Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Relationships of Late Pleistocene giant deer as revealed by Sinomegaceros mitogenomes from East Asia.

The giant deer, widespread in northern Eurasia during the Late Pleistocene, have been classified as western Megaloceros and eastern Sinomegaceros through morphological studies. While Megaloceros's evolutionary history has been unveiled through mitogenomes, Sinomegaceros remains molecularly unexplored. Herein, we generated mitogenomes of giant deer from East Asia. We find that, in contrast to the morphological differences between Megaloceros and Sinomegaceros, they are mixed in the mitochondrial phylogeny, and Siberian specimens suggest a range contact or overlap between these two groups. Meanwhile, one deep divergent clade and another surviving until 20.1 thousand years ago (ka) were detected in northeastern China, the latter implying this area as a potential refugium during the Last Glacial Maximum (LGM). Moreover, stable isotope analyses indicate correlations between climate-introduced vegetation changes and giant deer extinction. Our study demonstrates the genetic relationship between eastern and western giant deer and explores the promoters of their extirpation in northern East Asia.

Full-profile pharmacokinetics, anticancer activity and toxicity of an extended release trivalent PEGylated irinotecan prodrug.

Irinotecan is an anticancer topoisomerase I inhibitor that acts as a prodrug of the active metabolite, SN-38. Unfortunately, the limited utility of irinotecan is attributed to its pH-dependent stability, short half-life and dose-limiting toxicity. To address this problem, a novel trivalent PEGylated prodrug (PEG-[Irinotecan]3) has been synthesized and its full-profile pharmacokinetics, antitumor activity and toxicity compared with those of irinotecan. The results show that after intravenous administration to rats, PEG-[Irinotecan]3 undergoes stepwise loss of irinotecan to form PEG-[Irinotecan]3‒x (x = 1,2) and PEG-[linker] during which time the released irinotecan undergoes conversion to SN-38. As compared with conventional irinotecan, PEG-[Irinotecan]3 displays extended release of irinotecan and efficient formation of SN-38 with significantly improved AUC and half-life. In a colorectal cancer-bearing model in nude mice, the tumor concentrations of irinotecan and SN-38 produced by PEG-[Irinotecan]3 were respectively 86.2 and 2293 times higher at 48 h than produced by irinotecan. In summary, PEG-[Irinotecan]3 displays superior pharmacokinetic characteristics and antitumor activity with lower toxicity than irinotecan. This supports the view that PEG-[Irinotecan]3 is a superior anticancer drug to irinotecan and it has entered the phase II trial stage.

Ancient mitogenomes reveal a high maternal genetic diversity of Pleistocene woolly rhinoceros in Northern China.

BACKGROUND: Woolly rhinoceros (Coelodonta antiquitatis) is a typical indicator of cold-stage climate that was widely distributed in Northern Hemisphere during the Middle-Late Pleistocene. Although a plethora of fossils have been excavated from Northern China, their phylogenetic status, intraspecific diversity and phylogeographical structure are still vague. RESULTS: In the present study, we generated four mitogenomes from Late Pleistocene woolly rhinoceros in Northern China and compared them with published data. Bayesian and network analyses indicate that the analyzed individuals contain at least four maternal haplogroups, and Chinese samples fall in three of them. One of our samples belongs to a previously unidentified early diverging clade (haplogroup D), which separated from other woolly rhinoceros around 0.57 Ma (95% CI: 0.76-0.41 Ma). The timing of this clade's origin coincides with the first occurrence of woolly rhinoceros, which are thought to have evolved in Europe. Our other three samples cluster in haplogroup C, previously only identified from one specimen from Wrangel Island (ND030) and initially considered to be an isolated clade. Herein, our findings suggest that ND030 is likely descended from a northward dispersal of the individuals carrying haplogroup C from Northern China. Additionally, Chinese woolly rhinoceros specimens exhibit higher nucleotide diversity than those from Siberia. CONCLUSION: Our findings highlight Northern China as a possible refugium and a key evolution center of the Pleistocene woolly rhinoceros.

A genetic glimpse of the Chinese straight-tusked elephants.

