α was used to establish a model of inflammation, the Sirtuin1 selective inhibitor Selisistat (EX527) was used to inhibit Sirtuin1 activity, and small interfering RNA was used to silence cortistatin expression. The results showed that pre-treatment with resveratrol could time- and dose-dependently inhibit TNF-α induced cellular interleukin (IL)-1ß and IL-6 secretion, and upregulate Sirtuin1 and cortistatin mRNA and protein expression in the range of 48 h, 100 µM. Selisistat (EX527) could attenuate resveratrol inhibited inflammatory response and upregulated cortistatin expression. Silencing cortistatin expression attenuated the effect of resveratrol on inhibiting inflammatory response, but did not affect its effect on upregulating Sirtuin1 expression. In conclusion, resveratrol effectively inhibited the TNF-α induced inflammatory response in fibroblast-like synoviocytes by a mechanism involving the Sirtuin1/cortistatin pathway.

Age-related asymmetry in left-right ears of sound lateralization with respect to four different rise times.

An experimental investigation was conducted to elucidate the auditory characteristics of the older adult population. The study involved 24 older adult and 24 young participants, with the aim of exploring their horizontal lateralization ability. This was achieved by presenting 1-kHz pure tones to the participants' right and left ears while introducing interaural time differences (ITDs). We examined the impact of four rise times (2, 5, 20, and 50 ms) on the onset of the test sound. The findings revealed that older adult participants exhibited lower levels of lateralization than young participants. Moreover, both older adult and young participants demonstrated diminished recognition of the onset portion as the rise time increased. Of particular significance was the conspicuous presence of a right ear advantage (REA) among young participants as the rise time was extended (statistically significant between the left and right ears at the 1% level, considering an ITD of 0.8 ms and a rise time of 50 ms). In contrast, older adult participants did not exhibit REA, even with a prolonged rise time (not significant at the 5% level at the same condition). These results indicate that the REA is not only present in language, as previously observed, but also extends to a pure tone in young participants. The older adult participants exhibited reduced performance in both left-and right-ear sound recognition. The influence of hearing threshold and preferred ear on sound lateralization performance was minimal. Therefore, it can be inferred that factors other than hearing threshold or preferred ear contribute to the presence of REA in young participants or its decline with age. The central and/or corpus callosum functions may also contribute to this phenomenon.

Cysteine-Based Redox-Responsive Nanoparticles for Fibroblast-Targeted Drug Delivery in the Treatment of Myocardial Infarction.

Upon myocardial infarction (MI), activated cardiac fibroblasts (CFs) begin to remodel the myocardium, leading to cardiac fibrosis and even heart failure. No therapeutic approaches are currently available to prevent the development of MI-induced pathological fibrosis. Most pharmacological trials fail from poor local drug activity and side effects caused by systemic toxicity, largely due to the lack of a heart-targeted drug delivery system that is selective for activated CFs. Here, we developed a reduced glutathione (GSH)-responsive nanoparticle platform capable of targeted delivering of drugs to activated CFs within the infarct area of a post-MI heart. Compared with systemic drug administration, CF-targeted delivery of PF543, a sphingosine kinase 1 inhibitor identified in a high-throughput antifibrotic drug screening, had higher therapeutic efficacy and lower systemic toxicity in a MI mouse model. Our results provide a CF-targeted strategy to deliver therapeutic agents for pharmacological intervention of cardiac fibrosis.

Proteomic and metabolomic profiling of acupuncture for migraine reveals a correlative link via energy metabolism.

