MicroRNA-200c coordinates HNF1 homeobox B and apolipoprotein O functions to modulate lipid homeostasis in alcoholic fatty liver disease.

Hepatic steatosis is an initial manifestation of alcoholic liver disease. An imbalance of hepatic lipid processes including fatty acid uptake, esterification, oxidation, and triglyceride secretion leads to alcoholic fatty liver (AFL). However, the precise molecular mechanisms underlying the pathogenesis of AFL remain elusive. Here, we show that mice deficient in microRNAs (miRs)-141 and -200c display resistance to the development of AFL. We found that miR-200c directly targets HNF1 homeobox B (Hnf1b), a transcriptional activator for microsomal triglyceride transfer protein (Mttp), as well as apolipoprotein O (ApoO), an integral component of the mitochondrial contact site and cristae organizing system complex. We show that expression of these miRs is significantly induced by chronic ethanol exposure, which is accompanied by reduced HNF1B and APOO levels. Furthermore, miR-141/200c deficiency normalizes ethanol-mediated impairment of triglyceride secretion, which can be attributed to the restored levels of HNF1B and MTTP, as well as phosphatidylcholine abundance. Moreover, we demonstrate that miR-141/200c deficiency restores ethanol-mediated inhibition of APOO expression and mitochondrial dysfunction, improving mitochondrial antioxidant defense capacity and fatty acid oxidation. Taken together, these results suggest that miR-200c contributes to the modulation of lipid homeostasis in AFL disease by cooperatively regulating Hnf1b and ApoO functions.

Insulin Prevents Hypercholesterolemia by Suppressing 12α-Hydroxylated Bile Acids.

BACKGROUND: The risk of cardiovascular disease in type 1 diabetes remains extremely high, despite marked advances in blood glucose control and even the widespread use of cholesterol synthesis inhibitors. Thus, a deeper understanding of insulin regulation of cholesterol metabolism, and its disruption in type 1 diabetes, could reveal better treatment strategies. METHODS: To define the mechanisms by which insulin controls plasma cholesterol levels, we knocked down the insulin receptor, FoxO1, and the key bile acid synthesis enzyme, CYP8B1. We measured bile acid composition, cholesterol absorption, and plasma cholesterol. In parallel, we measured markers of cholesterol absorption and synthesis in humans with type 1 diabetes treated with ezetimibe and simvastatin in a double-blind crossover study. RESULTS: Mice with hepatic deletion of the insulin receptor showed marked increases in 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol. This phenotype was entirely reversed by hepatic deletion of FoxO1. FoxO1 is inhibited by insulin and required for the production of 12α-hydroxylated bile acids, which promote intestinal cholesterol absorption and suppress hepatic cholesterol synthesis. Knockdown of Cyp8b1 normalized 12α-hydroxylated bile acid levels and completely prevented hypercholesterolemia in mice with hepatic deletion of the insulin receptor (n=5-30), as well as mouse models of type 1 diabetes (n=5-22). In parallel, the cholesterol absorption inhibitor, ezetimibe, normalized cholesterol absorption and low-density lipoprotein cholesterol in patients with type 1 diabetes as well as, or better than, the cholesterol synthesis inhibitor, simvastatin (n=20). CONCLUSIONS: Insulin, by inhibiting FoxO1 in the liver, reduces 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol levels. Thus, type 1 diabetes leads to a unique set of derangements in cholesterol metabolism, with increased absorption rather than synthesis. These derangements are reversed by ezetimibe, but not statins, which are currently the first line of lipid-lowering treatment in type 1 diabetes. Taken together, these data suggest that a personalized approach to lipid lowering in type 1 diabetes may be more effective and highlight the need for further studies specifically in this group of patients.

MiR-200c-3p targets SESN1 and represses the IL-6/AKT loop to prevent cholangiocyte activation and cholestatic liver fibrosis.

