Background and Purpose
Finding new indications for existing drugs, also known as drug repositioning or repurposing, is a powerful approach to accelerate drug discovery and development. The unfolded protein response pathways have been proposed to be a viable target for developing new anticancer drugs.
We screened the Johns Hopkins Drug Library for inhibitors of prostate cancer cell proliferation to identify new antiprostate cancer treatments among known drugs. We systematically investigated the mechanism underlying the anticancer activity of a hit and assessed its efficacy in blocking prostate tumour growth in a mouse model.
The antibacterial drug clofoctol was identified as a novel inhibitor of prostate cancer cell proliferation. Morphologically, cells treated with clofoctol were found to undergo massive vacuolization, reminiscent of endoplasmic reticulum stress. Indeed, all three unfolded protein response pathways including inositol requiring enzyme 1, double-stranded RNA-activated PK-like ER kinase and activating transcription factor 6 were found to be activated by clofoctol. Activation of unfolded protein response pathways by clofoctol led to the inhibition of protein translation in cells and the induction of G1 cell cycle arrest in prostate cancer cells. Clofoctol also inhibited prostate cancer xenograft growth in vivo without apparent toxicity.
Conclusion and Implications
Our findings revealed clofoctol as a novel activator of the unfolded protein response pathways and a promising inhibitor of prostate cancer. As clofoctol has been used in the clinic for years, it is ready for clinical evaluation as a novel antiprostate cancer drug candidate.
AIM: To explore the potential of β-elemene as a radiosensitizer for gastric cancer cells and the underlying mechanisms.
METHODS: SGC7901, MKN45, MKN28, N87, and AGS human gastric cancer cell lines were used to screen for radioresistant gastric cancer cell lines. A 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was used to determine the effects of β-elemene and IPA-3 on cell viability in MKN45 and SGC7901 gastric cancer cell lines. A clonogenic survival assay and annexin V-FITC/PI apoptosis detection assay were used to evaluate cellular radiosensitivity and radiation-induced cell death, respectively. A proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), was employed to screen the proteins regulated by β-elemene pretreatment prior to ionizing radiation (IR) in SGC7901 gastric cancer cell line. IPA-3 was used as a specific small molecule inhibitor of p21-activated protein kinase 1 (Pak1) to target Pak1 signaling. Protein levels of PAK1IP1 (p21-activated protein kinase-interacting protein 1), total Pak1 (t-Pak1), phospho-Pak1 (T423), phospho-ERK1/2 (Thr202/Tyr204), and cleaved caspase-3 (17 kDa) were assessed by western blotting.
RESULTS: MKN45 and SGC7901 gastric cancer cell lines were relatively more resistant to IR. β-elemene pretreatment decreased clonogenic survival following IR in MKN45 and SGC7901 gastric cancer cell lines. Additionally, β-elemene pretreatment prior to IR increased radiation-induced cell death compared with IR alone in MKN45 (10.4% ± 0.9% vs 34.8% ± 2.8%, P < 0.05) and SGC7901 (11.6% ± 0.9% vs 46.7% ± 5.2%, P < 0.05) human gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Through iTRAQ analysis and western blot validation, we found that β-elemene upregulated PAK1IP1 and downregulated phospho-Pak1 (T423) and phospho-ERK1/2 in SGC7901 gastric cancer cells. IR increased the level of phospho-Pak1 (T423). Pretreatment with β-elemene decreased radiation-induced Pak1 and ERK1/2 phosphorylation. Inhibition of Pak1 using IPA-3 decreased clonogenic survival following IR. In addition, IPA-3 increased radiation-induced cell death in MKN45 (13.4% ± 0.3% vs 26.6% ± 1.0%, P < 0.05) and SGC7901 (16.0% ± 0.6% vs 37.3% ± 1.7%, P < 0.05) gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Western blotting showed that IPA-3 decreased radiation-induced Pak1 and ERK1/2 phosphorylation.
CONCLUSION: This is the first demonstration that β-elemene enhances radiosensitivity of gastric cancer cells, and that the mechanism involves inhibition of Pak1 signaling.