Straight-tusked elephants (genus: Palaeoloxodon) including their island dwarf forms are extinct enigmatic members of the Pleistocene megafauna and the most common Pleistocene elephants after the mammoths. Their taxonomic placement has been revised several times. Using palaeogenomic evidence, previous studies suggested that the European P. antiquus has a hybrid origin, but no molecular data have been retrieved from their Asian counterparts, leaving a gap in our knowledge of the global phylogeography and population dynamics of Palaeoloxodon. Here, we captured a high-quality complete mitogenome from a Pleistocene Elephantidae molar (CADG841) from Northern China, which was previously morphologically assigned to the genus Elephas (Asian elephant), and partial mitochondrial sequences (838 bp) of another Palaeoloxodon sp. specimen (CADG1074) from Northeastern China. We found that both Chinese specimens cluster with a 244 000-year-old P. antiquus (specimen name: WE) from Western Europe, suggesting that this clade may represent a population with a large spatial span across Eurasia. Based on the fossil record and the molecular dating of both the divergences of different Palaeoloxodon mitochondrial clades and previously determined hybridization events, we propose that this Eurasian-wide WE clade provides evidence for an earlier migration and/or another hybridization event that happened in the evolutionary history of straight-tusked elephants.

Ancient Mitogenomes Reveal Stable Genetic Continuity of the Holocene Serows.

As one of the remaining species of Caprinae only found in Asia, serows (Capricornis) and their classification and conservation have received increasing attention in recent years. However, their evolutionary history and population dynamics are not yet clear. To shed light on these topics, we report the first near-complete ancient mitochondrial genomes from two serow sub-fossils (CADG839 and CADG946) dating to 8860 ± 30 years and 2450 ± 30 years, and incorporate the newly obtained mitogenomes into the dataset of living serows (18 complete mitochondrial genomes drawn from National Center for Biotechnology Information, NCBI) to investigate their relationships and evolution. Phylogenetic results support four clades of serows that can be further divided into five subclades, indicating higher genetic diversity than previously thought. Notably, our two ancient samples do not form a separate branch but belong to Capricornis sumatraensis clade A together with modern individuals, which suggests genetic continuity between ancient and modern serows. Furthermore, our results suggest that the maternal divergences of serows occurred at the beginning of the Pleistocene. Bayesian estimation indicates that the first divergence among all serows happened approximately 2.37 Ma (95% highest posterior density, HPD: 2.74-2.02 Ma) when Japanese serow (Capricornis crispus) appeared, while the last divergence occurred within the Sumatran serow (C. sumatraensis clade A and B) around 0.37-0.25 Ma. Additionally, we found the effective maternal population size of C. sumatraensis increased around 225-160 and 90-50 ka, then remained stable since 50 ka. Overall, our study provides new insights into serow phylogeny and evolutionary history.

Colonic Epithelial Circadian Disruption Worsens Dextran Sulfate Sodium-Induced Colitis.

BACKGROUND: Disruption of central circadian rhythms likely mediated by changes in microbiota and a decrease in gut-derived metabolites like short chain fatty acids (SCFAs) negatively impacts colonic barrier homeostasis. We aimed to explore the effects of isolated peripheral colonic circadian disruption on the colonic barrier in a mouse model of colitis and explore the mechanisms, including intestinal microbiota community structure and function. METHODS: Colon epithelial cell circadian rhythms were conditionally genetically disrupted in mice: TS4Cre-BMAL1lox (cBMAL1KO) with TS4Cre as control animals. Colitis was induced through 5 days of 2% dextran sulfate sodium (DSS). Disease activity index and intestinal barrier were assessed, as were fecal microbiota and metabolites. RESULTS: Colitis symptoms were worse in mice with peripheral circadian disruption (cBMAL1KO). Specifically, the disease activity index and intestinal permeability were significantly higher in circadian-disrupted mice compared with control animals (TS4Cre) (P < .05). The worsening of colitis appears to be mediated, in part, through JAK (Janus kinase)-mediated STAT3 (signal transducer and activator of transcription 3), which was significantly elevated in circadian-disrupted (cBMAL1KO) mice treated with DSS (P < .05). Circadian-disrupted (cBMAL1KO) mice also had decreased SCFA metabolite concentrations and decreased relative abundances of SCFA-producing bacteria in their stool when compared with control animals (TS4Cre). CONCLUSIONS: Disruption of intestinal circadian rhythms in colonic epithelial cells promoted more severe colitis, increased inflammatory mediators (STAT3 [signal transducer and activator of transcription 3]), and decreased gut microbiota-derived SCFAs compared with DSS alone. Further investigation elucidating the molecular mechanisms behind these findings could provide novel circadian directed targets and strategies in the treatment of inflammatory bowel disease.

