Anti-GAPDH Antibody [GA1R] (A85271)

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

We present the crystal structure and biochemical characterization of Escherichia coli YbiB, a member of the hitherto uncharacterized TrpD2 protein family. Our results demonstrate that the functional diversity of proteins with a common fold can be far greater than predictable by computational annotation. The TrpD2 proteins show high structural homology to anthranilate phosphoribosyltransferase (TrpD) and nucleoside phosphorylase class II enzymes but bind with high affinity (KD = 10-100 nM) to nucleic acids without detectable sequence specificity. The difference in affinity between single- and double-stranded DNA is minor. Results suggest that multiple YbiB molecules bind to one longer DNA molecule in a cooperative manner. The YbiB protein is a homodimer that, therefore, has two electropositive DNA binding grooves. But due to negative cooperativity within the dimer, only one groove binds DNA in in vitro experiments. A monomerized variant remains able to bind DNA with similar affinity, but the negative cooperative effect is eliminated. The ybiB gene forms an operon with the DNA helicase gene dinG and is under LexA control, being induced by DNA-damaging agents. Thus, speculatively, the TrpD2 proteins may be part of the LexA-controlled SOS response in bacteria.

Establishment of cell polarity--or symmetry breaking--relies on local accumulation of polarity regulators. Although simple positive feedback is sufficient to drive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. Here, by integrating mathematical modelling with quantitative experimental validations, we show that in the yeast Saccharomyces cerevisiae a combination of actin- and guanine nucleotide dissociation inhibitor-dependent recycling of the central polarity regulator Cdc42 is needed to establish robust cell polarity at a single site during yeast budding. The guanine nucleotide dissociation inhibitor pathway consistently generates a single-polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and is therefore sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but cannot restrict polarization to a single site. Our results demonstrate how cells optimize symmetry breaking through coupling between multiple feedback loops.

LINE-1 (L1) retrotransposons are mobile genetic elements comprising ~17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic β-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2(-/-) mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.

BACKGROUND:

Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra.

RESULTS:

We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants.

CONCLUSIONS:

In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7.

Glioblastoma is an aggressive, incurable brain cancer with poor five-year survival rates of around 13% despite multimodal treatment with surgery, DNA-damaging chemoradiotherapy and the recent addition of Tumour Treating Fields (TTFields). As such, there is an urgent need to improve our current understanding of cellular responses to TTFields using more clinically and surgically relevant models, which reflect the profound spatial heterogeneity within glioblastoma, and leverage these biological insights to inform the rational design of more effective therapeutic strategies incorporating TTFields. We have recently reported the use of preclinical TTFields using the inovitro^TM system within 2D glioma stem-like cell (GSC) models and demonstrated significant cytotoxicity enhancement when co-applied with a range of therapeutically approved and preclinical DNA damage response inhibitors (DDRi) and chemoradiotherapy. Here we report the development and optimisation of preclinical TTFields delivery within more clinically relevant 3D scaffold-based primary GSC models of spatial heterogeneity, and highlight some initial enhancement of TTFields potency with temozolomide and clinically approved PARP inhibitors (PARPi). These studies, therefore, represent an important platform for further preclinical assessment of TTFields-based therapeutic strategies within clinically relevant 3D GSC models, aimed towards accelerating clinical trial implementation and the ultimate goal of improving the persistently dire survival rates for these patients.

Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. Genetically encoded biocontainment systems may solve this issue. Here, we describe such a system based on conditional stability of essential proteins. We used a destabilizing domain degron stabilized by estradiol addition (ERdd). We ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of <5×10^-7. Removal of its C-terminal domain decreased the escape frequency further to <10^-8. Being based on conditional protein stability, the presented approach is mechanistically orthogonal to previously reported genetic biocontainment systems.

Lung cancer is the most common cause of cancer- associated mortality for men and women worldwide. An increasing number of studies have reported that the abnormal expression of microRNAs contributes to the pathogenesis of the majority of human cancer types, including non-small cell lung cancer (NSCLC). The present study aimed to measure microRNA-650 (miR-650) expression in NSCLC and evaluate its function in NSCLC cells. Reverse transcription-quantitative polymerase chain reaction was used to determine miR-650 expression in NSCLC tissue samples and cell lines. Assays for cell proliferation, migration and invasion were performed to investigate the roles of miR-650 on NSCLC progression. Furthermore, the mechanisms underlying the effects of miR-650 on NSCLC cell growth and metastasis were determined. In the current study, miR-650 was demonstrated to be highly expressed in NSCLC tissue samples and cell lines. Inhibition of expression of miR-650 suppressed NSCLC cell proliferation, migration and invasion in vitro. Additionally, large tumor suppressor kinase 2 (LATS2) was identified as a direct target gene of miR-650 in NSCLC. LATS2 was revealed to be significantly downregulated in NSCLC tissues and was negatively correlated with miR-650 expression. Notably, LATS2 re-expression decreased NSCLC cell proliferation, migration and invasion; similar to the effects induced by miR-650 underexpression. In conclusion, the results of the current study suggest that miR-650 may serve as an oncogene by direct targeting LATS2 in NSCLC formation and progression.