β-elemene; Radiosensitivity; Gastric cancer cells; Clonogenic survival; Apoptosis; p21-activated protein kinase 1
The Brief COPE instrument has been utilized to conduct research on various populations, including people living with HIV (PLWH). However, the questionnaire constructs when applied to PLWH have not been subjected to thorough factor validation.
A total of 258 PLWH were recruited from two provinces of China. They answered questions involving the scales of three instruments: the Brief COPE, the Perceived Social Support Scale, and the Perceived Discrimination Scale for PLWH. Confirmatory factor analysis (CFA) and exploratory factor analysis (EFA) were conducted.
The CFA found a poor goodness of fit to the data. The subsequent EFA identified six preliminary factors, forming subscales with Cronbach’s alphas, which ranged from 0.61 to 0.80. Significant correlation coefficients between the subscales and measures of perceived social support and perceived discrimination were reported, giving preliminary support to the validity of the new empirical factor structure.
This study showed that the original factor structure of the Brief COPE instrument, when applied to PLWH in China, did not fit the data. Thus, the Brief COPE should be applied to various populations and cultures with caution. The new factor structure established by the EFA is only preliminary and requires further validation.
Electronic supplementary material
The online version of this article (doi:10.1186/s40249-015-0074-9) contains supplementary material, which is available to authorized users.
People living with HIV; Brief COPE; Confirmatory factor analysis; Explanatory factor analysis; Perceived Social Support Scale; Perceived Discrimination Scale for PLWH; China
Nanoparticles (NPs) that target bone tissue were developed using poly(lactic-co-glycolic acid) (PLGA) copolymers and tetracycline (TC)-based bone-targeting moieties. These NPs are expected to enable the transport of drugs, such as simvastatin (SIM), for the treatment of osteoporosis.
The molecular structures of TC–PLGA were validated by 1H-NMR, and the SIM-loaded NPs were prepared using the solvent emulsification method. The surface properties, cytotoxicity, cellular uptake, cell mineralization, bone targeting potential, and animal pharmacodynamics of the TC–PLGA NPs were evaluated and compared to those of PLGA NPs.
It was confirmed that the average particle size of the NPs was approximately 220 nm. In phosphate-buffered saline (PBS, pH 7.4), the SIM-loaded NPs exhibited a cumulative release of up to 80% within 72 hours. An in vitro cell evaluation indicated that the NPs had an excellent cellular uptake capacity and showed great biocompatibility with MC3T3-E1 cells, thereby reducing the cytotoxic effects of SIM. The cell mineralization assay showed that the SIM-loaded NPs induced osteogenic differentiation and mineralized nodule formation in MC3T3-E1 cells, thereby achieving the same effect as SIM. Preliminary findings from in vitro and in vivo bone affinity assays indicated that the TC–PLGA NPs may display increased bone-targeting efficiency compared to PLGA NPs lacking a TC moiety. The use of SIM-loaded TC–PLGA NPs in treating osteoporosis was tested through animal pharmacodynamics analyses performed in ovariectomized rats, and the results suggested that the SIM-loaded TC–PLGA NPs can improve the curative effects of SIM on the recovery of bone mineral density compared to either SIM-loaded PLGA NPs or SIM alone.
Bone-targeting NPs, which were based on the conjugation of TC to PLGA copolymers, have the ability to target bone. These NPs may be developed as a delivery system for hydrophobic drugs, and they are expected to improve the curative effects of drugs, reduce the administered drug doses, and reduce side effects in other organs.
poly(lactic-co-glycolic acid); simvastatin; tetracycline; osteoporosis; bone targeting; nanoparticles
AIM: To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin (IL)-24 in hepatocellular carcinoma (HCC) cells.
METHODS: γ-secretase inhibitors (GSIs) were used to down-regulate Notch1. HepG2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h. Cell viability was measured by MTT assay. The cellular and nuclear morphology was observed under a fluorescence microscope. To further verify the apoptotic phenotype, cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining. The expression of Notch1, SNAIL1, SNAIL2, E-cadherin, IL-24, XIAP and VEGF was detected by Western blot. The invasion and migration capacities of HCC cells were detected by wound healing assays. Notch1 and Snail were down-regulated by RNA interference, and the target proteins were analyzed by Western blot. To investigate the mechanism of apoptosis, we analyzed HepG2 cells treated with siNotch1 or siCON plus IL-24 or not for 48 h by caspase-3/7 activity luminescent assay.
