Search

The benefits and long-term effects of extracurricular scientific research on undergraduate students in many countries have been intensively investigated, but it remains obscure for Chinese medical students. In this study, we investigated the outcome of 60 medical students who have participated in extracurricular scientific research at Jinan University Medical School over a period of 7 years (2011-2018). The results revealed that these students have contributed to 31 biomedical science articles in reputable academic journals, as first- or co-authors. Furthermore, they also independently procured various funding based on their research achievements, and smaller awards for achievements in conferences and competitions. Assessment of the grade point average score of these students revealed that conducting extracurricular scientific research did not affect their routine medical study and exam grades (P>0.05). The students benefited from participating in extracurricular research, by acquiring the ability to think scientifically and enhancing their communication skills. In addition, the medical students were motivated to enlist for postgraduate studies so that they could further embark in scientific research. In sum, Chinese medical students are capable of participating in scientific research and make a significant contribution to science.

BRE (Brain and Reproductive Organ-Expressed) is an anti-apoptotic protein and a core component of DNA-repair BRCA1-A complex. Microarray-detected high BRE gene expression has been found to be associated with better patient survival in AML (acute myeloid leukemia) with MLL-AF9 translocation, and radiotherapy-treated non-familial breast cancer. A recent finding suggests that the high BRE gene expression in MLL-AF9 AML could be attributed to the additional expression of a transcript variant encoding a novel C-terminal BRE isoform. Using THP-1 as the MLL-AF9 AML cell model, we found that ectopic expression of the C-terminal BRE, which could not form an intact BRCA1-A complex, indeed increased cellular sensitivity to chemotherapeutic drugs and inhibited cell proliferation, while the complete opposite was achieved by the ectopic expression of full-length BRE. Our findings suggest that the C-terminal BRE-encoding transcript could be responsible for better patient survival and may have therapeutic potential for cancer.

SummaryFibroblast growth factor (FGF) signalling acts as one of modulators that control neural crest cell (NCC) migration, but how this is achieved is still unclear. In this study, we investigated the effects of FGF signalling on NCC migration by blocking this process. Constructs that were capable of inducing Sprouty2 (Spry2) or dominant-negative FGFR1 (Dn-FGFR1) expression were transfected into the cells making up the neural tubes. Our results revealed that blocking FGF signalling at stage HH10 (neurulation stage) could enhance NCC migration at both the cranial and trunk levels in the developing embryos. It was established that FGF-mediated NCC migration was not due to altering the expression of N-cadherin in the neural tube. Instead, we determined that cyclin D1 was overexpressed in the cranial and trunk levels when Sprouty2 was upregulated in the dorsal neural tube. These results imply that the cell cycle was a target of FGF signalling through which it regulates NCC migration at the neurulation stage.

Glioblastoma multiforme (GBM), a malignant tumor of the central nervous system, has a high mortality rate. No curative treatment is presently available, and the most commonly used chemotherapeutic drug, the alkylating agent temozolomide (TMZ), is only able to increase life expectancy and is often associated with drug resistance. Therefore, an urgent need does exist for novel drugs aimed at treating gliomas. In the present study, we obtained three major results using corilagin: (a) demonstrated that it inhibits the growth of U251 glioma cells through activation of the apoptotic pathway; (b) demonstrated that it is also active on TMZ-resistant T98G glioma cells; and (c) demonstrated that when used in combination with TMZ on T98G glioma cells, a higher level of proapototic and antiproliferative effects is observed. Our study indicates that corilagin should be investigated in more detail to determine whether it can be developed as a potential therapeutic agent. In addition, our results suggest that corilagin could be used in combination with low doses of other standard anticancer chemotherapeutic drugs against gliomas (such as TMZ) with the aim of obtaining enhanced anticancer effects.

BRISC and BRCA1-A complex member 2 (Babam2) plays an essential role in promoting cell cycle progression and preventing cellular senescence. Babam2-deficient fibroblasts show proliferation defect and premature senescence compared with their wild-type (WT) counterpart. Pluripotent mouse embryonic stem cells (mESCs) are known to have unlimited cell proliferation and self-renewal capability without entering cellular senescence. Therefore, studying the role of Babam2 in ESCs would enable us to understand the mechanism of Babam2 in cellular aging, cell cycle regulation, and pluripotency in ESCs. For this study, we generated Babam2 knockout (Babam2&minus, /&minus, ) mESCs to investigate the function of Babam2 in mESCs. We demonstrated that the loss of Babam2 in mESCs leads to abnormal G1 phase retention in response to DNA damage induced by gamma irradiation or doxorubicin treatments. Key cell cycle regulators, CDC25A and CDK2, were found to be degraded in Babam2&minus, mESCs following gamma irradiation. In addition, Babam2&minus, mESCs expressed p53 strongly and significantly longer than in control mESCs, where p53 inhibited Nanog expression and G1/S cell cycle progression. The combined effects significantly reduced developmental pluripotency in Babam2&minus, mESCs. In summary, Babam2 maintains cell cycle regulation and pluripotency in mESCs in response to induced DNA damage.

