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Supplementary Data from Aberrant Splicing of Hugl-1 Is Associated with Hepatocellular Carcinoma Progression

Purpose: Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC).Experimental Design: Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay.Results: In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short “direct repeat” sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice.Conclusions: We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.

Objective To investigate the outcome of the rapid lowering of elevated blood pressure in patients with intracerebral hemorrhage and to understand its association with clinical outcome. Materials and methods Between July 2014 and June 2018, a total of 1,500 patients diagnosed with cerebral hemorrhage were randomized and assessed for their neurological symptoms and diagnosed with CT scan. 1,500 (42%) patients received intensive treatment, while 1,645 (58%) patients were assigned the guideline-recommended therapy. The systolic blood pressure of these patients was measured every half hour during the first day of admission. The intensive-treatment group was further categorized into five different subgroups in 10-mmHg intervals. On the other hand, the clinical outcome, as represented by the volume of hematoma, adverse events, modified Rankin scale etc., was measured and analyzed. Results The volume of hematoma varied with a p-value of 0.014 among the investigated groups. There was no direct correlation among the five groups based on the systolic blood pressure groups and modified Rankin scale 4 - 6. The 140 - 150 mmHg group observed an elevated risk compared to the 120 - 130 mmHg group in the modified Rankin scale ((OR = 1.59; 95% CI (0.98 - 2.61)). The hematoma enlargement increased significantly with a p-value of 0.012. There was no direct association or statistical significance between the occurrence of the clinical outcome and the multivariate relationship between the five groups based on the multivariates (p = 0.513). Conclusion Systolic blood pressure ranging between 120 and 130 mmHg serves as an optimal goal for acute intracerebral hemorrhage by reducing the hematoma enlargement. It is also evident that the lowering of high mean systolic blood pressure after blood pressure-lowering therapy usually leads to cardiorenal injury.

YAP and TAZ (YAP/TAZ), two major effectors of the Hippo signaling pathway, are frequently activated in human cancers. The activity of YAP/TAZ is strictly repressed upon phosphorylation by LATS1/2 tumor suppressors. However, it is unclear how LATS1/2 are precisely regulated by upstream factors such as Hippo kinases MST1/2. Here, we show that WWC proteins (WWC1/2/3) directly interact with LATS1/2 and SAV1, and SAV1, in turn, brings in MST1/2 to phosphorylate and activate LATS1/2. Hence, WWC1/2/3 play an organizer role in a signaling module that mediates LATS1/2 activation by MST1/2. Moreover, we have defined a minimum protein interaction interface on WWC1/2/3 that is sufficient to activate LATS1/2 in a robust and specific manner. The corresponding minigene, dubbed as SuperHippo, can effectively suppress tumorigenesis in multiple tumor models. Our study has uncovered a molecular mechanism underlying LATS1/2 regulation and provides a strategy for treating diverse malignancies related to Hippo pathway dysregulation.

To compare the efficacy of honey mouthwash 12.5% and chlorhexidine solution 0.2% to reduce the rate of oropharyngeal bacterial colonization in mechanically-ventilated patients.This study was a randomized, single blind, phase Ⅲ controlled clinical trial. Sixty patients newly admitted to internal and trauma Intensive Care Units of the two educational hospitals of Sanandaj city affiliated with Kurdistan University of Medical Sciences were selected by convenience sampling and allocated to two groups of 30 patients using random blocks design. In each group, the mouthwash was applied twice a day for four consecutive days. Swab samples were taken from the mouth and throat of all patients three times a day (pre- intervention, two days, and four days after the intervention) and then the samples were transferred onto the blood agar and eosin methylene blue (EMB) culture plates and investigated for bacterial growth and colonization after 24-48 h.The findings showed that oropharyngeal colonization was not significantly different between the two groups, pre-intervention, two days, and four days after the intervention (P0.05). Rinsing with honey mouthwash 12.5% led to the inhibition of Staphylococcus aureus and Pseudomonas aeruginosa on the fourth day of the intervention in all samples.None of the studied solutions contributed to the reduction of oropharyngeal bacterial colonization. It seems that the growth inhibition of Staphylococcus aureus and Pseudomonas aeruginosa by the honey 12.5% mouthwash in mechanically-ventilated patients need further investigation.