Disruption of peripheral circadian rhythms of the colon epithelium results in worse colitis and increased intestinal permeability in mice when given dextran sulfate sodium. This may be mediated through alterations in microbiota, butyrate levels, and STAT3.

An extinct and deeply divergent tiger lineage from northeastern China recognized through palaeogenomics.

Tigers (Panthera tigris) are flagship big cats and attract extensive public attention due to their charismatic features and endangered status. Despite this, little is known about their prehistoric lineages and detailed evolutionary histories. Through palaeogenomic analyses, we identified a Pleistocene tiger from northeastern China, dated to beyond the limits of radiocarbon dating (greater than 43 500 years ago). We used a simulated dataset and different reads processing pipelines to test the validity of our results and confirmed that, in both mitochondrial and nuclear phylogenies, this ancient individual belongs to a previously unknown lineage that diverged prior to modern tiger diversification. Based on the mitochondrial genome, the divergence time of this ancient lineage was estimated to be approximately 268 ka (95% CI: 187-353 ka), doubling the known age of tigers' maternal ancestor to around 125 ka (95% CI: 88-168 ka). Furthermore, by combining our findings with putative mechanisms underlying the discordant mito-nuclear phylogenetic placement for the South China tigers, we proposed a more complex scenario of tiger evolution that would otherwise be missed using data from modern tigers only. Our study provides the first glimpses of the genetic antiquity of tigers and demonstrates the utility of aDNA-based investigation for further understanding tiger evolution.

Ancient Mitogenomes Provide New Insights into the Origin and Early Introduction of Chinese Domestic Donkeys.

Both molecular data and archaeological evidence strongly support an African origin for the domestic donkey. Recent genetic studies further suggest that there were two distinct maternal lineages involved in its initial domestication. However, the exact introduction time and the dispersal process of domestic donkeys into ancient China are still unresolved. To address these questions, we retrieved three near-complete mitochondrial genomes from donkey specimens excavated from Gaoling County, Shaanxi Province, and Linxia Basin, Gansu Province, China, dated at 2,349-2,301, 469-311, and 2,160-2,004 cal. BP, respectively. Maximum-likelihood and Bayesian phylogenetic analyses reveal that the two older samples fall into the two different main lineages (i.e., clade Ⅰ and clade Ⅱ) of the domestic donkey, suggesting that the two donkey maternal lineages had been introduced into Midwestern China at least at the opening of Silk Road (approximately the first century BC). Bayesian analysis shows that the split of the two donkey maternal lineages is dated at 0.323 Ma (95% CI: 0.583-0.191 Ma) using root-tip dating calibrations based on near-complete mitogenomes, supporting the hypothesis that modern domestic donkeys go back to at least two independent domestication events. Moreover, Bayesian skyline plot analyses indicate an apparent female population increase between 5,000 and 2,500 years ago for clade I followed by a stable population size to the present day. In contrast, clade II keeps a relatively stable population size over the past 5,000 years. Overall, our study provides new insights into the early domestication history of Chinese domestic donkeys.

Experimental investigation of femtosecond laser through-hole drilling of stainless steel with and without transverse magnetic assistance.

An experimental investigation of femtosecond laser through-hole drilling of stainless-steel 304 with and without transverse magnetic assistance was conducted. The characteristics of the through-hole geometry and sidewall as well as the chemical composition of the through-hole sidewall surface were analyzed. In addition, a theoretical analysis of magnetic-field-assisted femtosecond laser through-hole drilling is proposed. The results showed that transverse magnetic assistance could improve both the femtosecond laser through-hole drilling quality (through-hole geometry and sidewall characteristics) and efficiency. The primary reason is that transverse magnetic assistance changes the distribution of plasma and reduces the plasma density, which weakens the shielding effect of the plasma. However, compared with nanosecond laser drilling, the effect of the magnetic field on the femtosecond laser through-hole drilling was not obvious. A noticeable thermal effect appeared near the through-hole entrance at a pulse repetition rate of 500 kHz, and a heat affected zone and oxidation zone were produced, which is disadvantageous to laser drilling. This research has good prospects for industrial applications.