Baicalin is a flavone glycoside that possesses numerous pharmacological properties. but its protective mode of action in kidney injury induced by diabetes mellitus remains incompletely understood. Using a streptozotocin (STZ)-induced diabetic mouse model, we found that baicalin could ameliorate diabetes-induced the pathological changes of the kidney function and morphology through suppressing inflammation and oxidative stress. Furthermore, baicalin treatment could alleviate interstitial fibrosis in the diabetic kidney via inhibiting epithelial-to-mesenchymal transition (EMT), which was accompanied by a sharp upregulation of Klotho, the endogenous inhibitor of renal fibrosis. We further verified that baicalin-rescued expression of Klotho was associated with Klotho promoter hypomethylation due to aberrant methyltransferase 3a expressions. Klotho knockdown via RNA interferences largely abrogated the anti-renal fibrotic effects of Baicalin in HK2 cells. These findings suggested that baicalin could alleviate renal injury-induced by diabates through partly modulating Klotho promoter methylation, which provides new insights into the treatment of diabetic nephropathy.

Background: The functions of microRNA-1 (miR-1) in cardiac hypertrophy, and cardiomyocyte differentiation have been investigated. However, the mechanism on how miR-1 could repress cardiomyocyte proliferation has not been fully elucidated. Methods: We address this issue by investigating whether miR-1 affected the proliferation of neonatal cardiomyocyte and identify some of the genes targeted by miR-1. miR-1 was over-expressed in neonatal cardiomyocytes and the effect on cell cycle and growth were analyzed by flow cytometry and Brdu-incorporation assay. Relevant vectors carrying the luciferase reporter were constructed for validation of miR-1 binding to its matching sites on the 3’-untranslated region of the predicated target mRNAs. Cardiomyocytes were co-transfected with the vectors and miR-1 mimics, then luciferase reporter assay was performed. Lastly, we examined the expression of target genes in cardiomyocytes after transfection with miR-1 mimics, as well as their normal expression pattern in 2- and 13-day-old mice hearts. Results: We have demonstrated that miR-1 was the most significantly upregulated miRNA in 13-day-old mouse hearts compared with 2-day-old hearts. We also showed that miR-1 could repress cardiomyocyte G1/S phase transition, proliferation and viability. IGF1 and CCND1 were identified as candidate target genes regulated by miR-1. In addition, overexpression of miR-1 could suppress the expression of these two genes at the mRNA level. It could also correspondingly inhibit CCND1 expression at the protein level but not for IGF1. Conclusions: Our results suggest that miR-1 plays an important role in inhibiting cardiomyocyte proliferation in the developing neonatal mouse heart by directly suppressing the cell-cycle regulator, CCND1.

Suspension spheroid cultures of anchorage-dependent cell types have been widely used in cancer and stem cell research, as well as for producing organoids. It is believed that the 3-dimensional spheroid presents cells with a more physiological microenvironment to grow so that they behave more like cells in vivo, which is lacking in conventional 2-dimensional monolayer cultures. Recently, it has been reported that cancer cells grown as spheroids could express stem cell-associated genes. Hence, it is investigated whether normal mouse and human fibroblasts cultured as spheroids could also be induced to express stem cell-associated genes. The transcriptomes of human fibroblasts cultured as a monolayer and spheroids are compared and analyzed using real-time RT-qPCR, RNA-sequencing, and bioinformatics. The results reveal that the spheroid transcriptome resemble somatic cells being reprogramed into stem cells, including the induced expression of stemness-associated genes, increased expression of mesenchymal-to-epithelial transition-associated genes, and decreased expression of epithelial-to-mesenchymal transition-associated genes. In this context, it is hypothesized that during the process of spheroid formation, matrix-cell signaling is lost in favor of cell-cell contact signaling and that this subsequently increases the activity of the PI3K/Akt pathway that then upregulates Tbdx3 and stemness-associated genes.

The incidence of gestational diabetes mellitus (GDM) has increased dramatically amongst multiethnic population. However, how gestational diabetes mellitus damages the developing embryo is still unknown. In this study, we used yolk sac membrane (YSM) model to investigate angiogenesis in the developing chick embryo. We determined that in the presence of high glucose, it retarded the growth and extension of the embryonic vascular plexus and it also reduced the density of the vasculature in yolk sac membrane model. Using the same strategy, we used the chorioallantoic membrane (CAM) as a model to investigate the influence of high glucose on the vasculature. We established that high glucose inhibited development of the blood vessel plexus and the blood vessels formed had a narrower diameter than control vessels. Concurrent with the abnormal angiogenesis, we also examined how it impacted cardiogenesis. We determined the myocardium in the right ventricle and left atrium were significantly thicker than the control and also there was a reduction in glycogen content in cardiomyocytes. The high glucose also induced excess reactive oxygen species (ROS) production in the cardiomyocytes. We postulated that it was the excess reactive oxygen species that damaged the cardiomyocytes resulting in cardiac hyperplasia.

Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose‐dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up‐regulation of downstream osteoclast‐related target genes expression. The baicalin‐induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p‐ERK (U0126) and the Mitf‐specific siRNA, respectively. Protein–ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p‐ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p‐ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.

High oxidative stress can occur during ischemic reperfusion and chronic inflammation. It has been hypothesized that such oxidative challenges could contribute to clinical risks such as deep tissue pressure ulcers. Skeletal muscles can be challenged by inflammation-induced or reperfusion-induced oxidative stress. Oxidative stress reportedly can lower the compressive damage threshold of skeletal muscles cells, causing actin filament depolymerization, and reduce membrane sealing ability. Skeletal muscles thus become easier to be damaged by mechanical loading under prolonged oxidative exposure. In this study, we investigated the preventive effect of poloxamer 188 (P188) on skeletal muscle cells against extrinsic oxidative challenges (H2O2). It was found that with 1 mM P188 pre-treatment for 1 h, skeletal muscle cells could maintain their compressive damage threshold. The actin polymerization dynamics largely remained stable in term of the expression of cofilin, thymosin beta 4 and profilin. Laser photoporation demonstrated that membrane sealing ability was preserved even as the cells were challenged by H2O2. These findings suggest that P188 pre-treatment can help skeletal muscle cells retain their normal mechanical integrity in oxidative environments, adding a potential clinical use of P188 against the combined challenge of mechanical-oxidative stresses. Such effect may help to prevent deep tissue ulcer development.