Migraine is a neurovascular disease with a high disability rate. Acupuncture treatment has emerged as a safe and viable alternative prophylactic therapy that can effectively alleviate the duration and frequency of migraine attacks. However, the therapeutic mechanisms underlying the effects of acupuncture are yet to be systematically elucidated. In this study, we enrolled female patients with migraine without aura (n = 20) and healthy controls (n = 10). Patients received acupuncture treatment on DU20, DU24, bilateral GB13, GB8, and GB20, applied three times per week over the course of 4 weeks for 12 sessions in total. Blood samples were collected from the median cubital vein before and after acupuncture treatment. Proteomic and metabolomic profiling was performed using liquid chromatography-mass spectrometry to determine the characteristics of differentially expressed molecules and expression of their corresponding biological pathways as well as to elucidate the pathogenesis of migraine and the biological effects underlying the treatment of migraine with acupuncture. Proteomic and metabolomic profiling of plasma samples from patients with migraine without aura before and after acupuncture treatment revealed enrichment of immune-related pathway functions and the arginine synthesis pathway. Joint pathway analyses revealed significant enrichment of the pentose phosphate and glycolysis/gluconeogenesis pathways in patients with migraine. The glycolysis/gluconeogenesis and riboflavin metabolism pathways were significantly enriched after acupuncture treatment. The expression levels of various key proteins and metabolites, including α-D-glucose, flavin adenine dinucleotide, biliverdin reductase B, and L-glutamate, were significantly differentially expressed before and after acupuncture treatment in patients with migraine without aura. Treatment of migraine with acupuncture was associated with significant changes in key molecules and pathways, indicative of physiological changes in the trigeminovascular system, glutamate neurotoxicity, and other migraine-related physiological changes. Overall, our comprehensive analysis using proteomic and metabolomic profiling demonstrates that energy metabolism may serve as a key correlative link in the occurrence of migraine and the therapeutic effects of acupuncture treatment. Our findings may facilitate the identification of diagnostic and therapeutic modalities in the ongoing search for effective treatments for migraine attacks.

Severity of Anemia During Pregnancy and Adverse Maternal and Fetal Outcomes.

Importance: Anemia is the most widespread nutritional deficiency among pregnant females in the world. Despite numerous studies on anemia, evidence is limited about the association of severity of anemia with maternal and fetal health. Objective: To investigate the association between severity of anemia during pregnancy and risk of maternal and fetal adverse outcomes. Design, Setting, and Participants: This retrospective cohort study used data from China's Hospital Quality Monitoring System from January 1, 2016, to December 31, 2019, for pregnant females aged 15 to 49 years with birth outcomes reported at 1508 hospitals with maternity services in mainland China. Exposures: Anemia of varying severity during pregnancy was identified from daily standardized electronic inpatient discharge records using corresponding codes of the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision. Mild anemia was defined as a hemoglobin concentration of 100 to 109 g/L (to convert g/L to g/dL, divide by 10.0); moderate anemia, as 70 to 99 g/L; and severe anemia, as less than 70 g/L. Main Outcomes and Measures: The main outcomes included 6 maternal outcomes (placental abruption, preterm birth, severe postpartum hemorrhage, shock, admission to the intensive care unit [ICU], and maternal mortality) and 3 neonatal outcomes (fetal growth restriction, malformation, and stillbirth). Multivariable logistic regression models were used to estimate the odds ratios (ORs) and 95% CIs of these outcomes among pregnant females with varying severity of anemia. Results: Among 18 948 443 pregnant females aged 15 to 49 years (mean [SD] age, 29.42 [4.87] years), 17.78% were diagnosed with anemia during pregnancy, including 9.04% with mild anemia, 2.62% with moderate anemia, 0.21% with severe anemia, and 5.90% with anemia of unknown severity. Compared with no anemia, anemia severity during pregnancy was associated with increased risks of placental abruption (mild: adjusted OR [aOR], 1.36 [95% CI, 1.34-1.38]; moderate: aOR, 1.98 [95% CI, 1.93-2.02]; severe: aOR, 3.35 [95% CI, 3.17-3.54]), preterm birth (mild: aOR, 1.08 [95% CI, 1.07-1.08]; moderate: aOR, 1.18 [95% CI, 1.17-1.19]; severe: aOR, 1.36 [95% CI, 1.32-1.41]), severe postpartum hemorrhage (mild: aOR, 1.45 [95% CI, 1.43-1.47]; moderate: aOR, 3.53 [95% CI, 3.47-3.60]; severe: 15.65 [95% CI, 15.10-16.22]), and fetal malformation (mild: aOR, 1.15 [95% CI, 1.14-1.17]; moderate: aOR, 1.19 [95% CI, 1.16-1.21]; severe: aOR, 1.62 [95% CI, 1.52-1.73]). Compared with no anemia, moderate or severe anemia were associated with increased risks of maternal shock (moderate: aOR, 1.50 [95% CI, 1.41-1.60]; severe: aOR, 14.98 [95% CI, 13.91-16.13]), ICU admission (moderate: aOR, 1.08 [95% CI, 1.01-1.16]; severe: aOR, 2.88 [95% CI, 2.55-3.25]), maternal death (moderate: aOR, 0.45 [95% CI, 0.30-0.65]; severe: aOR, 1.56 [95% CI, 0.97-2.48], fetal growth restriction (moderate: aOR, 0.80 [95% CI, 0.78-0.82]; severe: aOR, 1.08 [95% CI, 1.00-1.17]), and stillbirth (moderate: aOR,0.79 [95% CI, 0.76-0.81]; severe: aOR, 1.86 [95% CI, 1.75-1.98]), and mild anemia was associated with decreased risks (maternal shock: aOR, 0.67 [95% CI, 0.63-0.71]; ICU admission: aOR, 0.80 [95% CI, 0.76-0.84]; maternal death: aOR, 0.37 [95% CI, 0.29-0.49]; fetal growth restriction: aOR, 0.79 [95% CI, 0.77-0.80]; stillbirth: aOR, 0.59 [95% CI, 0.58-0.61]) after adjusting for sociodemographic characteristics and other complications during pregnancy. Conclusions and Relevance: The findings suggest that anemia during pregnancy is associated with maternal and fetal health outcomes and that mild anemia is associated with improved maternal and fetal survival and fetal growth. Further work is needed to validate the concentration of hemoglobin at which optimal maternal and fetal health are achieved.