BACKGROUND MiR-301a and miR-301b are 2 oncomiRs involved in multiple types of cancer. In this study, we explored the expression change of miR-301a and miR-301b in prostate cancer cells in hypoxia and studied their regulation of autophagy and radiosensitivity of prostate cancer cells. MATERIAL AND METHODS QRT-PCR was performed to quantify the expression change of miR-301a and miR-301b in hypoxia. Their effects on autophagy were measured by Western blot analysis, and their effects on radiosensitivity were measured by clonogenic assay and flow cytometry. In addition, the regulation of miR-301a and miR-301b on NDRG2, a tumor-suppressor gene in prostate cancer, was also studied. The effect of miR-301a/b-NDRG2 axis on autophagy and radiosensitivity of prostate cancer cells was further investigated. RESULTS MiR-301a and miR-301b are 2 hypoxia responsive miRNAs that are significantly upregulated in hypoxia in prostate cancer cells. Higher level of miR-301a and miR-301b expression results in elevated autophagy and increased radioresistance in LNCaP cells. MiR-301a and miR-301b simultaneously target NDRG2 and decrease its expression. Knockdown of NDRG2 leads to increased autophagy and radioresistance. CONCLUSIONS MiR-301a and miR-301b are 2 hypoxia-responsive miRNAs that decrease autophagy of prostate cancer cells in hypoxia by targeting NDRG2. Through downregulating NDRG2, miR-301a and miR-301b can promote radioresistance of prostate cancer cells.

Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border.

Although microRNA-155 (miR-155) is known to play an important role in many cancers, its expression and function in oral squamous cell carcinoma (OSCC) was not fully understood. Thus, in the present study, we investigated the expression of miR-155 and also the role this miR plays in OSCC. We used the OSCC cell line (CAL27) and paired tumor and non-tumor tissue samples from patients with OSCC in order to detect the expression of miR-155. Cell proliferation, migration and invasion assays were then undertaken in order to determine the effect of miR-155 on the biological behavior of CAL27 cells following transient transfection with miR-155 mimic and antagomir. The regulatory effect of miR-155 on its target gene B-cell CLL/lymphoma 6 (BCL6) and downstream gene cyclin D2 (CCND2) was also analyzed. We found that miR-155 expression in OSCC cell and tumor tissues was significantly higher than that of the controls. We noted that the miR-155 mimic enhanced CAL27 cell proliferation, migration and invasion ability, downregulated BCL6 levels, and increased cyclin D2 expression. However, we noted that abrogating miR-155 with the miR-155 antagomir suppressed CAL27 cell proliferation, migration and invasion, upregulated BCL6 and reduced cyclin D2 expression. These results indicate that miR-155 plays a tumor-promoting role in OSCC by regulating the BCL6/cyclin D2 axis.

MicroRNA (miR)-27b has been reported to participate in glioma. However, a detailed role of miR-27b and the underlying mechanism remain largely unknown. The present study found that the expression of miR-27b was significantly increased in glioma tissues compared with normal adjacent tissues. In addition, miR-27b was also upregulated in the U87, U251 and SHG44 glioma cell lines compared with normal human astrocytes. Sprouty homolog 2 (Spry2), which has been reported to be associated with invasive glioma, was identified as a novel target of miR-27b in U251 glioma cells, and the protein expression of Spry2 was negatively regulated by miR-27b in U251 cells. Additionally, inhibition of miR-27b and upregulation of Spry2 suppressed glioma cell invasion, while downregulation of Spry2 reversed the suppressive effect of miR-27b inhibition on glioma cell invasion. These data suggest that miR-27b may promote glioma cell invasion through direct inhibition of Spry2 expression. The data also suggest that miR-27b may become a promising molecular target for inhibiting the invasion and metastasis of glioma.