Parental exposure to perfluorobutane sulfonate disturbs the transfer of maternal transcripts and offspring embryonic development in zebrafish.

Parental exposure to perfluorobutane sulfonate (PFBS), an aquatic pollutant of emerging concern, is previously found to impair the embryonic development of offspring. However, the impairing mechanisms remain to clarify. In the present study, adult zebrafish were exposed to 0, 10 and 100 µg/L PFBS for 28 d, after which disturbances in maternal transcript transfer and offspring embryogenesis were investigated. Prior to zygotic genome activation, high-throughput transcriptomic sequencing revealed that parental PFBS exposure significantly altered the transcript profile of maternal origin in offspring eggs, while toxic actions varied as a function of PFBS concentrations. In offspring eggs derived from 10 µg/L exposure group, differential transcripts were mainly associated with the histone-DNA interaction of nucleosome, which would modify the compacted chromatin configuration and accessibility of transcriptional factors to DNA sequences. In this regard, the timing of zygotic genome activation was presumably disrupted. Parental exposure to 100 µg/L PFBS primarily interrupted the maternal transfer of adherens junction transcripts, which was supposed to dysregulate the cell-cell adhesion during early embryo formation. Development and growth of offspring embryos were significantly compromised by parental PFBS exposure, as exemplified by higher mortality, delayed hatching, slower heart rate, reduced body weight and neurobehavioral disorders. Overall, the present study presented the first toxicological evidence about the disturbances of PFBS in maternal transcript transfer, although the inherent linkage between maternal transcript modifications and offspring development defects still needs future works to construct.

Dietary administration of probiotic Lactobacillus rhamnosus modulates the neurological toxicities of perfluorobutanesulfonate in zebrafish.

Perfluorobutanesulfonate (PFBS), an aquatic pollutant of emerging concern, is found to disturb the neural signaling along gut-brain axis, whereas probiotic additives have been applied to improve neuroendocrine function of teleosts. Both PFBS and probiotics can commonly target nervous system. However, whether and how probiotic bacteria can modulate the neurotoxicities of PFBS remain not explored. It is thus necessary to elucidate the probiotic modulation of PFBS neurotoxicity, which can provide implications to the application of probiotic bacteria in aquaculture industry. In the present study, adult zebrafish were exposed to 0, 10 and 100 µg/L PFBS with or without dietary administration of probiotic Lactobacillus rhamnosus. Interaction between PFBS and probiotic along gut-brain axis was examined, covering three dominant pathways (i.e., neurotransmission, immune response and hypothalamic-pituitary-adrenal (HPA) axis). The results showed that, compared to the single effects, PFBS and probiotic coexposure significantly altered the acetylcholinesterase activity and neurotransmitter profiles in gut and brain of zebrafish, with mild effects on neuronal integrity. Neurotransmitters closely correlated reciprocally in intestines, which, however, was distinct from the correlation profile in brains. In addition, PFBS and probiotic were combined to impact brain health through absorption of bacterial lipopolysaccharides and production of inflammatory cytokines. Relative to neurotransmission and immune signaling, HPA axis was not involved in the neurotoxicological interaction between PFBS and probiotic. Furthermore, it needs to point out that interactive modes between PFBS and probiotic varied a lot, depending on exposure concentrations, sex and toxic indices. Overall, the present study provided the first evidence that probiotic supplement could dynamically modulate the neurotoxicities of PFBS in teleost.

Unexpected Observations: Probiotic Administration Greatly Aggravates the Reproductive Toxicity of Perfluorobutanesulfonate in Zebrafish.