Arginine deprivation alters microglial polarity and synergizes with radiation to eradicate non-arginine-auxotrophic glioblastoma tumors.

New approaches for the management of glioblastoma (GBM) are an urgent and unmet clinical need. Here, we illustrate that the efficacy of radiotherapy for GBM is strikingly potentiated by concomitant therapy with the arginine-depleting agent ADI-PEG20 in a non-arginine-auxotrophic cellular background (argininosuccinate synthetase 1 positive). Moreover, this combination led to durable and complete radiological and pathological response, with extended disease-free survival in an orthotopic immune-competent model of GBM, with no significant toxicity. ADI-PEG20 not only enhanced the cellular sensitivity of argininosuccinate synthetase 1-positive GBM to ionizing radiation by elevated production of nitric oxide (˙NO) and hence generation of cytotoxic peroxynitrites, but also promoted glioma-associated macrophage/microglial infiltration into tumors and turned their classical antiinflammatory (protumor) phenotype into a proinflammatory (antitumor) phenotype. Our results provide an effective, well-tolerated, and simple strategy to improve GBM treatment that merits consideration for early evaluation in clinical trials.

Interaction between the inflammasome and commensal microorganisms in gastrointestinal health and disease.

The inflammasome is a cytosolic multiprotein complex that plays a crucial role in inflammation and cell death. The sensor proteins in the inflammasome complex detect various microbial and endogenous stimuli, leading to subsequent caspase activation. The activation of caspases results in the maturation of pro-inflammatory cytokines IL-1ß and IL-18 or pyroptosis. Inflammasome dysfunction is associated with the pathogenesis of various diseases, including autoimmune disease and cancer. It appears that the interactions between the gut microbiota and the inflammasome play crucial roles in the gastrointestinal tract. The gut microbiota induces the expression and activation of inflammasome proteins, which contribute to both homeostasis and disease in the gut. Likewise, although controversial, mounting evidence suggests that inflammasome activation can modulate the composition of the gut microbiota, which, in turn, affects disease progression. In this review, we summarize the current concepts and recent insights linking the inflammasome and gut commensal microorganisms. We describe how the reciprocal interaction between the inflammasome and the commensal microbiota relates to physiological and pathophysiological consequences in the host.