Tumour growth is closely related to the development of new blood vessels to supply oxygen and nutrients to cancer cells. Without the neovascular formation, tumour volumes cannot increase and undergo metastasis. Antiangiogenesis is one of the most promising approaches for antitumour therapy. The exploration of new antiangiogenic agents would be helpful in antitumour therapy. Quinoline is an aromatic nitrogen compound characterized by a double-ring structure which exhibits a benzene ring fused to pyridine at two adjacent carbon atoms. The high stability of quinoline makes it preferable in a variety of therapeutic and pharmaceutical applications, including antitumour treatment. This work is to examine the potential antiangiogenic activity of the synthetic compound 2-Formyl-8-hydroxy-quinolinium chloride. We found that 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of human umbilical vein endothelial cells in vitro. Using the diethylnitrosamine-induced hepatocarcinogenesis model, 2-Formyl-8-hydroxy-quinolinium chloride showed strong antiangiogenic activity. Furthermore, 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of large Hep3B xenografted tumour from the nude mice. We assume that 2-Formyl-8-hydroxy-quinolinium chloride could be a potential antiangiogenic and antitumour agent and it is worthwhile to further study its underlying working mechanism.

Candida albicans (C. albicans) is an opportunistic fungal pathogen, particularly observed in immunocompromised patients. C. albicans accounts for 50–70% of cases of invasive candidiasis in the majority of clinical settings. Terbinafine, an allylamine antifungal drug, has been used to treat fungal infections previously. It has fungistatic activity against C. albicans. Traditional Chinese medicines can be used as complementary medicines to conventional drugs to treat a variety of ailments and diseases. Berberine is a quaternary alkaloid isolated from the traditional Chinese herb, Coptidis Rhizoma, while berberrubine is isolated from the medicinal plant Berberis vulgaris, but is also readily derived from berberine by pyrolysis. The present study demonstrates the possible complementary use of berberine and berberrubine with terbinafine against C. albicans. The experimental findings assume that the potential application of these alkaloids together with reduced dosage of the standard drug would enhance the resulting antifungal potency.

High salt intake has been known to cause hypertension and other side effects. However, it is still unclear whether it also affects fibrosis in the mature or developing liver. This study demonstrates that high salt exposure in mice (4% NaCl in drinking water) and chick embryo (calculated final osmolality of the egg was 300 mosm/L) could lead to derangement of the hepatic cords and liver fibrosis using HE, PAS, Masson, and Sirius red staining. Meanwhile, Desmin immunofluorescent staining of mouse and chick embryo livers indicated that hepatic stellate cells were activated after the high salt exposure. pHIS3 and BrdU immunohistological staining of mouse and chick embryo livers indicated that cell proliferation decreased; as well, TUNEL analyses indicated that cell apoptosis increased in the presence of high salt exposure. Next, dihydroethidium staining on the cultured chick hepatocytes indicated the excess ROS was generated following high salt exposure. Furthermore, AAPH (a known inducer of ROS production) treatment also induced the liver fibrosis in chick embryo. Positive Nrf2 and Keap1 immunohistological staining on mouse liver suggested that Nrf2/Keap1 signaling was involved in high salt induced ROS production. Finally, the CCK8 assay was used to determine whether or not the growth inhibitory effect induced by high salt exposure can be rescued by antioxidant vitamin C. Meanwhile, the RT-PCR result indicated that the Nrf2/Keap1 downsteam genes including HO-1, NQO-1, and SOD2 were involved in this process. In sum, these experiments suggest that high salt intake would lead to high risk of liver damage and fibrosis in both adults and developing embryos. The pathological mechanism may be the result from an imbalance between oxidative stress and the antioxidant system.

Metals and metal complexes are promising antimicrobials due to their powerful inhibitory activity against microbes. However, the potential for metal toxicity to life remains unsolved. Mercury is a toxic element with no known positive biological functions, and its toxicity to humans is well known. With the known toxicity of Hg and its complexes, their usefulness for antimicrobial therapy should not be neglected. In this work, we investigated the antimicrobial activity of bis-(alkynyl)mercury(II) complexes with oligothiophene and bithiazole linking units against MRSA and C. albicans (pathogens commonly co-inhabit infected sites), and their cytotoxicity was tested on NIH 3T3 cells. Among the Hg(II) complexes, complex 2 showed potent microcidal activity against MRSA (MIC and MBC values = 0.2 μg mL−1) and C. albicans (MIC and MBC values = 0.4 μg mL−1). Complex 2 displayed a highly distinguished zone of clearance with comparably sharp zone edges on microbial agar plates. Time–kill kinetic studies showed that MRSA colonies were reduced by approximately 5 logs during the initial 3 h exposure to complex 2 at an 8× MIC level. The possible antimicrobial mechanism might be due to the intracellular ROS generation by complex 2 that caused the loss in MRSA viability. Complex 2 exhibited a higher IC50 value (0.8 μg mL−1) towards NIH 3T3 cells that was greater than its corresponding MIC values against tested microbes. Hg(II) complexes would be potent antimicrobials, but a challenge is to fully utilize their potential inhibitory effects on microbes while lowering their toxic levels on human health.