To observe the effect of acupuncture or electroacupuncture (EA) combined with usual medical care for treating knee osteoarthritis (KOA) .A total of 90 patients with KOA were randomly allocated to 3 groups: usual care group (UC group, n = 30) was treated by pharmacological treatment of non-steroidal anti-inflammatory drugs (NSAIDs) and drugs for activating blood circulation (Ds-ABC), acupuncture (AP) combined with usual care group (UC group) (AP + UC group, n = 30) and EA combined with UC group (EA + UC group, n = 30). The primary outcome measurements included pain visual analogue scale/score (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC Index) and its subscales. Secondary outcome measurement was Assessment of Quality of Life instrument version of the 36-item Short Form Health Survey (AQoL-SF36).By the end of the 1st week, AP + UC group and EA + UC group exhibited statistically significant improvements in primary outcome measures, except for WOMAC stiffness, compared with the UC group (P0.05). Moreover, the energy/fatigue domain of AQoL-SF36 in the AP + UC group showed better results than UC group (P0.05). By the end of the 2nd week, all the primary outcome measures revealed that either the AP + UC or EA + UC group demonstrating remarkable advantages compared with the UC group (P0.05). The social functioning and general health domains of AQoL-SF36 in the two acupuncture-intervention groups were improved significantly than UC group (P0.05). We also found the energy/fatigue and emotional wellbeing domains of AQoL-SF36 in the EA + UC group demonstrated better results than UC group (P0.05).AP or EA combined with usual care is more effective than usual care alone for the treatment of KOA, the intervention of electric current in the process of acupuncture may improve more domains of AQoL-SF36 in KOA patients.

Background: Scopoletin (SPT) is known to exert neuroprotective autophagy effect. However, the efficacy of SPT in the treatment of spinal cord injury (SCI) has yet not been explored. The investigation was intended to elucidate whether SPT can exert neuroprotective effect by triggering neuronal autophagy after SCI. The study was also directed to investigate the role of adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in the autophagy facilitated by SPT. Materials and Methods: The SCI was developed in female Sprague-Dawley rats by damaging the T10 spinal level using an impounder impact. Three animals groups were investigated – Sham group, SCI group, and SCI + SPT group. The SCI + SPT group was administered with SPT (100 mg/kg) intraperitoneally. Basso, Beattie, and Bresnahan scores and angle of incline test revealed that SPT administration improved the movement of hind limbs after SCI induction. Results: Results indicated that SPT imparted neuronal protection, alleviated neuronal apoptosis, and improved neuronal autophagy. SPT-induced autophagy was identified by increased Beclin-1 expression and LC3B-positive neuronal cells. Further investigations revealed that SPT triggers the pathway involving AMPK/mTOR signaling, thereby stimulating autophagy in SCI-induced rat model. Conclusion: The findings of the present investigation strongly advocate the beneficial effects of SPT in the treatment of the SCI. SPT ameliorates the AMPK/mTOR signaling-induced autophagy and thereby improves functional recovery in SCI-induced rats.

We describe the syntheses of 3,4-dihydropyrimidinones or analogous thioketones by a one-pot cyclocondensation of acetoacetates, aldehydes and urea or thiourea using gallium (III) chloride as catalyst under solvent-free conditions. The improved Biginelli reaction not only features a simple procedure, high yields and easy purification of production, but also the recycled catalyst could be directly reused for many times while the yields of reaction would not decrease.