Cholestasis causes ductular reaction in the liver where the reactive cholangiocytes not only proliferate but also gain a neuroendocrine-like phenotype, leading to inflammatory cell infiltration and extracellular matrix deposition and contributing to the development and progression of cholestatic liver fibrosis. This study aims to elucidate the role of miR-200c in cholestasis-induced biliary liver fibrosis and cholangiocyte activation. We found that miR-200c was extremely abundant in cholangiocytes but was reduced by cholestasis in a bile duct ligation (BDL) mouse model; miR-200c was also decreased by bile acids in vitro. Phenotypically, loss of miR-200c exacerbated cholestatic liver injury, including periductular fibrosis, intrahepatic inflammation, and biliary hyperplasia in both the BDL model and the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model. We identified sestrin 1 (SESN1) as a target of miR-200c. Sesn1-/--BDL mice showed mitigation of cholestatic liver injury. On a molecular level, the pro-proliferative IL-6/AKT feedback loop was activated in Mir200c-/- livers but was inhibited in Sesn1-/- livers upon cholestasis in mice. Furthermore, rescuing expression of miR-200c by the adeno-associated virus serotype 8 ameliorated BDL-induced liver injury in Mir200c-/- mice. Taken together, this study demonstrates that miR-200c restrains the proliferative and neuroendocrine-like activation of cholangiocytes by targeting SESN1 and inhibiting the IL-6/AKT feedback loop to protect against cholestatic liver fibrosis. Our findings provide mechanistic insights regarding biliary liver fibrosis, which may help to reveal novel therapeutic targets for the treatment of cholestatic liver injury and liver fibrosis.

A Potential Role for SerpinA3N in Acetaminophen-Induced Hepatotoxicity.

Acetaminophen (APAP) is a commonly used pain and fever reliever but is also the most frequent cause of drug-induced liver injury. The mechanism pertaining acetaminophen toxicity has been well documented, whereas mechanisms of hepatotoxicity are not well established. Serine (or cysteine) peptidase inhibitor, clade A, member 3N (SerpinA3N), a serine protease inhibitor, is synthesized in the liver but the role of SerpinA3N in relation to APAP-induced liver injury is not known. Wild-type and hepatocyte-specific SerpinA3N knockout (HKO) mice were injected intraperitoneally with a single dose of PBS or APAP (400 mg/kg) for 12 hours, and markers of liver injury, cell death, and inflammation were assessed. SerpinA3N expression was highly induced in mice with APAP overdose. SerpinA3N HKO mice had diminished liver injury and necrosis as shown by lower alanine aminotransferase and interleukin-6 levels, accompanied by suppressed inflammatory cytokines and reduced neutrophil infiltration. The reduced oxidative stress was associated with enhanced antioxidant enzyme capabilities. Taken together, hepatocyte SerpinA3N deficiency reduced APAP-induced liver injury by ameliorating inflammation and modulating the 5' AMP-activated protein kinase-unc-51-like autophagy activating kinase 1 signaling pathway. Our study provides novel insights into a potential role for SerpinA3N in APAP-induced liver injury. SIGNIFICANCE STATEMENT: Our studies indicate that serine (or cysteine) peptidase inhibitor, clade A, member 3N (SerpinA3N) may have a pathophysiological role in modulating acetaminophen (APAP)-induced liver injury. More specifically, mice with hepatic deletion of SerpinA3N suppressed inflammation and liver injury to reduce APAP-induced hepatotoxicity. Controlling the inflammatory response offers possible approaches for novel therapeutics; therefore, understanding the pathophysiological role of SerpinA3N in inducing liver injury may add to the development of more efficacious treatments.

Evaluation of leaching characteristic and kinetic study of lithium from lithium aluminum silicate glass-ceramics by NaOH.

The behavior and mechanism of Li leaching from lithium aluminum silicate glass-ceramics which can be used as a secondary source of Li using aqueous NaOH solution was investigated. The Li leaching efficiency is increased with increasing concentration of NaOH, specific surface area, and reaction temperature. When leached under optimum conditions, 2 mol/L NaOH, 53 µm particle undersize, 1:10 solid/liquid ratio, 250 r/min stirring speed, 100°C reaction temperature, 12 hr, the Li leaching efficiency was approximately 70%. However, when the leaching experiment was performed for 48 hr, the concentration of Li+ ions contained in the leach liquor decreased from 1160 to 236 mg/L. To investigate the origin of this phenomenon, the obtained leach residue was analyzed by X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. These analyses show that zeolite was formed around the lithium aluminum silicate glass-ceramics, which affected the leaching of by adsorbing Li+ ions. In addition, using the shrinking-core model and the Arrhenius equation, the leaching reaction with NaOH was found to depends on the chemical reaction of the two reactants, with a higher than 41.84 kJ/mol of the activation energy.