Quantification of Intact O-Glycopeptides on Haptoglobin in Sera of Patients With Hepatocellular Carcinoma and Liver Cirrhosis.

Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC. Furthermore, label-free quantification based on MS was performed. A total of 26 intact O-glycopeptides were identified on Hp, and most of them were elevated in HCC as compared to LC. Among them, the intensity of HYEGS 316TVPEK (H1N1S1) on Hp was the highest in HCC patients. Increased HYEGS 316TVPEK (H1N1S1) in HCC was quantified and confirmed using the MS method based on 18O/16O C-terminal labeling and multiple reaction monitoring. This study provided a comprehensive understanding of the glycosylation of Hp in liver diseases.

PVAT targets VSMCs to regulate vascular remodelling: angel or demon.

Vascular remodelling refers to abnormal changes in the structure and function of blood vessel walls caused by injury, and is the main pathological basis of cardiovascular diseases such as atherosclerosis, hypertension, and pulmonary hypertension. Among them, the neointimal hyperplasia caused by abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of vascular remodelling. Perivascular adipose tissue (PVAT) can release vasoactive substances to target VSMCs and regulate the pathological process of vascular remodelling. Specifically, PVAT can promote the conversion of VSMCs phenotype from contraction to synthesis by secreting visfatin, leptin, and resistin, and participate in the development of vascular remodelling-related diseases. Conversely, it can also inhibit the growth of VSMCs by secreting adiponectin and omentin to prevent neointimal hyperplasia and alleviate vascular remodelling. Therefore, exploring and developing new drugs or other treatments that facilitate the beneficial effects of PVAT on VSMCs is a potential strategy for prevention or treatment of vascular remodelling-related cardiovascular diseases.

Microbial adaptation to the healthy and inflamed gut environments.

There are 100 trillion diverse bacterial residents in the mammalian gut. Commensal bacterial species/strains cooperate and compete with each other to establish a well-balanced community, crucial for the maintenance of host health. Pathogenic bacteria hijack cooperative mechanisms or use strategies to evade competitive mechanisms to establish infection. Moreover, pathogenic bacteria cause marked environmental changes in the gut, such as the induction of inflammation, which fosters the selective growth of pathogens. In this review, we summarize the latest findings concerning the mechanisms by which commensal bacterial species/strains colonize the gut through cooperative or competitive behaviors. We also review the mechanisms by which pathogenic bacteria adapt to the inflamed gut and thrive at the expense of commensal bacteria. The understanding of bacterial adaptation to the healthy and the inflamed gut may provide new bacteria-targeted therapeutic approaches that selectively promote the expansion of beneficial commensal bacteria or limit the growth of pathogenic bacteria.

Study on the Relationship Between Microbial Composition and Living Environment in Important Medical Mites Based on Illumina MiSeq Sequencing Technology.