Otomycosis is a superficial infection of the ear caused by fungi. The disease may occur in the pinna, external auditory canal and tympanic membrane of humans and animals. Aspergillus niger and Candida albicans are the most prevalent fungal species responsible for otomycosis. In our study, the potential antifungal properties of a series of Schiff bases was examined. The synthesized compounds (1–14) were investigated for their minimum inhibitory concentration, minimum fungicidal concentration and zone of clearance against Aspergillus niger and Candida albicans. Compounds 5 and 6 with alkyl chain containing 6 or 7 carbon atoms exhibited an obvious fungicidal activity with the MIC/MFC ratio of 2. Zone of clearance study also demonstrated that both compounds 5 and 6 produced noticeable clear zones against the tested fungi as compared to fluconazole. The time-kill kinetic study showed that Candida albicans colonies were reduced by more than 3 logs after 24 h exposure to compound 5 at 4 and 8 × MIC levels. Both compounds 5 and 6 at a concentration of 50 μg mL−1 exhibited satisfactory cell viability towards human skin keratinocytes. Our results suggest that these compounds may be considered as potential drug candidates for the treatment of Aspergillus niger and Candida albicans ear infections.

Excess alcohol consumption during pregnancy has been acknowledged to increase the incidence of congenital disorders, especially the cardiovascular system. However, the mechanism involved in ethanol-induced cardiac malformation in prenatal fetus is still unknown. We demonstrated that ethanol exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Previously, we reported that autophagy was involved in heart tube formation. In this context, we demonstrated that ethanol exposure increased ATG7 and LC3 expression. mTOR was found to be inhibited by ethanol exposure. We activated autophagy using exogenous rapamycin (RAPA) and observed that it induced cardiac bifida and increased GATA5 expression. RAPA beads implantation experiments revealed that RAPA restricted ventricular myosin heavy chain (VMHC) expression. In vitro explant cultures of anterior primitive streak demonstrated that both ethanol and RAPA treatments could reduce cell differentiation and the spontaneous beating of cardiac precursor cells. In addition, the bead experiments showed that RAPA inhibited GATA5 expression during heart tube formation. Semiquantitative RT-PCR analysis indicated that BMP2 expression was increased while GATA4 expression was suppressed. In the embryos exposed to excess ethanol, BMP2, GATA4 and FGF8 expression was repressed. These genes are associated with cardiomyocyte differentiation, while heart tube fusion is associated with increased Wnt3a but reduced VEGF and Slit2 expression. Furthermore, the ethanol exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. In sum, our results revealed that disrupting autophagy and excess ROS generation are responsible for inducing abnormal cardiogenesis in ethanol-treated chick embryos.

Bag1 transcribes a multifunctional protein that participates in many important biological processes such as cell apoptosis, proliferation, differentiation and embryo development. Despite numerous published studies, the role of Bag1 in the context of embryonic stem (ES) cells, has not been explored. To investigate the function of Bag1 in ES cells, we generated mutant Bag1−/− ES cells using the CRISPR/Cas9 system. We established that the Bag1 double knockout ES cell line maintained their pluripotency, possessed a normal karyotype and the ability to differentiate into all three germ layers.

Baicalin is a flavone glycoside that possess numerous pharmacological properties including anti-inflammatory, anti-cancer and anti-pruritic effects. Based on these properties, we investigated whether baicalin could be used to alleviate kidney fibrosis induced by diabetes mellitus. Using a streptozotocin (STZ)-induced diabetic mouse model, we found that baicalin treatment could suppress diabetes-enhanced water consumption, urine volume production, blood urea nitrogen (BUN), serum creatinine and urinary albumin/urinary creatinine (UACR). Correspondingly, baicalin treatment could significantly maintain the morphology of the glomerulus and renal tubules in diabetes-induced kidney lesions by repressing WT1 and Nephrin expressions in the glomeruli, as well as LTL, caspases-9 and Fas expressions in the renal tubules. We also determined that baicalin could suppress inflammation responses and reduced MDA activities that were enhnaced by diabetes while increasing SOD, CAT, GPX and Nrf2 expressions that were inversely lowered. We demonstrated the presence of interstitial fibrosis in the diabetic kidney but this could be alleviated by baicalin which downregulated ɑ-SMA and TGF-β1 expressiond (key pro-fibrotic markers). We investigated Klotho a transmembrane protein that is expressed by renal tubule epithelial cells. Secreted form of Klotho could bind to TGF-β1 receptors and block the TGFβ1 signaling pathway. We determined that hypermethylation of the Klotho promoter plays an important role in baicalin's ability to rescue renal fibrosis induced by diabetes mellitus. In sum, our results showed that baicalin could alleviate renal fibrosis-induced by diabetes through partly modulating Klotho promoter methylation. Funding Statement: This study was supported by NSFC grant (31771331, 81741016), Science and Technology Planning Project of Guangdong Province (2017A050506029, 2017A020214015, 2016B030229002), Science and Technology Program of Guangzhou (201710010054), Guangdong Natural Science Foundation (2016A030311044), Project of National University Students "Challenge Cup" (18112003). Declaration of Interests: The authors declare that there are no competing financial interests. Ethics Approval Statement: This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Ethic Committee at Jinan University.