Long noncoding RNA H19 interacts with polypyrimidine tract-binding protein 1 to reprogram hepatic lipid homeostasis.

H19 is an imprinted long noncoding RNA abundantly expressed in embryonic liver and repressed after birth. We show that H19 serves as a lipid sensor by synergizing with the RNA-binding polypyrimidine tract-binding protein 1 (PTBP1) to modulate hepatic metabolic homeostasis. H19 RNA interacts with PTBP1 to facilitate its association with sterol regulatory element-binding protein 1c mRNA and protein, leading to increased stability and nuclear transcriptional activity. H19 and PTBP1 are up-regulated by fatty acids in hepatocytes and in diet-induced fatty liver, which further augments lipid accumulation. Ectopic expression of H19 induces steatosis and pushes the liver into a "pseudo-fed" state in response to fasting by promoting sterol regulatory element-binding protein 1c protein cleavage and nuclear translocation. Deletion of H19 or knockdown of PTBP1 abolishes high-fat and high-sucrose diet-induced steatosis. CONCLUSION: Our study unveils an H19/PTBP1/sterol regulatory element-binding protein 1 feedforward amplifying signaling pathway to exacerbate the development of fatty liver. (Hepatology 2018;67:1768-1783).

Bile Acid-Activated Receptors: A Review on FXR and Other Nuclear Receptors.

Nuclear receptors (NRs) are ligand-dependent transcription factors that are involved in various biological processes including metabolism, reproduction, and development. Upon activation by their ligands, NRs bind to their specific DNA elements, exerting their biological functions by regulating their target gene expression. Bile acids are detergent-like molecules that are synthesized in the liver. They not only function as a facilitator for the digestion of lipids and fat-soluble vitamins but also serve as signaling molecules for several nuclear receptors to regulate diverse biological processes including lipid, glucose, and energy metabolism, detoxification and drug metabolism, liver regeneration, and cancer. The nuclear receptors including farnesoid X receptor (FXR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), vitamin D receptor (VDR), and small heterodimer partner (SHP) constitute an integral part of the bile acid signaling. This chapter reviews the role of the NRs in bile acid homeostasis, highlighting the regulatory functions of the NRs in lipid and glucose metabolism in addition to bile acid metabolism.

YAP suppresses gluconeogenic gene expression through PGC1α.

Cell growth and proliferation are tightly coupled to metabolism, and dissecting the signaling molecules which link these processes is an important step toward understanding development, regeneration, and cancer. The transcriptional regulator Yes-associated protein 1 (YAP) is a key regulator of liver size, development, and function. We now show that YAP can also suppress gluconeogenic gene expression. Yap deletion in primary hepatocytes potentiates the gluconeogenic gene response to glucagon and dexamethasone, whereas constitutively active YAP suppresses it. The effects of YAP are mediated by the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1. YAP inhibits its ability to bind to and activate transcription from the promoters of its gluconeogenic targets, and the effects of YAP are blunted upon its knockdown. In vivo, constitutively active YAP lowers plasma glucose levels and increases liver size. CONCLUSION: YAP appears to reprogram cellular metabolism, diverting substrates away from the energy-consuming process of gluconeogenesis and toward the anabolic process of growth. (Hepatology 2017;66:2029-2041).