RESULTS: GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in HepG2 cells. The addition of 50 ng/mL IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%, respectively. Treatment with IL-24 alone did not induce any cytotoxic effect. In SMMC7721 cells with the addition of IL-24 to GSI-I (2.5 μmol/L), the reduction of cell viability was only about 25%. Following GSI-I/IL-24 combined treatment for 6 h, the apoptotic rate of HepG2 cells was 47.2%, while no significant effect was observed in cells treated with the compounds employed separately. Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in HepG2 cells. Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I. Furthermore, the increased GSI-I and IL-24 in HepG2 cell was associated with downregulation of MMP-2, XIAP and VEGF. In the absence of treatment, HepG2 cells could migrate into the scratched space in 24 h. With IL-24 or GSI-I treatment, the wound was still open after 24 h. And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I. Treatment of Notch-1 silenced HepG2 cells with 50 ng/mL IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination. Caspase-3/7 activity was increased in the presence of siNotch1 plus IL-24 treatment.
CONCLUSION: Down-regulation of Notch1 by GSI-I or siRNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of HepG2 cells.
Notch signaling pathway; Interleukin-24; γ-secretase inhibitor; Invasion; Migration; Hepatocellular carcinoma
The fungus Cronartium ribicola (Cri) is an economically and ecologically important forest pathogen that causes white pine blister rust (WPBR) disease on five-needle pines. To cause stem cankers and kill white pine trees the fungus elaborates a life cycle with five stages of spore development on five-needle pines and the alternate host Ribes plants. To increase our understanding of molecular WP-BR interactions, here we report genome-wide transcriptional profile analysis of C. ribicola using RNA-seq.
cDNA libraries were constructed from aeciospore, urediniospore, and western white pine (Pinus monticola) tissues post Cri infection. Over 200 million RNA-seq 100-bp paired-end (PE) reads from rust fungal spores were de novo assembled and a reference transcriptome was generated with 17,880 transcripts that were expressed from 13,629 unigenes. A total of 734 unique proteins were predicted as a part of the Cri secretome from complete open reading frames (ORFs), and 41 % of them were Cronartium-specific. This study further identified a repertoire of candidate effectors and other pathogenicity determinants. Differentially expressed genes (DEGs) were identified to gain an understanding of molecular events important during the WPBR fungus life cycle by comparing Cri transcriptomes at different infection stages. Large-scale changes of in planta gene expression profiles were observed, revealing that multiple fungal biosynthetic pathways were enhanced during mycelium growth inside infected pine stem tissues. Conversely, many fungal genes that were up-regulated at the urediniospore stage appeared to be signalling components and transporters. The secreted fungal protein genes that were up-regulated in pine needle tissues during early infection were primarily associated with cell wall modifications, possibly to mask the rust pathogen from plant defenses.
This comprehensive transcriptome profiling substantially improves our current understanding of molecular WP-BR interactions. The repertoire of candidate effectors and other putative pathogenicity determinants identified here are valuable for future functional analysis of Cri virulence and pathogenicity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1861-1) contains supplementary material, which is available to authorized users.
Cronartium ribicola; Effector; Pathogenicity; RNA-seq; Transcriptome profiling
Studies suggest T cells modulate arterial pressure. Since robust sex differences exist in the immune system and in hypertension, we investigated sex differences in T cell modulation of angiotensin II (Ang II)-induced increases in mean arterial pressure (MAP) in male (M) and female (F) wild type (WT) and recombination-activating-gene-1-deficient (Rag1−/−) mice. Sex-differences in peak MAP in WT were lost in Rag1−/− mice [mmHg: WT-F, 136±4.9 vs. WT-M, 153±1.7; P<0.02; Rag1−/−-F, 135±2.1 vs. Rag1−/−-M, 141±3.8]. Peak MAP was 13 mmHg higher after adoptive transfer of male (CD3M→Rag1−/−-M) vs. female (CD3F→Rag1−/−-M) T-cells. CD3M→Rag1−/−-M mice exhibited higher splenic frequencies of pro-inflammatory interleukin-17A (2.4-fold) and tumor-necrosis factor-α (2.2-fold)-producing T cells and lower plasma levels (13-fold) and renal mRNA expression (2.4-fold) of interleukin-10 while CD3F→Rag1−/−-M mice displayed a higher activation state in general and T-helper 1-biased renal inflammation. Greater T cell infiltration into perivascular adipose tissue and kidney associated with increased pressor responses to Ang II if the T cell donor was male but not female and these sex differences in T cell subset expansion and tissue infiltration were maintained for 7–8 weeks within the male host. Thus, the adaptive immune response and role of pro- and anti-inflammatory cytokine signaling in hypertension is distinct between the sexes and needs to be understood to improve therapeutics for hypertension-associated disease in both men and women.