The present study exposed adult zebrafish to 0, 10, and 100 µg/L perfluorobutanesulfonate (PFBS) with or without dietary supplement of probiotic Lactobacillus rhamnosus. Interaction between probiotic and PFBS on sex endocrine and reproduction was investigated. It was striking to find that PFBS and probiotic coexposures almost ceased the fecundity, which was accompanied by disturbances in sex hormones and oocyte maturation in females. In contrast, probiotic additive efficiently antagonized the estrogenic activity of PFBS in males. For the first time, this study reported that probiotic heavily depended on sex to modulate the endocrine disruption and reproductive toxicity of aquatic pollutants.

Interaction between hypoxia and perfluorobutane sulfonate on developmental toxicity and endocrine disruption in marine medaka embryos.

The co-occurrence of hypoxia and xenobiotics is extremely common in natural environments, highlighting the necessity to elicit their interaction on aquatic toxicities. In the present study, marine medaka embryos were exposed to various concentrations (nominal 0, 1, 3.3 and 10 mg/L) of perfluorobutane sulfonate (PFBS), an environmental pollutant of emerging concern, under either normoxia (6.9 mg/L) or hypoxia (1.7 mg/L) condition. After acute exposure till 15 days post-fertilization, single or combined toxicities of PFBS and hypoxia on embryonic development (e.g., mortality, hatching and heartbeat) and endocrine systems were investigated. Sex and thyroid hormones were measured by enzyme-linked immunosorbent assay. Transcriptional changes of endocrine genes were determined by quantitative real-time PCR assays. Co-exposure to 10 mg/L PFBS and hypoxia caused a further reduction in survival rate and heart beat compared to single exposure. PFBS induced a precocious hatching, while no larvae hatched under hypoxia condition. By disturbing the balance of sex hormones, either PFBS or hypoxia single exposure produced an anti-estrogenic activity in medaka larvae. However, PFBS and hypoxia combinations reversed to estrogenic activity in co-exposed larvae. Variation in disrupting pattern may be attributed to the interactive effects on steroidogenic pathway involving diverse cytochrome P450 enzymes. Regarding thyroid system, PFBS exposure caused detriments of multiple processes along thyroidal axis (e.g., feedback regulation, synthesis and transport of thyroid hormones, receptor-mediated signaling and thyroid gland development), while hypoxia potently impaired the development and function of thyroid gland. Combinations of PFBS and hypoxia interacted to dysregulate the function of thyroid endocrine system. In summary, the present study revealed the dynamic interaction of PFBS pollutant and hypoxia on aquatic developmental toxicities and endocrine disruption. Considering the frequent co-occurrence of xenobiotics and hypoxia, current results would be beneficial to improve our understanding about their interactive mechanisms and provide baseline evidences for accurate ecological risk evaluation.

Recent advances in the bioanalytical methods of polyethylene glycols and PEGylated pharmaceuticals.

Polyethylene glycols are synthetic polymers composed of repeating oxyethylene subunits, which have been known for non-toxic, non-immunogenic, non-antigenic, good solubility in water and therefore approved for pharmaceutical applications. Recently, attachment or amalgamation of polyethylene glycols to therapeutic small molecules, peptides, proteins, or nanoparticles has become a mature technology for the sake of improving their pharmacokinetic and pharmacological profiles, also referred to as PEGylation. By comparison, there are only a few PEGylated pharmaceuticals have been registered for further clinical trials and even less was approved for marketing. High failure rate of PEGylated pharmaceuticals in pre-clinical and clinical trials could be majorly attributed to their unclear pharmacokinetic behaviors. Therefore, the in vivo fate of the PEGylated pharmaceuticals for the various routes of administration needs to be thoroughly investigated An accurate in vivo pharmacological study thereof highly depends on the precise detection of polyethylene glycols as well as their fragments in biological matrixes. The goal of this review is to highlight the analytical methods that were developed and applied to evaluate the polyethylene glycols in pharmaceutical ingredients and excipients, which bring us closer to bridging the gap between the development of polyethylene glycol-based drug delivery systems and their clinical application.

Alcohol Feeding in Mice Promotes Colonic Hyperpermeability and Changes in Colonic Organoid Stem Cell Fate.