Programmed cell death 4 (PDCD4) is known to suppress neoplastic transformation, cell proliferation and metastasis, and to be downregulated by microRNA-21 (miR-21) in renal cell carcinoma (RCC) cell lines and tissues. The aim of the present study was to investigate the roles of and association between PDCD4 and miR-21 in a nude mouse renal cancer model. A total of 24 BALB/c male nude mice were randomly assigned into the following three groups: Negative control (NC; n=8), miR-21 inhibitor (n=8) and miR-21 mimic (n=8). Subsequently, renal cell adenocarcinoma 786-O cells were subcutaneously transplanted into the armpits of the mice, which were then injected daily with NC small interfering (si)RNA, precursor-miR-21 (mimic) or anti-miR-21 (inhibitor). Tumors were removed from the mice and weighed 16 days following 786-O cell transplantation. In addition, the expression of miR-21 and PDCD4 mRNA in cancer tissues was analyzed using reverse transcription-quantitative PCR. The expression of PDCD4 protein in cancer tissues was also examined using immunohistochemistry and western blotting. Furthermore, 786-O cells were transfected with PDCD4 siRNA or NC siRNA, and the effects of silencing PDCD4 on tumor cell growth, proliferation and invasion were investigated using soft agar colony formation, EdU cell proliferation assay and Transwell migration and invasion assays. Another 16 BALB/c male nude mice were randomly assigned into two groups as follows: NC (n=8) and PDCD4 siRNA (n=8). The 786-O cells were subcutaneously transplanted into the armpits of the mice, which were subsequently injected daily with NC siRNA or PDCD4 siRNA. The tumors were removed and weighed 16 days following transplantation. Compared with the NC group, tumor weight in the miR-21 mimic group was significantly increased. By contrast, tumor weight in the miR-21 inhibitor group was significantly decreased. Similar to the results observed in human renal cancer tissue and cell lines, miR-21 expression in the nude mouse renal cancer models was significantly upregulated in the miR-21 mimic group compared with the NC group, while it was significantly lower in the miR-21 inhibitor group. Furthermore, there was a significant reduction in PDCD4 protein levels in the miR-21 mimic group and a significant increase in the miR-21 inhibitor group compared with the NC, whereas PDCD4 mRNA expression was not significantly altered. In the EdU proliferation assay, the mean percentage of new cells that incorporated EdU was 28.6% in the NC siRNA group and significantly increased to 44.7% in PDCD4 siRNA transfected cells. In the soft agar colony formation assay, Transwell and migration and invasion assays, a significant increase in colony formation, migration and invasion capacity in PDCD4 siRNA-transfected cells was observed compared with the NC. Furthermore, compared with the NC group, tumor weight in the PDCD4 siRNA group was significantly increased. Similar to the results observed in human renal cancer tissue and cell lines, miR-21 promoted cancer cell hyperplasia and proliferation, and post-transcriptionally downregulated PDCD4 protein expression, in the nude mouse renal cancer model. The results of the present study and previous studies indicate that PDCD4 and miR-21 serve an important role in renal cancer. Thus, increasing PDCD4 expression or inhibiting miR-21 expression may constitute effective novel therapeutic strategies for the treatment of renal cancer.

The binding of insulin to its receptor triggers a signaling cascade regulated by protein complexes via tyrosine phosphorylation events on a multitude of associated proteins. To search novel phosphotyrosine proteins or associated proteins involved in insulin signaling pathway, we employed a method in which Rat1 cells stably expressing the human insulin receptor were stimulated with or without insulin and sub-fractionated prior to enrichment of phosphotyrosine proteins by immunoprecipitation and analysis by LC-MS/MS. Bioinformatic analysis and manual confirmation of peptide phosphorylation site assignments led to identification of 35 phosphotyrosine sites derived from 31 protein groups. Over 50% of these proteins were reported for the first time as tyrosine phosphorylated, including gigaxonin, XIAP and CDK10. In addition, we also found that calcium/calmodulin-dependent protein serine kinase (CASK), a key protein in protein-targeting and vesicle transport in neurons, forms a complex with two unidentified phosphotyrosine proteins pp100 and pp95 in response to insulin-stimulation, though CASK is not itself tyrosine phosphorylated. Furthermore, insulin was able to decrease CASK nuclear location, as well as down-regulate the expression of CASK targeted genes. Our results imply CASK as a novel joint knot connecting CASK-mediated pathways with the insulin signaling. Our data provide a wealth of information potentially paving the way to identify new components in the insulin signaling network.