Role of Insulin in the Regulation of Proprotein Convertase Subtilisin/Kexin Type 9.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds the low-density lipoprotein receptor and targets it for degradation, has emerged as an important regulator of serum cholesterol levels and cardiovascular disease risk. Although much work is currently focused on developing therapies for inhibiting PCSK9, the endogenous regulation of PCSK9, particularly by insulin, remains unclear. The objective of these studies was to determine the effects of insulin on PCSK9 in vitro and in vivo. APPROACH AND RESULTS: Using rat hepatoma cells and primary rat hepatocytes, we found that insulin increased PCSK9 expression and increased low-density lipoprotein receptor degradation in a PCSK9-dependent manner. In parallel, hepatic Pcsk9 mRNA and plasma PCSK9 protein levels were reduced by 55% to 75% in mice with liver-specific knockout of the insulin receptor; 75% to 88% in mice made insulin-deficient with streptozotocin; and 65% in ob/ob mice treated with antisense oligonucleotides against the insulin receptor. However, antisense oligonucleotide-mediated knockdown of insulin receptor in lean, wild-type mice had little effect. In addition, we found that fasting was able to reduce PCSK9 expression by 80% even in mice that lack hepatic insulin signaling. CONCLUSIONS: Taken together, these data indicate that although insulin induces PCSK9 expression, it is not the sole or even dominant regulator of PCSK9 under all conditions.

Genome-wide analysis of FoxO1 binding in hepatic chromatin: potential involvement of FoxO1 in linking retinoid signaling to hepatic gluconeogenesis.

The forkhead transcription factor FoxO1 is a critical regulator of hepatic glucose and lipid metabolism, and dysregulation of FoxO1 function has been implicated in diabetes and insulin resistance. We globally identified FoxO1 occupancy in mouse hepatic chromatin on a genome-wide level by chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq). To establish the specific functional significance of FoxO1 against other FoxO proteins, ChIP-seq was performed with chromatin from liver-specific FoxO1 knockout and wild-type mice. Here we identified 401 genome-wide FoxO1-binding locations. Motif search reveals a sequence element, 5' GTAAACA 3', consistent with a previously known FoxO1-binding site. Gene set enrichment analysis shows that the data from FoxO1 ChIP-seq are highly correlated with the global expression profiling of genes regulated by FoxO1, demonstrating the functional relevance of our FoxO1 ChIP-seq study. Interestingly, gene ontology analysis reveals the functional significance of FoxO1 in retinoid metabolic processes. We show here that FoxO1 directly binds to the genomic sites for the genes in retinoid metabolism. Notably, deletion of FoxO1 caused a significantly reduced induction of Pck1 and Pdk4 in response to retinoids. As Pck1 and Pdk4 are downstream targets of retinoid signaling, these results suggest that FoxO1 plays a potential role in linking retinoid metabolism to hepatic gluconeogenesis.

miR-141/200c contributes to ethanol-mediated hepatic glycogen metabolism.

OBJECTIVE: Hepatic glucose metabolism is profoundly perturbed by excessive alcohol intake. miR-141/200c expression is significantly induced by chronic ethanol feeding. This study aimed at identifying the role of miR-141/200c in glucose homeostasis during chronic ethanol exposure. METHODS: WT and miR-141/200c KO mice were fed a control or an ethanol diet for 30 days, followed by a single binge of maltose dextrin or ethanol, respectively. Untargeted metabolomics analysis of hepatic primary metabolites was performed along with analyses for liver histology, gene expression, intracellular signaling pathways, and physiological relevance. Primary hepatocytes were used for mechanistic studies. RESULTS: miR-141/200c deficiency rewires hepatic glucose metabolism during chronic ethanol feeding, increasing the abundance of glucose intermediates including G6P, an allosteric activator for GS. miR-141/200c deficiency replenished glycogen depletion during chronic ethanol feeding accompanied by reduced GS phosphorylation in parallel with increased expression of PP1 glycogen targeting subunits. Moreover, miR-141/200c deficiency prevented ethanol-mediated increases in AMPK and CaMKK2 activity. Ethanol treatment reduced glycogen content in WT-hepatocytes, which was reversed by dorsomorphin, a selective AMPK inhibitor, while KO-hepatocytes displayed higher glycogen content than WT-hepatocytes in response to ethanol treatment. Furthermore, treatment of hepatocytes with A23187, a calcium ionophore activating CaMKK2, lowered glycogen content in WT-hepatocytes. Notably, the suppressive effect of A23187 on glycogen deposition was reversed by dorsomorphin, demonstrating that the glycogen depletion by A23187 is mediated by AMPK. KO-hepatocytes exhibited higher glycogen content than WT-hepatocytes in response to A23187. Finally, miR-141/200c deficiency led to improved glucose tolerance and insulin sensitivity during chronic ethanol feeding. CONCLUSIONS: miR-141/200c deficiency replenishes ethanol-mediated hepatic glycogen depletion through the regulation of GS activity and calcium signaling coupled with the AMPK pathway, improving glucose homeostasis and insulin sensitivity. These results underscore miR-141/200c as a potential therapeutic target for the management of alcohol intoxication.