BACKGROUND: Alcohol increases intestinal permeability to proinflammatory microbial products that promote liver disease, even after a period of sobriety. We sought to test the hypothesis that alcohol affects intestinal stem cells using an in vivo model and ex vivo organoids generated from jejunum and colon from mice fed chronic alcohol. METHODS: Mice were fed a control or an alcohol diet. Intestinal permeability, liver steatosis-inflammation, and stool short-chain fatty acids (SCFAs) were measured. Jejunum and colonic organoids and tissue were stained for stem cell, cell lineage, and apical junction markers with assessment of mRNA by PCR and RNA-seq. ChIP-PCR analysis was carried out for Notch1 using an antibody specific for acetylated histone 3. RESULTS: Alcohol-fed mice exhibited colonic (but not small intestinal) hyperpermeability, steatohepatitis, and decreased butyrate/total SCFA ratio in stool. Stem cell, cell lineage, and apical junction marker staining in tissue or organoids from jejunum tissue were not impacted by alcohol. Only chromogranin A (Chga) was increased in jejunum organoids by qPCR. However, colonic tissue and organoid staining exhibited an alcohol-induced significant decrease in cytokeratin 20+  (Krt20+) absorptive lineage enterocytes, a decrease in occludin and E-cadherin apical junction proteins, an increase in Chga, and an increase in the Lgr5 stem cell marker. qPCR revealed an alcohol-induced decrease in colonic organoid and tissue Notch1, Hes1, and Krt20 and increased Chga, supporting an alteration in stem cell fate due to decreased Notch1 expression. Colonic tissue ChIP-PCR revealed alcohol feeding suppressed Notch1 mRNA expression (via deacetylation of histone H3) and decreased Notch1 tissue staining. CONCLUSIONS: Data support a model for alcohol-induced colonic hyperpermeability via epigenetic effects on Notch1, and thus Hes1, suppression through a mechanism involving histone H3 deacetylation at the Notch1 locus. This decreased enterocyte and increased enteroendocrine cell colonic stem cell fate and decreased apical junctional proteins leading to hyperpermeability.

Development of a UPLC-MS/MS method for the determination of lomefloxacin in rabbit aqueous humor and its application to a pharmacokinetic study.

Lomefloxacin is a kind of synthetic fluoroquinolone antibiotic, which is used for the treatment of infectious diseases. In this study, a rapid and efficient liquid chromatography-tandem mass spectrometric assay was developed to determine the concentration of lomefloxacin in rabbit aqueous humor quantitatively. Aqueous humor samples were extracted by protein precipitation. Ofloxacin was chosen as internal standard. The chromatographic separation was achieved on a Kinetex C18 (50mm×2.10mm, 2.6µm, Phenomenex Corp, USA) column, with a gradient of methanol (0.1% formic acid) and water (0.1% formic acid). Multiple reaction monitoring (MRM) with positive ionization mode was used for the mass analysis. The validation of this method was based on the European Medicines Agency (2011) [1] and US FDA Guidelines (2001) [2]. The calibration range of aqueous humor samples was 5-1200ng/mL with r=0.9990 (n=6). For all QC samples, Inter-and intra-run precisions were less than 15% and accuracies were between 80%-120%. In conclusion, the assay was rapid, sensitive and able to determinate the lomefloxacin in rabbit aqueous humor accurately. At the same time, this method was successfully applied to study the pharmacokinetics of lomefloxacin hydrochloride eye drops and lomefloxacin hydrochloride ophthalmic gel in rabbit aqueous humor.

The Role of miR-212 and iNOS in Alcohol-Induced Intestinal Barrier Dysfunction and Steatohepatitis.

BACKGROUND: Alcoholic liver disease is commonly associated with intestinal barrier dysfunction. Alcohol-induced dysregulation of intestinal tight junction proteins, such as Zonula Occludens-1 (ZO-1), plays an important role in alcohol-induced gut leakiness. However, the mechanism of alcohol-induced disruption of tight junction proteins is not well established. The goal of this study was to elucidate this mechanism by studying the role of microRNA 212 (miR-212) and inducible nitric oxide synthase (iNOS) in alcohol-induced gut leakiness. METHODS: The permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance and flux of fluorescein sulfonic acid. miR-212 was measured by real-time polymerase chain reaction. The wild-type, iNOS knockout, and miR-212 knockdown mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. The LNA-anti-miR-212 was used to inhibit miR-212 expression in mice. The alcohol-induced intestinal permeability, miR-212 expression, and liver injuries in mice were measured. RESULTS: Our in vitro monolayer and in vivo mice studies showed that: (i) alcohol-induced overexpression of the intestinal miR-212 and intestinal hyperpermeability is prevented using miR-212 knockdown techniques; and (ii) iNOS is up-regulated in the intestine by alcohol and that iNOS signaling is required for alcohol-induced miR-212 overexpression, ZO-1 disruption, gut leakiness, and steatohepatitis. CONCLUSIONS: These studies thus support a novel miR-212 mechanism for alcohol-induced gut leakiness and a potential target that could be exploited for therapeutic intervention to prevent leaky gut and liver injury in alcoholics.