hypertension; angiotensin; T cells; Rag1−/−-M mice; sex differences
Drainage is a routine practice used to reduce hematoma and blood loss following total hip arthroplasty. The aim of this study was to assess the effect of clamped drainage on blood loss and wound healing after total hip arthroplasty.
A prospective cohort of 44 patients with hip osteoarthritis or femur head necrosis undergoing total hip arthroplasty was randomized equally into two groups: 6-h postoperative clamped or non-clamped suction tube drainage. Body mass index, gender distribution, preoperative hemoglobin, hip pathology, and affected side were comparable between the two groups. Blood loss, hemoglobin levels, and wound healing complications were recorded and compared between groups.
The drainage blood loss and calculated blood loss volumes were higher for the non-clamped group. About 100 mL more blood loss was noticed in the non-clamped group. There was no significant difference in adverse events or need for transfusion.
The present study showed a statistically significant reduction in postoperative drainage amount between clamped and unclamped drainage groups, but this difference was not large enough to warrant increased blood transfusion requirements in patients with unclamped drainage. Further studies are essential to define the critical period of clamping that is compatible with the dual objectives of reduced blood loss and lack of wound complications from hematoma.
Total hip arthroplasty; Clamped drainage; Blood loss; Wound complications
The exact mechanism through which elevated serum ferritin promotes the development of type 2 diabetes is unknown. This study showed that ferritin concentration in impaired glucose regulation and newly diagnosed diabetes mellitus subjects of nonobesity already significantly increased when compared with normal glucose tolerant subjects of nonobesity. Elevated serum ferritin levels are associated with insulin resistance and may be not associated with the decline of insulin beta cells in different status of glucose tolerance in nonobese Han adults.
The accurate localization of intraocular foreign bodies (IOFBs) is very important for the management of ocular trauma patients. B-scan ultrasonography is usually used to detect IOFBs in the posterior segment. Here, we report three cases with IOFBs in the anterior segment near the posterior lens capsule, which were accurately localized by B-scan ultrasonography under dynamic transversal scanning.
All three patients had a history of ocular trauma, and their clinical symptoms were compatible with the persistence of IOFBs. It was difficult to get a direct visualization of IOFBs with slit-lamp biomicroscopy because of opacities of the cornea and traumatic cataract. A computed tomography scan detected IOFBs in the anterior segment, but could not determine the exact location. Ultrasound biomicroscopy was performed but failed to show any IOFBs owing to the limited depth of penetration. B-scan ultrasonography was further applied but also failed to show any intraocular foreign bodies using axial scanning, a routine procedure of B-scan ultrasonography examination. However, using dynamic transversal scanning of B-scan ultrasonography, the accurate location of IOFBs was eventually shown to be embedded in the posterior lens cortex in case 1, adjacent to the posterior lens capsule in case 2, and located in the anterior vitreous close to the posterior lens capsule in case 3. Different surgical procedures were designed according to localization by B-scan ultrasonography, and all IOFBs were successfully removed.
B-scan ultrasonography is a simple and effective imaging modality in the localization of IOFBs in traumatic cataract. Transversal scanning is more suitable than axial scanning to detect IOFBs in the anterior segment near the posterior lens capsule.
Ocular trauma; Intraocular foreign bodies; B-scan ultrasonography; Localization
Novel therapeutic approaches for the treatment of malignant peripheral nerve sheath tumors (MPNSTs) are critically needed. Tyrosine kinase receptors are commonly deregulated in cancer and constitute attractive targets. We assessed the protein expression level of a panel of ‘drugable’ TKRs in a relatively large cohort of human plexiform and MPNST surgical specimens.