Regulation of protein activity by phosphorylation is central in many cellular processes. Phosphorylation of serine, threonine and tyrosine residues is well documented and studied. In addition, other amino acids, like histidine can be phosphorylated, but neither the mechanism nor the function of this modification is well understood. Nevertheless, there is a 14 kDa enzyme with phosphohistidine phosphatase activity, named PHPT1, found in most animals, but not in bacteria, plant or fungi. There are a few splice variant transcripts formed from the human PHPT1 locus and some of them are predicted to form variant proteins, but studies of these proteins are lacking. In order to get insight into the possible function of the variant transcripts encoded at the PHPT1 locus, ectopic expression of PHPT1 transcript variant 6, predicted to be degraded by the non-sense mediated mRNA decay pathway, in HeLa cells was undertaken. In HeLa cells the splice variant protein was degraded by the proteasome, unlike the wild type protein. Using an in silico modeling approach the variant C-terminal end of the proteins were predicted to form different secondary structures that might explain the different properties of the two proteins. The specific degradation of the PHPT1 splice variant indicates that at least for the PHPT1 protein, the quality control and the self-guarding of the cellular system works at two levels, first at the RNA level, aberrant transcripts are degraded by the non-sense mediated mRNA decay pathway, and the small amount of proteins that are formed will be degraded by the proteasome.

OBJECTIVE:

Both miR-21 and miR-183 are upregulated in hepatocellular carcinoma (HCC) and are considered as oncomiR. However, their oncogenic roles are still not fully understood. This study aimed to explore the regulative role of miR-21 and miR-183 over suppressors of cytokine signaling 6 (SOCS6), a negative regulator of cytokine receptor signaling.

MATERIALS AND METHODS:

qRT-PCR analysis was performed to assess miR-21 and miR-183 expression in tumor tissues obtained from HCC patients and in HCC cell lines HepG2 and Hep3B. Their regulation over SOCS6 is verified using dual luciferase assay and Western blot analysis. The function of miR-21/miR-183-SOCS6 axis in cell growth, invasion and apoptosis was studied.

RESULTS:

MiR-21 and miR-183 expression in HCC tissues than in adjacent normal tissues. Knockdown of miR-21 and miR-183 in HepG2 and Hep3B cells could decrease cell viability, increase cell apoptosis and decrease cell invasion. Based on the dual luciferase assay and Western blot analysis, we confirmed that both miR-21 and miR-183 can simultaneously target SOCS6 and modulate its expression at protein level. Overexpression of SOCS6 without 3'UTR could significantly lower cell growth rate and invasion capability, but increase relative caspase 3/7 activity and the ratio of apoptotic cells. However, these effects could not be blocked by miR-21 or miR-183 mimics.

CONCLUSIONS:

This study revealed a novel miR-21/miR-183-SOCS6 axis that might play an important role in modulating cell growth and invasion of HCC cells.

BACKGROUND:

Although Helicobacter pylori infection is necessary for development of gastric adenocarcinoma (GAC), the underlying mechanism remains poorly defined. This study aimed to explore how miR-221 and miR-222 are dysregulated after H. pylori infection and how these 2 miRNAs are involved in pathological development of gastric cancer.

MATERIAL AND METHODS:

qRT-PCR analysis was performed to quantify miR-221 and miR-222 expression in patients with H. pylori - induced chronic gastritis, H. pylori-negative healthy controls, and in gastric cancer tissues and the corresponding adjacent normal tissues. Cell models were used to verify the expression profile. Dual luciferase assay was performed to verify putative binding between miR-221 or miR-222 and RECK. A loss-and-gain function study was performed to assess the miR-221/miR-222-RECK axis in gastric cancer cells.

RESULTS:

H. pylori infection leads to significantly higher miR-221 and miR-222 expression. MiR-221 and miR-222 can bind the same sequence of RECK 3'UTR, thereby modulating its expression. Through simultaneous regulation over RECK, miR-221 and miR-222 can promote gastric cancer cell growth and invasion.

CONCLUSIONS:

The miR-221/miR-222-RECK axis might be an important path modulating H. pylori infection-related gastric cancer development.

BACKGROUND:

MiR-27a is significantly overexpressed in triple-negative breast cancer (TNBC). However, the exact biological function of MiR-27a in TNBC is not fully understood. In this study, we verified miR-27a expression in TNBC cells and explored how its overexpression modulates radiosensitivity of the cells.

MATERIAL/METHODS:

qRT-PCR analysis was performed to study miR-27a expression in TNBC lines MDA-MB-435 and MDA-MB-231 and in normal human breast epithelial cell line MCF10A. Dual luciferase assay was performed to verify a putative downstream target of miR-27a, CDC27. CCK-8 assay was used to assess the influence of miR-27a-CDC27 axis on cell proliferation under irradiation (IR) treatment.

RESULTS:

We confirmed significantly higher miR-27a expression in 2 TNBC cell lines--MDA-MB-435 and MDA-MB-231--than in human breast epithelial cell line MCF10A. miR-27a could modulate proliferation and radiosensitivity of TNBC cells. CDC-27 is a direct target of miR-27a and its downregulation conferred increased radioresistance of the cells.

CONCLUSIONS:

The miR-27a-CDC27 axis might play an important role in modulating response to radiotherapy in TNBC cells. Testing miR-27a expression might be a useful way to identify a subgroup of patients who will benefit from an IR-based therapeutic approach.