Disruption of the Circadian Clock in Mice Increases Intestinal Permeability and Promotes Alcohol-Induced Hepatic Pathology and Inflammation.

The circadian clock orchestrates temporal patterns of physiology and behavior relative to the environmental light:dark cycle by generating and organizing transcriptional and biochemical rhythms in cells and tissues throughout the body. Circadian clock genes have been shown to regulate the physiology and function of the gastrointestinal tract. Disruption of the intestinal epithelial barrier enables the translocation of proinflammatory bacterial products, such as endotoxin, across the intestinal wall and into systemic circulation; a process that has been linked to pathologic inflammatory states associated with metabolic, hepatic, cardiovascular and neurodegenerative diseases - many of which are commonly reported in shift workers. Here we report, for the first time, that circadian disorganization, using independent genetic and environmental strategies, increases permeability of the intestinal epithelial barrier (i.e., gut leakiness) in mice. Utilizing chronic alcohol consumption as a well-established model of induced intestinal hyperpermeability, we also found that both genetic and environmental circadian disruption promote alcohol-induced gut leakiness, endotoxemia and steatohepatitis, possibly through a mechanism involving the tight junction protein occludin. Circadian organization thus appears critical for the maintenance of intestinal barrier integrity, especially in the context of injurious agents, such as alcohol. Circadian disruption may therefore represent a previously unrecognized risk factor underlying the susceptibility to or development of alcoholic liver disease, as well as other conditions associated with intestinal hyperpermeability and an endotoxin-triggered inflammatory state.

Growth factor osteogenic protein-1: differing effects on cells from three distinct zones in the bovine intervertebral disc.

OBJECTIVE: The objective of this study was to determine if cells isolated from three distinct zones in the bovine intervertebral disc (IVD) differ in their response to growth factor osteogenic protein-1 (OP-1) because of inherent biological differences. The new knowledge gained will help determine if treatment of degenerative disc disease with purified growth factors is effective and will provide guidance in terms of the injection technique and frequency of treatments required. DESIGN: This was an in vitro study measuring the effects of OP-1 on proteoglycan accumulation and synthesis by cells from the nucleus pulposus, inner-annulus fibrosus (inner-AF), and outer-annulus fibrosus (outer-AF) in the bovine IVD. RESULTS: Growth factor OP-1, at 100 ng/ml, stimulated proteoglycan accumulation and resulted in a statistically significant increase in the proteoglycan content of cells derived from three zones of the bovine IVD: 97% in the nucleus pulposus, 40% in the inner-AF, and 75% in the outer-AF. To elucidate the mechanism of enhanced proteoglycan accumulation in response to OP-1, we studied the rate of proteoglycan synthesis and cell proliferation. OP-1 stimulation resulted in a statistically significant increase in the DNA content in cultures containing cells from all three zones of the IVDs: 79% in the nucleus pulposus, 100% in the inner-AF, and 73% in the outer-AF. After dividing by DNA content, OP-1 resulted in a statistically significant increase in the rate of proteoglycan synthesis in the nucleus pulposus (78%) and outer-AF (17%) cells, but the increase in inner-AF cells (23%) did not achieve statistical significance. CONCLUSIONS: OP-1 stimulates proteoglycan accumulation by bovine IVD cells isolated from all three zones of the bovine IVDs. Cells from all three zones proliferated significantly. Individual cells derived from nucleus pulposus and outer-AF, but not those from the inner-AF, synthesized proteoglycans at a significantly faster rate with OP-1 stimulation.