Methods and Results
Immunohistochemistry for HER2, PDGFRA, PDGFRB, KIT, IGF-1R, MET, and AXL was performed on an MPNST tissue microarray, yielding data from 99 tumors (plexiform/atypical neurofibroma = 26 and MPNST =73). PDGFRA, PDGFRB, MET, IGFR, and AXL were found to be highly expressed in human MPNST and all but AXL were significantly higher in MPNST as compared to neurofibroma. No HER2 expression was found. KIT expression in tumor cells was uncommon, but highlighted mast intratumoral cells in both neurofibroma and MPNST.
Several TKRs were overexpressed in MPNSTs, exhibiting tumor-to-tumor heterogeneity. When designing future MPNST clinical trials, pre-treatment molecular analysis may help in ‘smart’ patient selection. Furthermore, utilizing single compounds blocking multiple TKRs or therapeutic combinations could constitute a superior anti-MPNST treatment approach.
Pachymic acid (PA), a lanostane-type triterpenoid from Poria cocos, has been reported to possess anti-emetic, anti-inflammatory, and anti-cancer properties. Nonetheless, the anti-tumor effect of PA in lung cancer cells remains unclear. Herein, we report the chemotherapeutic effects and underlying mechanisms of PA against human lung cancer.
The anti-proliferative ability of PA on lung cancer cells was assessed by MTT, colony formation and EdU proliferation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by annexin V/PI double-staining and the DNA ladder formation assays. The expressions of the apoptosis-related proteins were analysed by western blot. The in vivo efficacy of PA was measured using a NCI-H23 xenograft model in nude mice.
PA exhibited anti-tumor effects in vitro accompanied by induction of G2/M phase arrest and apoptosis in NCI-H23 and NCI-H460 lung cancer cells. Mechanistically, our data showed that PA induced reactive oxygen species (ROS) production, resulting in the activation of both c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress apoptotic pathways in lung cancer cells. Moreover, blockage of ROS production reversed PA-induced JNK and ER stress activation. Finally, PA inhibited the growth of NCI-H23 xenograft tumors without causing any host toxicity, and inhibited cell proliferation and induction of apoptosis of tumor cells in tumor xenograft tissues.
In summary, our study demonstrates that PA induces apoptosis through activation of the JNK and ER stress pathways in human lung cancer cells. Our findings provide a rationale for the potential application of PA in lung cancer therapy.
Pachymic acid; Lung cancer; Apoptosis; JNK; ER stress
Micropowder (20–250 µm) made from ground dry waste sludge from a municipal sewage treatment plant was added in a sequencing batch reactor (R2), which was fed by synthetic wastewater with acetate as carbon source. Compared with the traditional SBR (R1), aerobic sludge granulation time was shortened 15 days in R2. Furthermore, filamentous bacteria in bulking sludge were controlled to accelerate aerobic granulation and form large granules. Correspondingly, the SVI decreased from 225 mL/g to 37 mL/g. X-ray Fluorescence (XRF) analysis demonstrated that Al and Si from the micropowder were accumulated in granules. A mechanism hypotheses for the acceleration of aerobic granulation by adding dry sludge micropowder is proposed: added micropowder acts as nuclei to induce bacterial attachment; dissolved matters from the micropowder increase abruptly the organic load for starved sludge to control overgrown filamentous bacteria as a framework for aggregation; increased friction from the movement of micropowder forces the filaments which extend outwards to shrink for shaping granules.
aerobic granulation; granule; filamentous bacteria; micropowder; dry sewage sludge
15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.
15,16-dihydrotanshinone I; acute myeloid leukemia; c-Jun N-terminal kinase; Fas ligand; apoptosis
is affecting the life of millions of people. A large proportion
of diabetic patients suffer from severe complications such as neuropathic
pain, and current treatments for these complications have deleterious
side effects. Thus, alternate therapeutic strategies are needed. Recently,
the elevation of epoxy-fatty acids through inhibition of soluble epoxide
hydrolase (sEH) was shown to reduce diabetic neuropathic pain in rodents.
In this report, we describe a series of newly synthesized sEH inhibitors
with at least 5-fold higher potency and doubled residence time inside
both the human and rodent sEH enzyme than previously reported inhibitors.