The microbiota of mites is closely related to their growth, development, and pathogenicity. Therefore, it is necessary to study the bacteria in mites. Here, for the first time, based on 16s rRNA V3-V4 region, the microbiota of 45 samples of nine species in six families of medically important mites were analyzed using Illumina MiSeq sequencing technique. The results showed that, at the phylum level, Proteobacteria (56.20-86.40%) were the dominant, followed by Firmicutes (6.41-19.43%), Bacteroidetes (5.56-13.38%) and Actinobacteria (1.93-28.07%). But at the genera the microbiota of mites are different, showing four characteristics: 1) The microbiota is related to the parasitic host. Demodex folliculorum (Acariforms: Demodicidae) and D. brevis (Acariforms: Demodicidae), both parasitizing humans, showed similar microbial composition, as did D. canis (Acariforms: Demodicidae) and Sarcoptes scabiei canis (Acariforms: Sarcoptidae) parasitizing dogs, but D. caprae (Acariforms: Demodicidae) parasitizing sheep showed unique microbial community; 2) The microbiota is related to mite's species. Dermatophagoides farinae and Cheyletus malaccensis (Acariforms: Cheyletidae), both collecting from flour, show respective microbial composition; 3) The microbiota is related to the life stage. There were differences in microbiota between adults and larvae of D. farinae, but no differences observed in Psoroptes cuniculi (Acariforms: Psoroptidae); and 4) The microbiota is related to the blood-feeding state. The microbiota of blood-fed Ornithonyssus bacoti (Parasitiformes: Macronyssidae) adults was significantly higher than that of unfed adults. This indicates that the microbiota of mites is affected by mite species, parasitic host, growth stage and habitat. Therefore, understanding these influencing factors will have a very important guiding significance for the prevention and control of mite-borne diseases.

Decreased Expression of CHST-12, CHST-13, and UST in the Proximal Interphalangeal Joint Cartilage of School-Age Children with Kashin-Beck Disease: an Endemic Osteoarthritis in China Caused by Selenium Deficiency.

The objective of this study is to investigate changes in the expression of enzymes involved in chondroitin sulfate (CS) sulfation in distal articular surface of proximal interphalangeal joint isolated from school-age children patients with Kashin-Beck disease (KBD), using normal children as controls. Articular cartilage samples were collected from four normal and four KBD children (7-12 years old), and these children were assigned to control and KBD groups. Hematoxylin and eosin (H&E), toluidine blue (TB), and immunohistochemical (IHC) stainings were utilized to evaluate changes in joint pathology and expression of enzymes involved in CS sulfation, including carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), and uronyl 2-O-sulfotransferase (UST). The correspondence results were examined by semi-quantitative analysis. Compared with the control group, the KBD group showed the following: a significant decrease of total chondrocytes in superficial, middle, and deep layers and deposition of sulfated glycosaminoglycans in extracellular matrix of KBD cartilage were observed; positive staining chondrocytes of CHST-12, CHST-13, and UST were significantly less in superficial zone of KBD cartilage; and CHST-13 positive staining chondrocytes was reduced in deep zone of KBD cartilage. In contrast, the positive staining rates of CHST-12, CHST-13, and UST in KBD were significantly higher than those in the control group. The decreased expression of these enzymes and the physiologic compensatory reaction may be the signs of early-stage KBD. The alterations of CS structure modifying sulfotransferases in finger articular cartilage might play an important role in the onset and pathogenesis of school-age KBD children.

A bovine myeloid antimicrobial peptide (BMAP-28) and its analogs kill pan-drug-resistant Acinetobacter baumannii by interacting with outer membrane protein A (OmpA).

Antimicrobial peptides (AMPs) exhibit multiple activities against bacteria and fungi. A bovine myeloid antimicrobial peptide (BMAP-28) belongs to the cathelicidin-derived AMPs and has antimicrobial activity. Due to the rapidly increasing number of infections and outbreaks caused by pan-drug-resistant Acinetobacter baumannii (PDRAB), we sought to determine whether BMAP-28 and its 4 analog peptides (A837, A838, A839, and A840) have antimicrobial activity against PDRAB. Furthermore, we clarified the possible mechanism of inhibition by which of BMAP-28 acts against PDRAB. In the current study, we examined the inhibitory effect of BMAP-28 and its 4 analog peptides on the growth of PDRAB through minimal inhibitory concentration (MIC) analysis and short time killing assays. We also evaluated the effects of BMAP-28 and its analogs on the bacterial cell surface through the use of field emission scanning electron microscopy (FE-SEM). In order to determine the inhibitory mechanism of BMAP-28, we examined the interaction between BMAP-28 and outer membrane proteins (OMPs), especially the interaction between BMAP-28 and A. baumannii OmpA (AbOmpA), which is the main component of OMPs, by using a quartz crystal microbalance (QCM). BMAP-28 and its 4 analogs were effective in inhibiting the growth of PDRAB and had rapid killing ability. BMAP-28 showed exceptionally strong and rapid inhibitory effects on PDRAB when compared to the other peptides and was also shown to cause damage to the cell surface of PDRAB. Moreover, QCM analysis provided evidence of potential interaction between BMAP-28 and AbOmpA. These data indicate that BMAP-28 is a promising candidate for the treatment of PDRAB infections and that its inhibitory effects were related with its binding to AbOmpA.

