DNA Origami-Based Protein Manipulation Systems: From Function Regulation to Biological Application.

Proteins directly participate in tremendous physiological processes and mediate a variety of cellular functions. However, precise manipulation of proteins with predefined relative position and stoichiometry for understanding protein-protein interactions and guiding cellular behaviors is still challenging. With superior programmability of DNA molecules, DNA origami technology is able to construct arbitrary nanostructures that can accurately control the arrangement of proteins with various functionalities to solve these problems. Herein, starting from the classification of DNA origami nanostructures and the category of assembled proteins, we summarize the existing DNA origami-based protein manipulation systems (PMSs), review the advances on the regulation of their functions, and discuss their applications in cellular behavior modulation and disease therapy. Moreover, the limitations and potential directions of DNA origami-based PMSs are also presented, which may offer guidance for rational construction and ingenious application.

Isolation and evaluation of strong endogenous promoters for the heterologous expression of proteins in Pichia pastoris.

BACKGROUND: The heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production usually requires to express more than one gene in the host cells. In eukaryotes, the pathway flux is typically balanced by controlling the transcript levels of the genes involved. It is difficult to balance the stoichiometric fine-tuning of the reaction steps of the pathway by acting on one or two promoters. Furthermore, the promoter used should not be identical to avoid loss of inserted genes by recombination or dilute its transcription factors. RESULTS: Based on RNA-seq data, 18 candidate genes with the highest transcription levels at three carbon sources (glucose, glycerol and methanol) were selected and their promoter regions were isolated from GS115 genome. The performance of these promoters on the level of protein production was evaluated using LacZ and EGFP genes as the reporters, respectively. These isolated promoters all exhibited activity to express LacZ gene. Using LacZ as a reporter, of the 18 promoter candidates, 9 promoters showed higher expression levels for the reporter compare to pGAP, a strong promoter widely used for constitutive expression of heterologous proteins in Pichia pastoris. These promoters with high expression levels were further employed to evaluate secreted expression using EGFP as a reporter. 6 promoters exhibited stronger protein expression compare to pGAP. Interestingly, the protein expression driven by pFDH1 was slightly higher than that of commonly used pAOX1 at methanol, and methanol-induced expression of pFDH1 was not repressed by glycerol. CONCLUSION: The various promoters identified in this study could be used for heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production. the methanol-induced pFDH1 that is not repressed by glycerol is an attractive alternative to pAOX1 and may provide a novel way to produce heterologous proteins in Pichia pastoris.

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris.

BACKGROUND: Codon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias. RESULTS: In this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris. CONCLUSIONS: Codon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

Death-associated protein kinase 1 suppresses hepatocellular carcinoma cell migration and invasion by upregulation of DEAD-box helicase 20.

Death-associated protein kinase 1 (DAPK) is a calcium/calmodulin kinase that plays a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in hepatocellular carcinoma (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using a proteomics approach, we identified DEAD-box helicase 20 (DDX20) as an important downstream target of DAPK in HCC cells and critical for DAPK-mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion through the CDC42-integrin pathway, which was previously reported as an important downstream pathway of DAPK in cancer. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and identifies new potential therapeutic targets for HCC.

Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris.

BACKGROUND: Proprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications. However, it is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides. RESULTS: In this study, we evaluated the ability of P. pastoris to process furin cleavage sites and how to improve the cleavage efficiencies of furin cleavage sites in P. pastoris. The results showed that P. pastoris can process furin cleavage sites but the cleavage efficiencies are not high. Arg residue at position P1 or P4 in furin cleavage sites significantly affect cleavage efficiency in P. pastoris. Kex2 protease, but not YPS1, in P. pastoris is responsible for processing furin cleavage sites. Heterologous expression of furin or overexpression of Kex2 in P. pastoris effectively increased cleavage efficiencies of furin cleavage sites. CONCLUSIONS: Our investigation on the processing of furin cleavage sites provides important information for recombinant expression of furin precursors in P. pastoris. Furin or Kex2 overexpressing strains may be good choices for expressing precursors processed by furin in P. pastoris.

Motor Imagery EEG Classification for Patients with Amyotrophic Lateral Sclerosis Using Fractal Dimension and Fisher's Criterion-Based Channel Selection.