These inhibitors also have better physical properties and optimized
pharmacokinetic profiles. The optimized inhibitor selected from this
new series displayed improved efficacy of almost 10-fold in relieving
pain perception in diabetic neuropathic rats as compared to the approved
drug, gabapentin, and previously published sEH inhibitors. Therefore,
these new sEH inhibitors could be an attractive alternative to treat
diabetic neuropathy in humans.
Previous studies have indicated that acupuncture can alleviate the symptoms of Tourette syndrome (TS), but the evidence is insufficient. So far, there have been no reports on plum-blossom needle therapy for TS. Here we present a protocol for a randomized controlled trial using plum-blossom needle therapy to treat TS.
Sixty patients will be randomly allocated into either the plum-blossom needle therapy group or the habit reversal training (HRT) group. All patients in each group will be given 12 weeks of treatment, with follow-up at the 24th week. The primary outcome measure will be the mean change from baseline in the total tic score on the Yale Global Tic Severity Scale (YGTSS) at the 12th week. Secondary outcome measures will include the scores on the TS Clinical Global Impression Scale (CGI) and the mean changes from baseline in the YGTSS score and the Children and Adolescents’ Quality of Life Scale (CAQOL) at other time points. Safety will also be evaluated.
This trial will evaluate the effectiveness and safety of plum-blossom needle therapy for TS compared with HRT. A limitation of this trial is that patients and acupuncturists cannot be blinded.
ClinicalTrials.gov Identifier: NCT02403258 (Date of registration: March 31, 2015).
Acupuncture; Plum-blossom needle; Tourette syndrome; Randomized controlled trial
Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes.
Type 1 diabetes is caused by the body incorrectly destroying the cells in the pancreas—known as β cells—that produce insulin and so control the amount of sugar found in the bloodstream. Drugs that increase the rate at which new β cells form could therefore help to treat this disease.
High-throughput screening is a technique that uses automated systems to rapidly test the effects of large numbers of drug-like compounds on living cells. Unfortunately, drugs sometimes produce different effects in animals than those they produce in isolated cells or other more simplified screening systems.
Zebrafish are often used in biological studies because the larvae are transparent, making it easier to study what goes on inside them. Wang et al. have now developed a high-throughput screening system that uses genetically engineered zebrafish. The zebrafish contain ‘reporter’ genes that fluoresce when a gene is activated, and the intensity of the fluorescence can be interpreted to work out the effects of an applied drug.
To search for compounds that cause β cells to grow, Wang et al. created two reporter genes: one that glows yellow when new β cells form, and one that glows red when other pancreatic cells are stimulated. An initial screen tested the effects of over 3000 drugs, most of which have been approved for use in humans. This screen identified and confirmed 24 drugs that trigger the growth of new β cells or other pancreatic cells in zebrafish larvae. Further investigation uncovered new roles for two signaling pathways that had not previously been linked to pancreatic growth. One pathway—the serotonin pathway, which is better known for transmitting signals in the brain—selectively stimulates the growth of new β cells.
The work of Wang et al. therefore presents a number of possible drugs and pathways that could be targeted in the search for a new treatment for type 1 diabetes. Furthermore, this new whole-organism, high-throughput screening system could be used in the future to search for drugs that affect a range of other biological processes.
high-throughput screening; whole-organism drug discovery; beta cell; diabetes; NF-κB; serotonin; mouse; zebrafish
Patient: Female, 76
Final Diagnosis: Peritoneal mesothelioma epithelioid type
Symptoms: Alternating bowel habits • ascites • small bowel obstruction • weight loss
Clinical Procedure: Exploratory laparotomy • excisional biopsy
Peritoneal mesothelioma is a rare malignancy that affects the serosal surfaces of the peritoneum. The peritoneum is the second most common site of mesothelium affected following the pleura. The aggressive nature and vague presentation pose many obstacles in not only diagnosis but also the treatment of patients with this disease.
We present a case of a 76-year-old woman who presented with small bowel obstruction secondary to carcinomatosis secondary to primary peritoneal mesothelioma. The patient had multiple risk factors with asbestos exposure and prior therapeutic radiation.