The epididymis is a key structure of the male reproductive system; its function is to mature, transport, and store sperm. Most of the research examining the epididymis to date has been limited to the study of the secreted proteins involved in the maturation of spermatozoa. However, it is also very important to understand the protein components, regulation and function of the tissue itself since these are the basis for all of its physiological processes. We investigated the differential expression of proteins among the caput, corpus, and cauda regions of rat epididymis and considered the possible links between the localization of these proteins and the different functions of these epididymal regions. High-resolution 2-D gel electrophoresis followed by mass spectrometry (MS) revealed 28 distinct proteins whose expression levels varied from the caput to the cauda epididymis. Sixteen of them were reported for first time to be expressed in the epididymis. Expression patterns of some proteins were validated by Northern blot or Western blot. Immunohistochemistry revealed that inducible carbonyl reductase (iCR), an important enzyme in the anti-oxidative system, exhibits primary and cell-type specific distribution in the distal cauda region. Moreover, analysis of iCR transcription in castrated animals showed that its expression is androgen-dependent. Together with its known functions, iCR may also be involved in androgen metabolism and maintaining a steady microenvironment in the duct of epididymis.

A facile approach to the diazotransfer reagent of imidazole-1-sulfonyl azide was reported. The procedure was well optimized to clarify potential explosion risks. A high production yield as well as small batch variation was achieved even without careful pretreatment of reagents and solvents. HPLC and NMR methods to monitor the process were provided. These features made this protocol suitable for large scale preparation in academia and industry as well.

Purpose: Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC).Experimental Design: Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay.Results: In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short “direct repeat” sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice.Conclusions: We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.

Cdc42, a member of the Rho guanosine triphosphatase (GTPase) family, plays important roles in the regulation of the cytoskeleton, cell proliferation, cell polarity, and cellular transport, but little is known about its specific function in mammalian liver. We investigated the function of Cdc42 in regulating liver regeneration. Using a mouse model with liver-specific knockout of Cdc42 (Cdc42LK), we studied liver regeneration after partial hepatectomy. Histological analysis, immunostaining, and western blot analysis were performed to characterize Cdc42LK livers and to explore the role of Cdc42 in liver regeneration. In control mouse livers, Cdc42 became activated between 3 and 24 hours after partial hepatectomy. Loss of Cdc42 led to a significant delay of liver recovery after partial hepatectomy, which was associated with reduced and delayed DNA synthesis indicated by 5-bromo-2′-deoxyuridine staining. Consistent with this, expression of cyclins D1, A, and E was markedly delayed or reduced in Cdc42LK livers during regeneration. As a potential effector of Cdc42, Rac1 activation was dramatically attenuated in Cdc42LK livers after partial hepatectomy, suggesting it is regulated in a Cdc42-dependent manner. Activation of certain proliferative signaling pathways, such as extracellular signal–regulated kinase, c-Jun N-terminal kinase, and p70S6 kinase pathways, was delayed in Cdc42LK livers. In addition, dilated bile canaliculi and excessive lipid accumulation were observed in mutant livers during liver regeneration, which may result from impaired cytoskeletal organization and intracellular trafficking in hepatocytes. Conclusion: Our results revealed important roles of Cdc42 in the regulation of proliferative signaling during liver regeneration. (HEPATOLOGY 2009:49:240-249.)

SummaryCell polarity proteins regulate tight junction formation and directional migration in epithelial cells. To date, the mechanism by which these polarity proteins assemble at the leading edge of migrating epithelial cells remains unclear. We report that occludin, a transmembrane protein, is localized at the leading edge of migrating cells and regulates directional cell migration. During migration, occludin knockdown disrupted accumulation of aPKC-Par3 and PATJ at the leading edge, and led to a disorganized microtubule network and defective reorientation of the microtubule organization center (MTOC). Phosphorylation of occludin at tyrosine 473 residue allowed recruitment of p85α to the leading edge via association with its C-terminal SH2 domain. Loss of occludin attenuated activation of PI3K, leading to disorganization of the actin cytoskeleton and reduced cell protrusions. Our data indicate that occludin is required for the leading-edge localization of polarity proteins aPKC-Par3 and PATJ and promotes cell protrusion by regulating membrane-localized activation of PI3K.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.