Motor imagery is based on the volitional modulation of sensorimotor rhythms (SMRs); however, the sensorimotor processes in patients with amyotrophic lateral sclerosis (ALS) are impaired, leading to degenerated motor imagery ability. Thus, motor imagery classification in ALS patients has been considered challenging in the brain-computer interface (BCI) community. In this study, we address this critical issue by introducing the Grassberger-Procaccia and Higuchi's methods to estimate the fractal dimensions (GPFD and HFD, respectively) of the electroencephalography (EEG) signals from ALS patients. Moreover, a Fisher's criterion-based channel selection strategy is proposed to automatically determine the best patient-dependent channel configuration from 30 EEG recording sites. An EEG data collection paradigm is designed to collect the EEG signal of resting state and the imagination of three movements, including right hand grasping (RH), left hand grasping (LH), and left foot stepping (LF). Five late-stage ALS patients without receiving any SMR training participated in this study. Experimental results show that the proposed GPFD feature is not only superior to the previously-used SMR features (mu and beta band powers of EEG from sensorimotor cortex) but also better than HFD. The accuracies achieved by the SMR features are not satisfactory (all lower than 80%) in all binary classification tasks, including RH imagery vs. resting, LH imagery vs. resting, and LF imagery vs. resting. For the discrimination between RH imagery and resting, the average accuracies of GPFD in 30-channel (without channel selection) and top-five-channel configurations are 95.25% and 93.50%, respectively. When using only one channel (the best channel among the 30), a high accuracy of 91.00% can still be achieved by the GPFD feature and a linear discriminant analysis (LDA) classifier. The results also demonstrate that the proposed Fisher's criterion-based channel selection is capable of removing a large amount of redundant and noisy EEG channels. The proposed GPFD feature extraction combined with the channel selection strategy can be used as the basis for further developing high-accuracy and high-usability motor imagery BCI systems from which the patients with ALS can really benefit.

Development of all-in-one multicistronic Tet-On lentiviral vectors for inducible co-expression of two transgenes.

Inducible co-expression of multiple genes is often needed in research. Here we describe a single-vector-based Tet-On inducible system for co-expression of two transgenes. The two transgenes (DsRed1 and eGFP as model genes) and reverse tetracycline-controlled transactivator were separated by internal ribosomal entry sites and 2A sequences, and their transcription was controlled by the same tetracycline responsive element. Two novel vectors with different internal ribosomal entry sites and 2A positions on the vectors were constructed. The DsRed1 and eGFP in cells transduced with both vectors are undetectable in the absence of doxycycline and can be efficiently induced in the presence of doxycycline in vitro and in vivo. These two vectors can be useful tools when regulated co-expression of two ecotopic genes is needed.

Evaluating DAPK as a therapeutic target.

Death associated protein kinase 1 (DAPK) is an important serine/theoreine kinase involved in various cellular processes such as apoptosis, autophagy and inflammation. DAPK expression and activity are misregulated in multiple diseases including cancer, neuronal death, stoke, et al. Methylation of the DAPK gene is common in many types of cancer and can lead to loss of DAPK expression. In this review, we summarize the pathological status and functional roles of DAPK in disease and compare the published reagents that can manipulate the expression or activity of DAPK. The pleiotropic functions of DAPK make it an intriguing target and the barriers and opportunities for targeting DAPK for future clinical application are discussed.

Expression of codon optimized human bone morphogenetic protein 4 in Pichia pastoris.

Bone morphogenetic proteins (BMPs) are TGF-ß family member proteins that have therapeutic potential. The amount of BMPs from natural resources is limited, and the production of biologically active BMPs in heterologous protein expression systems remains an obstacle for their clinical application. In this study, the DNA sequence of human BMP4 mature domain (hBMP4) was optimized according to the codon relative synonymous codon usage values in Pichia pastoris, and the A+T content in the sequence after optimization was within the range of 30% to 55%. In Pichia pastoris cultured in shake-flask, the expression level of hBMP4 protein from the optimized sequence (48 mg/L) increased fourfold in comparison with that from the native sequence (12 mg/L). Recombinant hBMP4 protein was purified by SP Sepharose and heparin affinity chromatography. The biological activities of recombinant hBMP4 were examined by measuring proliferation stimulation in cells and induction of ectopic cartilage formation in mouse models. Our results demonstrated that the optimized DNA sequence could significantly enhance hBMP4 protein expression in Pichia pastoris compared with the native sequence and produce biologically active recombinant hBMP4; this indicates the potential of this optimized sequence for bulk production of hBMP4 protein in future clinical applications.

Asymmetric silicon phthalocyanine based nanoparticle with spatiotemporally targeting of mitochondria for synergistic apoptosis-ferroptosis antitumor treatment.