Antimicrobial resistance of disease-related microorganisms is considered a worldwide prevalent and serious issue which increases the failure of treatment outcomes and leads to high mortality. Considering that the increased resistance to systemic antimicrobial therapy often needs of the use of more toxic agents, topical antimicrobial therapy emerges as an attractive route for the treatment of infectious diseases. The topical antimicrobial therapy is based on the absorption of high drug doses in a readily accessible skin surface, resulting in a reduction of microbial proliferation at infected skin sites. Topical antimicrobials retain the following features: (a) they are able to escape the enzymatic degradation and rapid clearance in the gastrointestinal tract or the first-pass metabolism during oral administration; (b) alleviate the physical discomfort related to intravenous injection; (c) reduce possible adverse effects and drug interactions of systemic administrations; (d) increase patient compliance and convenience; and (e) reduce the treatment costs. Novel antimicrobials for topical application have been widely exploited to control the emergence of drug-resistant microorganisms. This review provides a description of antimicrobial resistance, common microorganisms causing skin and soft tissue infections, topical delivery route of antimicrobials, safety concerns of topical antimicrobials, recent advances, challenges and future prospective in topical antimicrobial development.

It is now known that excess alcohol consumption during pregnancy can cause fetal alcohol syndrome to develop. However, it is not known whether excess ethanol exposure could directly affect angiogenesis in the embryo or angiogenesis being indirectly affected because of ethanol-induced fetal alcohol syndrome. Using the chick yolk sac membrane (YSM) model, we demonstrated that ethanol exposure dramatically inhibited angiogenesis in the YSM of 9-day-old chick embryos, in a dose-dependent manner. Likewise, the anti-angiogenesis effect of ethanol could be seen in the developing vessel plexus (at the same extra-embryonic regions) during earlier stages of embryo development. The anti-angiogenic effect of ethanol was found associated with excess reactive oxygen species (ROS) production; as glutathione peroxidase activity increased while superoxide dismutase 1 and 2 activities decreased in the YSMs. We further validated this observation by exposing chick embryos to 2,2′-azobis-amidinopropane dihydrochloride (a ROS inducer) and obtained a similar anti-angiogenesis effect as ethanol treatment. Semiquantitative reverse transcription–polymerase chain reaction analysis of the experimental YSMs revealed that expression of angiogenesis-related genes, vascular endothelial growth factor and its receptor, fibroblast growth factor 2 and hypoxia-inducible factor, were all repressed following ethanol and 2,2′-azobis-amidinopropane dihydrochloride treatment. In summary, our results suggest that excess ethanol exposure inhibits embryonic angiogenesis through promoting superfluous ROS production during embryo development. Copyright © 2015 John Wiley & Sons, Ltd.

It is now known that over-consumption of caffeine by pregnant mothers could have detrimental effects on normal fetal development. However, it remains obscure how caffeine's harmful effect impacts directly or indirectly on the developing embryo/fetus through damaging placenta development. In this study, we demonstrated the morphological similarities between the yolk sac and chorioallantoic membranes (CAM) of chick embryos and the villi of the mammalian placenta. Using the chick yolk sac and the CAM as a model, we found that 5–15 µmol per egg of caffeine exposure inhibited angiogenesis. Under the same condition, cell proliferation in extraembryonic mesoderm was reduced while apoptosis was enhanced. Semi-quantitative RT-PCR analysis revealed that caffeine treatment down-regulated VEGF, VEGFR2, PIGF, IGF2 and NRP1 expression, but up-regulated Ang1 and Ang2 expression. We performed in situ hybridization to show VE-cadherin expression and as to demonstrate the blood vessels in the CAM and yolk sac membranes. This distribution of the VE-cadherin+ blood vessels was determined to be reduced after caffeine treatment. Furthermore, MDA activity was induced after caffeine exposure, but GSH-PX activity was inhibited after caffeine exposure; SOD activity was unchanged as compared with the control. In summary, our results suggest that caffeine exposure could negatively impact on angiogenesis in the chick yolk sac and CAM by targeting angiogenesis-related genes. Some of these genes are also involved in regulating excess ROS generation. The results implied that the negative impact of caffeine on fetal development was partly attributed to impaired placental angiogenesis. Copyright © 2015 John Wiley & Sons, Ltd.

Many human disorders induce high salinity in tissues and organs, interfering with their normal physiological functions. Using a mouse model, we demonstrated that high salt intake caused infertility. Specifically, we established that high salinity dramatically affects ovarian follicle development and the extent of follicular atresia. However, it did not significantly influence the primordial follicles. TUNEL assays revealed that high salt intake inhibited follicle development by inducing the granulosa and theca cells that surround the oocytes to undergo apoptosis. Furthermore, immunohistological staining for the proliferation markers Ki67 and PH3 showed that high salt intake also repressed granulosa cell proliferation. In vitro testing of granulosa cells also confirmed that high salt significantly repressed cell proliferation and promoted cell apoptosis. In summary, high salt consumption negatively impacts reproductive functions in female mice by interfering with ovarian folliculogenesis.

Cell membranes are susceptible to biophysical damages. These biophysical damages often present themselves in challenging oxidative environments, such as in chronic inflammation. Here we report the damage evolution after single myoblasts were individually subjected to femtosecond (fs) laser photoporation on their plasma membranes under normal and oxidative conditions. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscope. The post-damage wound evolution was documented by real-time imaging. The fs laser could generate a highly focused hole at a targeted site of the myoblast plasma membrane. The initial hole size depended on the laser dosage in terms of power and exposure duration. With the same laser power and irradiation duration, photoporation invoked bigger holes in the oxidative groups than in the control. Myoblasts showed difficulty in repairing holes with initial size beyond certain threshold. Within the threshold, holes could apparently be resealed within 100 s under the normal condition; while in oxidative condition, the resealing process could take 100-300 s. The hole-resealing capacity of myoblasts was compromised under oxidative stress particularly when the oxidative exposure was chronic. It is interesting to note that brief exposure to oxidative stress apparently could promote resealing in myoblasts after photoporation.