The Novel Hooked Kirschner Wire Technique for Ulna Coronoid Process Fractures.

Background: The aim of this study was to introduce a novel technique to improve the ease of fixing of even small fragments of the coronoid process and report the clinical outcomes of this method. Methods: Forty-nine patients with ulnar coronoid process fractures fixed using the hooked Kirschner wire (K-wire) technique at our hospital from 2007 to 2019 were reviewed. Radiological features and fracture union were assessed using simple radiographs. Functional outcomes of the treated elbows were evaluated at the final follow-up visit using the Mayo Elbow Performance Score (MEPS). Results: All patients were examined at a mean follow-up of 17.7 months (range, 6-62 months). We observed bony union in patients at a mean of 10.9 weeks (range, 6-22 weeks). The mean flexion and extension ranges of the elbow were 132.0° (range, 106° -151°) and 4.5° (range, -20° to 30°), respectively. The mean pronation and supination ranges of the forearm were 81.1° (range, 60°-90°) and 88.3° (range, 60°-120°), respectively. The mean arc of the elbow was 127.4° (range, 78°-160°). All patients were evaluated using the MEPS at the final follow-up visit, with a mean score of 96.9 points (range, 80-100 points). One case of coronoid nonunion was observed and re-fixation was performed. One case of infection was observed and also treated with additional surgery. Three patients complained of ulnar nerve symptoms and 1 patient underwent surgical release for tardy ulnar nerve palsy. Conclusions: Despite its limitations, the hooked K-wire technique was a useful method for even smaller coronoid process fractures. K-wires were also a useful temporary intraoperative fixation method and could provide permanent fixation.

Avascular necrosis of the capitellum after surgical treatment of a coronal shear fracture of the distal humerus: A case report.

INTRODUCTION AND IMPORTANCE: Coronal shear fractures of the distal humerus are rare and are expected to have a high incidence of avascular necrosis (AVN) due to the avascular nature of the capitellar bone fragment and limited soft tissue attachment. However, according to the literature published thus far, AVN is infrequently reported, and some studies suggest that it does not have a significant impact on clinical outcomes. CASE PRESENTATION: Two female patients, one aged 72 and the other 70, presented with coronal shear fractures of distal humerus. Both patients were diagnosed with AVN of the capitellum 7 and 10 months after undergoing open reduction and internal fixation. One patient underwent hardware removal, while the other patient declined due to the absence of discomfort. However, at their final follow-up, both patients exhibited good clinical results. CLINICAL DISCUSSION: The occurrence of AVN may be related to the severity of the initial injury, including posterior comminution. While some studies suggest that AVN of the capitellum may not affect clinical outcomes, hardware removal may be required in cases where there is intra-articular protrusion of the hardware. CONCLUSION: Although AVN is a rare occurrence, even when it does occur, it may not significantly affect clinical outcomes. In this study, AVN may be associated with initial injury severity, and surgical treatment may make it possible to develop AVN. Moreover, considering the timing of the occurrence of AVN, it is believed that a close follow-up of more than one year will be required.

SerpinA3N deficiency attenuates steatosis and enhances insulin signaling in male mice.