A promising therapeutic strategy in cancer treatment merges photodynamic therapy (PDT) induced apoptosis with ferroptosis, a form of programmed cell death governed by iron-dependent lipid peroxidation. Given the pivotal role of mitochondria in ferroptosis, the development of photosensitizers that specifically provoke mitochondrial dysfunction and consequentially trigger ferroptosis via PDT is of significant interest. To this end, we have designed and synthesized a novel nanoparticle, termed FECTPN, tailored to address this requisite. FECTPN harnesses a trifecta of critical attributes: precision mitochondria targeting, photoactivation capability, pH-responsive drug release, and synergistic apoptosis-ferroptosis antitumor treatment. This nanoparticle was formulated by conjugating an asymmetric silicon phthalocyanine, Chol-SiPc-TPP, with the ferroptosis inducer Erastin onto a ferritin. The Chol-SiPc-TPP is a chemically crafted entity featuring cholesteryl (Chol) and triphenylphosphine (TPP) functionalities bonded axially to the silicon phthalocyanine, enhancing mitochondrial affinity and leading to effective PDT and subsequent apoptosis of cells. Upon cellular uptake, FECTPN preferentially localizes to mitochondria, facilitated by Chol-SiPc-TPP's targeting mechanics. Photoactivation induces the synchronized release of Chol-SiPc-TPP and Erastin in the mitochondria's alkaline domain, driving the escalation of both ROSs and lipid peroxidation. These processes culminate in elevated antitumor activity compared to the singular application of Chol-SiPc-TPP-mediated PDT. A notable observation is the pronounced enhancement in glutathione peroxidase-4 (GPX4) expression within MCF-7 cells treated with FECTPN and subjected to light exposure, reflecting intensified oxidative stress. This study offers compelling evidence that FECTPN can effectively induce ferroptosis and reinforces the paradigm of a synergistic apoptosis-ferroptosis pathway in cancer therapy, proposing a novel route for augmented antitumor treatments.

Optimizing Pichia pastoris protein secretion: Role of N-linked glycosylation on the α-mating factor secretion signal leader.

The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.

A Casein kinase 1/Checkpoint kinase 1 pyrazolo-pyridine protein kinase inhibitor as novel activator of the p53 pathway.

Reactivation of the wild-type p53 pathway is one key goal aimed at developing targeted therapeutics in the cancer research field. Although most p53 protein kinases form 'p53-activating' signals, there are few kinases whose action can contribute to the inhibition of p53, as Casein kinase 1 (CK1) and Checkpoint kinase 1 (CHK1). Here we report on a pyrazolo-pyridine analogue showing activity against both CK1 and CHK1 kinases that lead to p53 pathway stabilisation, thus having pharmacological similarities to the p53-activator Nutlin-3. These data demonstrate the emerging potential utility of multivalent kinase inhibitors.

Expression of canonical WNT/ß-CATENIN signaling components in the developing human lung.

BACKGROUND: The WNT/ß-CATENIN signaling cascade is crucial for the patterning of the early lung morphogenesis in mice, but its role in the developing human lung remains to be determined. In this study, expression patterns of canonical WNT/ß-CATENIN signaling components, including WNT ligands (WNT2, WNT7B), receptors (FZD4, FZD7, LRP5, LRP6), transducers (DVL2, DVL3, GSK-3ß, ß-CATENIN, APC, AXIN2), transcription factors (TCF4, LEF1) and antagonists (SOSTDC1) were examined in human embryonic lung at 7, 12, 17 and 21 weeks of gestation (W) by real-time qRT-PCR and in situ hybridization. RESULTS: qRT-PCR analysis showed that some of these components were gradually upregulated, while some were significantly downregulated from the 7 W to the 12 W. However, most components reached a high level at 17 W, with a subsequent decrease at 21 W. In situ hybridization showed that the canonical WNT ligands and receptors were predominantly located in the peripheral epithelium, whereas the canonical WNT signal transducers and transcription factors were not only detected in the respiratory epithelium, but some were also scattered at low levels in the surrounding mesenchyme in the developing human lung. Furthermore, Western blot, qRT-PCR and histological analysis demonstrated that the ß-CATENIN-dependent WNT signaling in embryonic human lung was activated in vitro by CHIR 99021 stimulation. CONCLUSIONS: This study of the expression patterns and in vitro activity of the canonical WNT/ß-CATENIN pathways suggests that these components play an essential role in regulation of human lung development.

The α-mating factor secretion signals and endogenous signal peptides for recombinant protein secretion in Komagataella phaffii.