Sorafenib has been used as an oral anti-cancer drug because of its ability to inhibit tumor growth. However, the pharmacological effect of sorafenib is still the lack of in vivo experimental evidence. Tumor and embryonic cells share some similar features, so we investigated the effects of sorafenib on the development of gastrulating chick embryos. We found that sorafenib exposure was markedly attributed to the number of embryonic cell in proliferation and apoptosis. We also detected sorafenib significantly interfered with epithelial-mesenchymal transition (EMT). Furthermore, sorafenib treatment impaired the production and migration of neural crest cells.

n-Hexane is widely used in industry and its metabolite, 2,5-hexanedione (2,5-HD), has been implicated as a neural toxin in the developing fetus. Using the chick embryo model, we have previously revealed the neurotoxicity of 2,5-HD during development and established that high dose of 2,5-HD was embryo lethal. In view of the close linkage in biology for neurogenesis and angiogenesis, we speculated that it was most likely caused by cardiovascular dysplasia, therefore in this study, we investigated the effects of 2,5-HD on the development of the vasculature, which involves vasculogenesis and angiogenesis. Using gastrulating chick embryos as a model, we demonstrated that the hemangioblasts (precursor of hematopoietic and endothelial cells) migrated to the area opaca where they form the blood islands. However, this process was impaired when the embryos were treated with 2,5-HD, suggesting that 2,5-HD is capable of impairing vasculogenesis. To study the effect of 2,5-HD exposure on angiogenesis, we used the chick yolk-sac membrane (YSM) and chorioallantoic membrane (CAM) models. We found that, at low (0.02M) concentration, 2,5-HD stimulated angiogenesis while at higher concentrations (>0.1M) it inhibited this process. This biphasic response of angiogenesis to 2,5-HD exposure was found to be associated with altered expression of the VEGF-R, FGF-2 and angiogenin. Moreover, we also determined that 2,5-HD exposure increased the reactive oxygen species (ROS) production. In conclusion, 2,5-HD could induce dysplasia in the developing vasculature, which in turn could cause extravascular hemolysis and the embryos to die.

This is the first study of the role of BRE in embryonic development using early chick embryos. BRE is expressed in the developing neural tube, neural crest cells, and somites. BRE thus plays an important role in regulating neurogenesis and indirectly somitogenesis during early chick embryo development., The brain and reproductive expression (BRE) gene is expressed in numerous adult tissues and especially in the nervous and reproductive systems. However, little is known about BRE expression in the developing embryo or about its role in embryonic development. In this study, we used in situ hybridization to reveal the spatiotemporal expression pattern for BRE in chick embryo during development. To determine the importance of BRE in neurogenesis, we overexpressed BRE and also silenced BRE expression specifically in the neural tube. We established that overexpressing BRE in the neural tube indirectly accelerated Pax7+ somite development and directly increased HNK-1+ neural crest cell (NCC) migration and TuJ-1+ neurite outgrowth. These altered morphogenetic processes were associated with changes in the cell cycle of NCCs and neural tube cells. The inverse effect was obtained when BRE expression was silenced in the neural tube. We also determined that BMP4 and Shh expression in the neural tube was affected by misexpression of BRE. This provides a possible mechanism for how altering BRE expression was able to affect somitogenesis, neurogenesis, and NCC migration. In summary, our results demonstrate that BRE plays an important role in regulating neurogenesis and indirectly somite differentiation during early chick embryo development.

Excess alcohol consumption during pregnancy could lead to fetal alcohol syndrome (FAS). However, the molecular mechanism leading to craniofacial abnormality, a feature of FAS, is still poorly understood. The cranial neural crest cells (NCCs) contribute to the formation of the craniofacial bones. Therefore, NCCs exposed to ethanol was investigated - using chick embryos and in vitro explant culture as experimental models. We demonstrated that exposure to 2% ethanol induced craniofacial defects, which includes parietal defect, in the developing chick fetus. Immunofluorescent staining revealed that ethanol treatment downregulated Ap-2ɑ, Pax7 and HNK-1 expressions by cranial NCCs. Using double-immunofluorescent stainings for Ap-2ɑ/pHIS3 and Ap-2ɑ/c-Caspase3, we showed that ethanol treatment inhibited cranial NCC proliferation and increased NCC apoptosis, respectively. Moreover, ethanol treatment of the dorsal neuroepithelium increased Laminin, N-Cadherin and Cadherin 6B expressions while Cadherin 7 expression was repressed. In situ hybridization also revealed that ethanol treatment up-regulated Cadherin 6B expression but down-regulated slug, Msx1, FoxD3 and BMP4 expressions. In summary, our experimental results demonstrated that ethanol treatment interferes with the production of cranial NCCs by affecting the proliferation and apoptosis of these cells. In addition, ethanol affected the delamination, epithelial-mesenchymal transition (EMT) and cell migration of cranial NCCs, which may have contributed to the etiology of the craniofacial defects.