We discuss the highly varied and elusive presentation of peritoneal mesothelioma. Cumulative asbestos exposure, either directly or indirectly, remains the leading cause of mesothelioma. However, there are other non-asbestos etiologies. Small bowel obstruction often is a late-presenting symptom of widespread tumor burden. A concise review of the current diagnostic and surgical treatment of primary peritoneal mesothelioma demonstrates that early diagnosis and implementation remains vital.
Mesothelioma; Peritoneal Cavity; Intestinal Obstruction
We aimed to evaluate the efficacy of combination of propranolol and sclerotherapy in treating parotid hemangiomas. Twenty-six parotid hemangiomas patients were subjected to combined treatment from January 2009 and June 2014. The effects of the therapy modality were evaluated. Nineteen patients were females and 7 were males. The median age of treatment initiation was 4.96 months. Twelve lesions were located on the left side parotid glands, while thirteen lesions affected the right side. One infant had bilateral lesions. One to six (average 2.04) injections were performed and the mean period for propranolol was 8.94 months. All the patients got satisfied aesthetic outcomes. No complications of propranolol or sclerotherapy occurred during the whole medication period. The study demonstrated that combination of propranolol and sclerotherapy was an effective and safe method for infantile parotid hemangiomas. Larger-scale studies should be performed to further investigate the long-term efficacy and results of the present combined method for infantile parotid hemangiomas.
Parotid hemangiomas; propranolol; sclerotherapy; complication
The preparative purification of liriodendrin from Sargentodoxa cuneata using macroporous resin combined with crystallization process was evaluated. The properties of adsorption/desorption of liriodendrin on eight macroporous resins were investigated systematically. X-5 resin was selected as the most suitable medium for liriodendrin purification. The adsorption of liriodendrin on X-5 resin fitted well with the pseudo-second-order kinetic model and Langmuir isotherm model. Dynamic adsorption/desorption tests were performed using a glass column packed with X-5 resin to optimize the separation process of liriodendrin. After one treatment with X-5 resin, the content of liriodendrin in the product was increased 48.73-fold, from 0.85% to 41.42%, with a recovery yield of 88.9%. 97.48% liriodendrin was obtained by further crystallization and determined by HPLC. The purified product possessed strong antioxidant activity. In conclusion, purification of liriodendrin might expend its further pharmacological researches and further applications in pharmacy.
We present a simple configuration incorporating a single polarization-sensitive phase-only liquid crystal spatial light modulator (LC-SLM) to facilitate polarization-insensitive spatial light modulation. The polarization-insensitive configuration is formed by a polarization beam splitter (PBS), a polarization-sensitive phase-only LC-SLM, a half-wave plate (HWP), and a mirror in a loop structure. We experimentally demonstrate polarization-insensitive spatial light modulations for incident linearly polarized beams with different polarization states and polarization-multiplexed beams. Polarization-insensitive spatial light modulations generating orbital angular momentum (OAM) beams are demonstrated in the experiment. The designed polarization-insensitive configuration may find promising applications in spatial light modulations accommodating diverse incident polarizations.
The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased—rather than monoallelic—expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.
Most cells in the human body contain two copies of each chromosome—one that was inherited from the individual's mother and one from the father—and therefore contain two copies of every gene. While both copies are usually used equally and simultaneously to produce proteins, in a minority of cases the gene from one parent is silenced in a process known as genomic imprinting. This is generally achieved via the addition of chemical marks onto the DNA, which prevent the molecular machinery that activates genes from accessing the genetic material.
Previous efforts to map imprinting in the brain throughout the mouse genome have yielded inconsistent results, due in part to the large number of factors that can affect gene expression. Perez, Rubinstein, Fernandez et al. have now addressed this issue by applying a combined approach, which includes developing a powerful statistical model that takes into account variation in age, sex, and mouse strain and extensively validating each imprinted gene candidate using an independent experimental technique.
Perez, Rubinstein, Fernandez et al. analyzed genomic imprinting initially in a part of the brain called the cerebellum in both young and adult mice. This analysis confirmed the occurrence of imprinting in 74 genes identified in previous studies, and revealed imprinting for the first time in a further 41 genes. The degree of imprinting varied between genes. In some genes only one copy was expressed and the other was completely silenced whereas others only deviated from the two copies being expressed equally. For individual genes, imprinting varied with age, tending to be more pronounced in young animals than in adults. It also varied between brain regions and typically genes were imprinted more in the brain compared to elsewhere in the body.