Aberrant hepatic lipid metabolism is the major cause of non-alcoholic fatty liver disease (NAFLD) and is associated with insulin resistance and type 2 diabetes. Serine (or cysteine) peptidase inhibitor, clade A, member 3N (SerpinA3N) is highly expressed in the liver; however, its functional role in regulating NAFLD and associated metabolic disorders are not known. Male wildtype and hepatocyte Serpina3N knockout (HKO) mice were fed a control diet, methionine- and choline-deficient diet or high-fat high-sucrose diet to induce NAFLD and markers of lipid metabolism and glucose homeostasis were assessed. SerpinA3N protein was markedly induced in mice with fatty livers. Hepatic deletion of SerpinA3N attenuated steatosis which correlated with altered lipid metabolism genes, increased fatty acid oxidation activity and enhanced insulin signaling in mice with NAFLD. Additionally, SerpinA3N HKO mice had reduced epididymal white adipose tissue mass, leptin, and insulin levels, improved glucose tolerance, and enhanced insulin sensitivity which was associated with elevated insulin-like growth factor binding protein-1 (IGFBP1) and activation of the leptin receptor (LEPR)-STAT3 signaling pathway. Our findings provide a novel insight into the functional role of SerpinA3N in regulating NAFLD and glucose homeostasis.

PDK4-Deficiency Reprograms Intrahepatic Glucose and Lipid Metabolism to Facilitate Liver Regeneration in Mice.

Liver regeneration requires intrahepatic and extrahepatic metabolic reprogramming to meet the high hepatic bioenergy demand for liver cell repopulation. This study aims to elucidate how pyruvate dehydrogenase kinase 4 (PDK4), a critical regulator of glucose and lipid metabolism, coordinates metabolic regulation with efficient liver growth. We found that hepatic Pdk4 expression was elevated after two-thirds partial hepatectomy (PHx). In Pdk4 -/- PHx mice, the liver/body weight ratio was more rapidly restored, accompanied by more aggressive hepatic DNA replication; however, Pdk4 -/- mice developed more severe hypoglycemia. In Pdk4 -/- PHx livers, the pro-regenerative insulin signaling was potentiated, as demonstrated by early peaking of the phosphorylation of insulin receptor, more remarkable induction of the insulin receptor substrate proteins, IRS1 and IRS2, and more striking activation of Akt. The hepatic up-regulation of CD36 contributed to the enhanced transient regeneration-associated steatosis in Pdk4 -/- PHx mice. Notably, CD36 overexpression in mice promoted the recovery of liver/body weight ratio and elevated intrahepatic adenosine triphosphate after PHx. CD36 expression was transcriptionally suppressed by FOXO1 (forkhead box protein O1), which was stabilized and translocated to the nucleus following AMPK (adenosine monophosphate-activated protein kinase) activation. PHx remarkably induced AMPK activation, which became incompetent to respond in Pdk4 -/- livers. Moreover, we defined that PDK4-regulated AMPK activation directly depended on intracellular adenosine monophosphate in vitro and in regenerative livers. Conclusion: PDK4 inhibition reprograms glucose and lipid metabolism to promote liver regeneration by enhancing hepatic insulin/Akt signaling and activating an AMPK/FOXO1/CD36 regulatory axis of lipid. These findings may lead to potential therapeutic strategies to prevent hepatic insufficiency and liver failure.

Analysis of early failure of the locking compression plate in osteoporotic proximal humerus fractures.

BACKGROUND: Although there has been continuous evolution in the management of fracture fixation, treatment for osteoporotic proximal humerus fractures is still challenging to trauma surgeons. The purpose of this study was to report early failure of the locking compression plate (LCP) in the treatment of osteoporotic proximal humerus fracture and characterize the mode of failure. METHODS: Nine patients, older than 65 years, underwent internal fixation with the use of a locking compression plate and had early failure within 4 weeks postoperatively. According to Neer's classification, five were included in a two-part surgical neck fracture, three in a three-part fracture, and one in a four-part fracture. RESULTS: All failures occurred with back-out of the plate-screw construct, leading to varus displacement in eight patients and plate breakage in one. Revision surgery was performed in six patients using replating and tension band wiring with a bone graft, and three patients underwent hemiarthroplasty. The average UCLA score was 25 points for the hemiarthroplasty group and 30 points for the reconstruction group. CONCLUSIONS: Early postoperative failure of the LCP developed within 4 weeks with a presentation of en bloc back-out of the plate-screw construct and plate breakage. Possible risk factors included malreduction, loss of medial support, and negligence of tension band sutures on the tuberosities.