Red nucleus interleukin-1ß evokes tactile allodynia through activation of JAK/STAT3 and JNK signaling pathways.

We previously reported that interleukin-1ß (IL-1ß) in the red nucleus (RN) is involved in pain modulation and exerts a facilitatory effect in the development of neuropathic pain. Here, we explored the actions of signaling pathways, including the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB (NF-κB) pathways, on RN IL-1ß-mediated pain modulation. After a single dose of recombinant rat IL-1ß (rrIL-1ß, 10 ng) injected into the RN in normal rats, a tactile allodynia was evoked in the contralateral but not ipsilateral hindpaw, commencing 75 min and peaking 120 min postinjection. Up-regulated protein levels of phospho-STAT3 (p-STAT3) and p-JNK were observed in the RN 120 min after rrIL-1ß injection, the increases of p-STAT3 and p-JNK were blocked by anti-IL-1ß antibody. However, the expression levels of p-ERK, p-p38 MAPK, and NF-κB in the RN were not affected by rrIL-1ß injection. RN neurons and astrocytes contributed to IL-1ß-evoked up-regulation of p-STAT3 and p-JNK. Further studies demonstrated that injection of the JAK2 antagonist AG490 or JNK antagonist SP600125 into the RN 30 min prior to the administration of rrIL-1ß could completely prevent IL-1ß-evoked tactile allodynia, while injection of the ERK antagonist PD98059, p38 MAPK antagonist SB203580, or NF-κB antagonist PDTC did not affect IL-1ß-evoked tactile allodynia. In conclusion, our data provide additional evidence that RN IL-1ß is involved in pain modulation, and that it exerts a facilitatory effect by activating the JAK/STAT3 and JNK signaling pathways.

Red nucleus interleukin-6 participates in the maintenance of neuropathic pain through JAK/STAT3 and ERK signaling pathways.

We previously reported that interleukin-6 (IL-6) in the red nucleus (RN) is up-regulated at 3weeks after spared nerve injury (SNI), and plays facilitated role in the later maintenance of neuropathic pain. The current study aimed to reveal the roles of different signaling pathways, including Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase/protein kinase B (PI3K/AKT), in RN IL-6-mediated pain modulation. In accord with the increase of IL-6 in the RN following SNI, the protein levels of phospho-STAT3 (p-STAT3), p-ERK and p-JNK were also up-regulated in the RN contralateral to the nerve injury side at 3weeks after SNI. The increases of p-STAT3 and p-ERK (but not p-JNK) were associated with IL-6 and could be blocked by anti-IL-6 antibody. Microinjection of JAK2 inhibitor AG490, ERK inhibitor PD98059 and also JNK inhibitor SP600125 into the RN significantly increased the paw withdrawal threshold (PWT) and alleviated SNI-induced mechanical allodynia. Further studies showed that microinjection of recombinant rat IL-6 (rrIL-6, 20ng) into the RN of normal rats significantly decreased the PWT of rats and increased the local protein levels of p-STAT3 and p-ERK, but not p-JNK. Pre-treatment with AG490 and PD98059 could prevent IL-6-induced mechanical allodynia. Whereas, p-p38 MAPK and p-AKT did not show any expression changes in the RN of rats with SNI or rats treated with rrIL-6. These results suggest that RN IL-6 participates in the later maintenance of SNI-induced neuropathic pain and plays facilitated role through activating JAK/STAT3 and ERK signaling pathways.