The external epithelial surfaces of plants and animals are frequently carpeted with small micro- and nanostructures, which broadens their adaptive capabilities in challenging physical habitats. Hairs and other shaped protuberances manage with excessive water, light contaminants, predators or parasites in innovative ways. We are interested in transferring these intricate architectures onto biomedical devices and daily-life surfaces. Such a project requires a very rapid and accurate small-scale fabrication process not involving lithography. In this study, we describe a simple benchtop biotemplating method using shed gecko lizard skin that generates duplicates that closely replicate the small nanotipped hairs (spinules) that cover the original skin. Synthetic replication of the spinule arrays in popular biomaterials closely matched the natural spinules in length. More significantly, the shape, curvature and nanotips of the synthetic arrays are virtually identical to the natural ones. Despite some small differences, the synthetic gecko skin surface resisted wetting and bacterial contamination at the same level as natural shed skin templates. Such synthetic gecko skin surfaces are excellent platforms to test for bacterial control in clinical settings. We envision testing the biocidal properties of the well-matched templates for fungal spores and viral resistance in biomedicine as well as co/multi-cultures.

Glucocorticoids are important regulators of cell differentiation and mesenchymal cell lineage commitment during skeletogenesis. In clinical practice, it has been difficult to study the effects of glucocorticoids on target tissues because patients taking glucocorticoids often suffer from adverse skeletal effects. Dexamethasone (Dex) is a long-acting synthetic corticosteroid hormone that ranks amongst the most widely used prescribed drugs, and it is a powerful medication that is increasingly employed during the perinatal and neonatal periods. However, Dex is a potential teratogen. In particular, it has been claimed that Dex exposure during pregnancy can affect osteogenesis in the developing embryo, although this claim remains highly controversial. In this review, we summarize the published data from numerous clinical follow-up, animal-based and in vitro studies on the effects of Dex exposure on embryonic skeletogenesis. These studies indicate that Dex may adversely affect skeletal progenitor cells during development. In addition, Dex can exert a number of effects on bone growth at different developmental stages. We also discuss how glucocorticoids influence the BMP, FGF, Hedgehog and Wnt signaling pathways, which are key regulators of skeletogenesis in the embryo. A fuller understanding of the negative, and perhaps teratogenic, effects of Dex on skeletogenesis will have important implications for the routine use of Dex in clinical practice.

Background The developing embryo is very sensitive to oxidative stress and excess reactive oxygen species (ROS) generation is often associated with cardiovascular malformation. However, little is known about the adverse effects of ROS during heart morphogenesis, especially during the formation of the atria and ventricles. Methods and Results We have treated early chick embryos with 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) to generate free radicals in the developing heart. We established that excess ROS induced by AAPH caused cardiomegaly to develop in 4-, 14- and 17-day-old embryos. The cardiomyocytes of these AAPH-treated hearts were hypertrophic, in both the compact and trabeculated myocardium. The weight of these hearts was also significantly increased in an AAPH dose-dependent fashion. We examined and compared the functions of the AAPH-treated and untreated hearts by echocardiography and determined that the ejection fraction was shortened. BrdU incorporation assay was performed and revealed that cell proliferation was not the main cause of cardiomegaly. However, we established that the cardiomyocytes exposed to excess ROS were distinctively larger than control cardiomyocytes — indicting that cardiomegaly was attributed to hypertrophy. We have also found that excess ROS inhibited Wnt signaling but enhanced VEGF signaling. Consequently, this promoted angiogenesis and caused larger coronary arteries to develop in the AAPH-treated hearts. Conclusions We have demonstrated that cardiomyocyte hypertrophy and changes in Wnt and VEGF signaling were the main contributing factors in the development of cardiomegaly induced by oxidative stress.

Angiogenesis is involved in the development, progression and metastasis of various human cancers. Herein, we report the discovery of glipizide, a widely used drug for type 2 diabetes mellitus, as a promising anticancer agent through the inhibition of tumor angiogenesis. By high-throughput screening (HTS) of an FDA approved drug library utilizing our in vivo chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, glipizide has been identified to significantly inhibit blood vessel formation and development. Moreover, glipizide was found to suppress tumor angiogenesis, tumor growth and metastasis using xenograft tumor and MMTV-PyMT transgenic mouse models. We further revealed that the anticancer capability of glipizide is not attributed to its antiproliferative effects, which are not significant against various human cancer cell lines. To investigate whether its anticancer efficacy is associated with the glucose level alteration induced by glipizide application, glimepiride, another medium to long-acting sulfonylurea antidiabetic drug in the same class, was employed for the comparison studies in the same fashion. Interestingly, glimepiride has demonstrated no significant impact on the tumor growth and metastasis, indicating that the anticancer effects of glipizide is not ascribed to its antidiabetic properties. Furthermore, glipizide suppresses endothelial cell migration and the formation of tubular structures, thereby inhibiting angiogenesis by up-regulating the expression of natriuretic peptide receptor A. These findings uncover a novel mechanism of glipizide as a potential cancer therapy, and also for the first time, provide direct evidence to support that treatment with glipizide may reduce the cancer risk for diabetic patients.

Heart formation commences from a single heart tube, which fuses from bilateral primordial heart fields. The developing heart tube is composed of outer-layer myocardial cells and inner-layer endocardial cells. Several distinct populations of precardiac cells contribute to cardiac morphogenesis. However, it still remains not very clear about the lineage of endocardium at gastrulation stage. Thereby, this study focused on ascertaining the correlation between the hypoblast in gastrulation and endocardium during cardiogenesis. Firstly, the fusing heart tube morphologically is closed to endoderm-derived pharynx floor, implying the possibility that pharynx floor might be wrapped into the formation of endoderm. Secondly, HNK1 is expressed in hypoblast strongly at gastrula stage and subsequently appeared in endocardium of cardiogenesis. Moreover, fate map data displayed that DiI labeled hypoblast was also present in endocardium later on. One more evidence is chick-quail chimera of hypoblast transplantation, in which quail-hypoblast derivative could be identified in endocardium of cardiogenesis by QCPN antibody. In sum, our current data suggests that endoderm in gastrula contribute at least partly to the formation of endocardium of cardiogenesis.