Crossbar Technique for the Failed Clavicular Hook Plate Fixation in an Acute Acromioclavicular Joint Dislocation: Salvage for Acromial Fracture after Clavicular Hook Plate.

We experienced acromial erosion and subsequent fracture after the treatment of Rockwood type V acromioclavicular dislocation with hook plate and coracoclavicular ligament augmentation. It was treated by using a surgical technique to address an acromial fracture and subsequent losses of reduction in acromioclavicular joint with two trans-acromial cortical screws (crossbar technique). The reduction state of acromioclavicular joint could be maintained by these two screws. Our crossbar technique could be considered as a good salvage procedure for the reduction loss caused by cutout or significant erosion of acromion after insertion of clavicular hook plate.

Genome-Wide Analysis for Identifying FOXO Protein-Binding Sites.

Forkhead box O (FOXO) proteins comprise a superfamily of transcription factors that play important roles in controlling various biological processes. Transcriptional control constitutes a crucial component in regulating complex biological processes. The identification of cis-regulatory elements is essential to understand the regulatory mechanism of gene expression. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is widely used to identify the cis-regulatory elements of transcription factors and other DNA-binding proteins on a genome-wide level. It is a powerful tool to analyze the regulatory networks underlying the biological processes. Here, we describe a detailed protocol for preparing ChIP-seq samples that are used for sequencing and subsequent data analyses.

A study on Zn recovery from other metals in the spent mixed batteries through a sequence of hydrometallurgical processes.

A study on selective separation of Zn from a leaching solution by disposal batteries including various type batteries was carried out to understand the recovery behaviour of Zn in leaching solution. Selective recovery of Zn in leaching solution including Mn, Cd, Cu ion was difficult due to its similar physicochemical behaviour. Experiment results by present leaching solution with 279 µm undersize indicated that the best condition for leaching is 1 M H2SO4, 250 rpm, 5 vol.% H2O2 and 353 K and the leaching efficient of Zn, Co and Mn is approximately 97%, respectively. The exclusive extraction behaviour of Zn by using D2EHPA is indicated that the best conditions for solvent extraction are to be 0.6 M D2EHPA diluted with kerosene, 30% saponification, 298 K, 5-min contact time and three-stage countercurrent extraction, and the O/A ratio 1, respectively. Recovery of Zn was with approximately 99.7% selectively from Mn, Co, Ni, Cd and Li. After scrubbing 5 times by pH 2 modified solution and single stripping experiment by 1.5 M H2SO4, the solution including Zn of 9.0 g/L can be produced.

Two uniquely arranged thyroid hormone response elements in the far upstream 5' flanking region confer direct thyroid hormone regulation to the murine cholesterol 7alpha hydroxylase gene.

Cholesterol 7alpha hydroxlyase (CYP7A1) is a key enzyme in cholesterol catabolism to bile acids and its activity is important for maintaining appropriate cholesterol levels. The murine CYP7A1 gene is highly inducible by thyroid hormone in vivo and there is an inverse relationship between thyroid hormone and serum cholesterol. Eventhough gene expression has been shown to be upregulated, whether the induction was mediated through a direct effect of thyroid hormone on the CYP7A1 promoter has never been established. Using gene targeted mice, we show that either of the two TR isoforms are sufficient to maintain normal hepatic CYP7A1 expression but a loss of both results in a significant decrease in expression. We also identified two new functional thyroid hormone receptor-binding sites in the CYP7A1 5' flanking sequence located 3 kb upstream from the transcription start site. One site is a DR-0, which is an unusual type of TR response element, and the other consists of only a single recognizable half site that is required for TR/retinoid X receptor (RXR) binding. These two independent TR-binding sites are closely spaced and both are required for full induction of the CYP7A1 promoter by thyroid hormone, although the DR-0 site was more crucial.