Mapping the activities of the imprinted genes revealed that many are involved in regulating the process of controlled cell death, or ‘apoptosis’. For one particular test gene, selectively deleting either the maternal or paternal copy had different effects on the mice, thereby confirming that imprinting can affect brain development and activity. With this in mind, the potential impact of imprinting should also be considered when evaluating the effects of inherited mutations on human health.
genomic imprinting; molecular neuroscience; cerebellum; RNA-seq; apoptosis; Bcl-x; mouse
Availability of diverse genomes makes it possible to predict gene function based on shared evolutionary history. This approach can be challenging, however, for pathways whose components do not exhibit a shared history, but rather, consist of distinct “evolutionary modules.” We introduce a computational algorithm, CLIME (clustering by inferred models of evolution), which inputs a eukaryotic species tree, homology matrix, and pathway (gene set) of interest. CLIME partitions the gene set into disjoint evolutionary modules, simultaneously learning the number of modules and a tree-based evolutionary history that defines each module. CLIME then expands each module by scanning the genome for new components that likely arose under the inferred evolutionary model. Application of CLIME to ∼1000 annotated human pathways, organelles and proteomes of yeast, red algae, and malaria, reveals unanticipated evolutionary modularity and novel, co-evolving components. CLIME is freely available and should become increasingly powerful with the growing wealth of eukaryotic genomes.
Chronic wasting disease (CWD) is a fatal prion disease of North American deer and elk and poses an unclear risk for transmission to humans. Human exposure to CWD occurs through hunting activities and consumption of venison from prion-infected animals. Although the amino acid residues of the prion protein (PrP) that prevent or permit human CWD infection are unknown, NMR-based structural studies suggest that the β2-α2 loop (residues 165–175) may impact species barriers. Here we sought to define PrP sequence determinants that affect CWD transmission to humans. We engineered transgenic mice that express human PrP with four amino acid substitutions that result in expression of PrP with a β2-α2 loop (residues 165–175) that exactly matches that of elk PrP. Compared with transgenic mice expressing unaltered human PrP, mice expressing the human-elk chimeric PrP were highly susceptible to elk and deer CWD prions but were concurrently less susceptible to human Creutzfeldt-Jakob disease prions. A systematic in vitro survey of amino acid differences between humans and cervids identified two additional residues that impacted CWD conversion of human PrP. This work identifies amino acids that constitute a substantial structural barrier for CWD transmission to humans and helps illuminate the molecular requirements for cross-species prion transmission.
Infectious disease; Neuroscience
Rheumatoid arthritis (RA) is an inflammatory joint disorder, the progression of which leads to the destruction of cartilage and bone. Chemokines are involved in RA pathogenesis. In this study, we investigated the chemokine signaling pathway associated with CCL2 in peripheral blood (PB) and synovial tissues (ST) of RA patients based on our previous work about chemokine signaling pathway involved in the activation of CCL2 production in collagen-induced arthritis rat ST.
Materials and Methods
Total RNA was isolated from PB leukocytes and synovium of the knee joint in both RA patients and control populations. Real-time polymerase chain reaction was used to determine CCL4, CCR5, c-Jun, c-Fos, and CCL2 expressions. Serum level of CCL2 was assessed by enzyme-linked immunosorbent assay, and the production of CCL2 in ST was analyzed immunohistochemically.
The expressions of CCL4, CCR5, c-Jun, c-Fos, and CCL2 messenger RNA in RA patients were significantly higher than those in healthy controls, both in ST and on PB leukocyte. Serum CCL2 levels were elevated in RA patients. Histological examination of rheumatoid joints revealed extensive CCL2 expression in RA ST.
CCL2, CCL4, c-Jun, c-Fos, and CCR5 may play an important role in the recruitment of PB leukocytes into the RA joints. These data provide evidence that the chemokine signaling pathway is involved in CCL2 expression in RA patient tissues, which may contribute to chronic inflammation associated with RA. Targeting this signaling pathway may provide a novel therapeutic avenue in RA.
CCL2; leukocytes; rheumatoid; arthritis; synovial membrane