Gelatin/Collagen-based matrix and reservoir nanoparticles require crosslinkers to stabilize the formed nanosuspensions, considering that physical instability is the main challenge of nanoparticulate systems. The use of crosslinkers improves the physical integrity of nanoformulations under the-host environment. Aldehyde-based fixatives, such as formaldehyde and glutaraldehyde, have been widely applied to the crosslinking process of polymeric nanoparticles. However, their potential toxicity towards human beings has been demonstrated in many previous studies. In order to tackle this problem, D-glucose was used during nanoparticle formation to stabilize the gelatin/collagen-based matrix wall and reservoir wall for the deliveries of Calendula officinalis powder and oil, respectively. In addition, therapeutic selectivity between malignant and normal cells could be observed. The C. officinalis powder loaded nanoparticles significantly strengthened the anti-cancer effect towards human breast adenocarcinoma MCF7 cells and human hepatoma SKHep1 cells when compared with the free powder. On the contrary, the nanoparticles did not show significant cytotoxicity towards normal esophageal epithelial NE3 cells and human skin keratinocyte HaCaT cells. On the basis of these evidences, D-glucose modified gelatin/collagen matrix nanoparticles containing C. officinalis powder might be proposed as a safer alternative vehicle for anti-cancer treatments.

Caffeine has been an integral component of our diet and medicines for centuries. It is now known that over consumption of caffeine has detrimental effects on our health, and also disrupts normal foetal development in pregnant mothers. In this study, we investigated the potential teratogenic effect of caffeine over-exposure on eye development in the early chick embryo. Firstly, we demonstrated that caffeine exposure caused chick embryos to develop asymmetrical microphthalmia and induced the orbital bone to develop abnormally. Secondly, caffeine exposure perturbed Pax6 expression in the retina of the developing eye. In addition, it perturbed the migration of HNK-1(+) cranial neural crest cells. Pax6 is an important gene that regulates eye development, so altering the expression of this gene might be the cause for the abnormal eye development. Thirdly, we found that reactive oxygen species (ROS) production was significantly increased in eye tissues following caffeine treatment, and that the addition of anti-oxidant vitamin C could rescue the eyes from developing abnormally in the presence of caffeine. This suggests that excess ROS induced by caffeine is one of the mechanisms involved in the teratogenic alterations observed in the eye during embryogenesis. In sum, our experiments in the chick embryo demonstrated that caffeine is a potential teratogen. It causes asymmetrical microphthalmia to develop by increasing ROS production and perturbs Pax6 expression.

Brain and Reproductive Organ Expressed (BRE), or BRCC45, is a death receptor-associated antiapoptotic protein, which is also involved in DNA-damage repair, and K63-specific deubiquitination. BRE overexpression attenuates both death receptor- and stress-induced apoptosis, promotes experimental tumor growth, and is associated with human hepatocellular and esophageal carcinoma. How BRE mediates its antiapoptotic function is unknown. Here we report based on the use of a mouse Lewis lung carcinoma cell line D122 that BRE has an essential role in maintaining the cellular protein level of XIAP, which is the most potent endogenous inhibitor of the caspases functioning in both extrinsic and intrinsic apoptosis. shRNA-mediated exhaustive depletion of BRE sensitized D122 cells to apoptosis induced not only by etopoxide, but also by TNF-α even in the absence of cycloheximide, which blocks the synthesis of antiapoptotic proteins by TNF-α-activated NF-κB pathway. In BRE-depleted cells, protein level of XIAP was downregulated, but not the levels of other antiapoptotic proteins, cIAP-1, 2, and cFLIP, regulated by the same NF-κB pathway. Reconstitution of BRE restored XIAP levels and increased resistance to apoptosis. XIAP mRNA level was also reduced in the BRE-depleted cells, but the level of reduction was less profound than that of the protein level. However, BRE could not delay protein turnover of XIAP. Depletion of BRE also increased tumor cell apoptosis, and decreased both local and metastatic tumor growth. Taken together, these findings indicate that BRE and its XIAP-sustaining mechanism could represent novel targets for anti-cancer therapy.

1,1-Dimethyl-4-phenyl piperazine iodide (DMPP) is a synthetic nicotinic acetylcholine receptor (nAChR) agonist that could reduce airway inflammation. In this study, we demonstrated that DMPP could dramatically inhibit glioma size maintained on the chick embryonic chorioallantoic membrane (CAM). We first performed MTT and BrdU incorporation experiments on U87 glioma cells in vitro to understand the mechanism involved. We established that DMPP did not significantly affect U87 cell proliferation and survival. We speculated that DMPP directly caused the tumor to regress by affecting the vasculature in and around the implanted tumor on our chick CAM model. Hence, we conducted detailed analysis of DMPP's inhibitory effects on angiogenesis. Three vasculogenesis and angiogenesis in vivo models were used in the study which included (1) early chick blood islands formation, (2) chick yolk-sac membrane (YSW) and (3) CAM models. The results revealed that DMPP directly suppressed all developmental stages involved in vasculogenesis and angiogenesis - possibly by acting through Ang-1 and HIF-2α signaling. In sum, our results show that DMPP could induce glioma regression grown on CAM by inhibiting vasculogenesis and angiogenesis.