BACKGROUND: The budding yeast Komagataella phaffii (Pichia pastoris) is widely employed to secrete proteins of academic and industrial interest. For secretory proteins, signal peptides are the sorting signal to direct proteins from cytosol to extracellular matrix, and their secretion efficiency directly impacts the yields of the targeted proteins in fermentation broth. Although the α-mating factor (MF) secretion signal from S. cerevisiae, the most common and widely used signal sequence for protein secretion, works in most cases, limitation exists as some proteins cannot be secreted efficiently. As the optimal choice of secretion signals is often protein specific, more secretion signals need to be developed to augment protein expression levels in K. phaffii. RESULTS: In this study, the secretion efficiency of 40 α-MF secretion signals from various yeast species and 32 endogenous signal peptides from K. phaffii were investigated using enhanced green fluorescent protein (EGFP) as the model protein. All of the evaluated α-MF secretion signals successfully directed EGFP secretion except for the secretion signals of the yeast D. hansenii CBS767 and H. opuntiae. The secretion efficiency of α-MF secretion signal from Wickerhamomyces ciferrii was higher than that from S. cerevisiae. 24 out of 32 endogenous signal peptides successfully mediated EGFP secretion. The signal peptides of chr3_1145 and FragB_0048 had similar efficiency to S. cerevisiae α-MF secretion signal for EGFP secretion and expression. CONCLUSIONS: The screened α-MF secretion signals and endogenous signal peptides in this study confer an abundance of signal peptide selection for efficient secretion and expression of heterologous proteins in K. phaffii.

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris.

The human chromogranin A-derived peptide CGA-N12, which is composed of 12 amino acid residues with the sequence ALQGAKERAHQQ, showed strong antifungal activity and the least hemolytic activity in previous studies. However, synthetic peptides are relatively expensive to produce. Recombinant expression of peptides in the host cells, such as bacteria or yeast, can fastly provide cost-efficient products of peptides. Here, we developed an innovative system to produce CGA-N12 peptides in the yeast Pichia pastoris GS115 using genetic engineering technology. In order to directly secret short CGA-N12 peptides into the culture media from GS115 cells and enhance its expression effect, the structure of the CGA-N12 coding sequence was designed to mimic that of native α-factor gene of Saccharomyces cerevisiae. Four long primer pairs with sticky end were used to synthesize CGA-N12 expression sequence which contains four copies of CGA-N12 flanked by a Lys-Arg pair and two Glu-Ala repeating units. Endogenous proteases Kex2 and Ste13 in Golgi apparatus recognize and excise Lys-Arg and Glu-Ala pair to release short CGA-N12 peptides from the tandem repeat sequences, respectively. The CGA-N12 peptides were successfully expressed in Pichia pastoris with a yield of up to 30 mg/L of yeast culture as determined using HPLC. Our study indicated that the strategy employed in this work may be a good way to express small-molecule peptides directly in the Pichia pastoris system.

Screening for functional IRESes using α-complementation system of ß-galactosidase in Pichia pastoris.

BACKGROUND: Pichia pastoris is becoming a promising chassis cell for metabolic engineering and synthetic biology after its whole genome and transcriptome sequenced. However, the current systems for multigene co-expression in P. pastoris are not efficient. The internal ribosome entry site (IRES) has an ability to recruit the ribosome to initiate protein synthesis by cap-independent translation manner. This study seeks to screen IRES sequences that are functional in P. pastoris, which will allow P. pastoris to express multiple proteins in a single mRNA and increase its efficacy as a platform for metabolic engineering and synthetic biology. RESULTS: In order to efficiently screen the IRES sequences, we first set out to create a screening system using LacZ gene. Due to the cryptic transcription of the LacZ gene, we established the α-complementation system of ß-galactosidase in P. pastoris with the optimum length of the α-complementing peptide at ~ 92 amino acids. The optimal α-complementing peptide was then used as the second reporter to screen IRESes in the engineered GS115 expressing the corresponding ω-peptide. A total of 34 reported IRESes were screened. After ruling out all false positive or negative IRESes, only seven IRESes were functional in P. pastoris, which were from TEV, PVY, RhPV, TRV, KSHV, crTMV viruses and the 5'-UTR of the YAP1 gene of S. cerevisiae. CONCLUSIONS: We showed here that α-complementation also works in P. pastoris and it can be used in a variety of in vivo studies. The functional IRESes screened in this study can be used to introduce multiple genes into P. pastoris via a prokaryotic-like polycistronic manner, which provided new efficient tools for metabolic engineering and synthetic biology researches in P. pastoris.

Distribution, Trafficking, and in Vitro Photodynamic Therapy Efficacy of Cholesterol Silicon(IV) Phthalocyanine and Its Nanoparticles in Breast Cancer Cells.

A cholesterol silicon(IV) phthalocyanine (Chol-Pc) and a water-soluble Chol-Pc based nanoparticle (DSPE@Chol-Pc), which was prepared using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG2000) as a nanocarrier were developed. Chol-Pc readily distributed within the cholesterol-rich domains and was preferentially localized in the Golgi apparatus after being transported into the cells. The trafficking of DSPE@Chol-Pc in breast cancer cells was visualized by tracking the fluorescence of Chol-Pc and FITC-labeled DSPE-PEG2000 through two-photonic imaging in real-time. It was discovered that Chol-Pc disassociated from the DSPE-PEG2000 on the plasma membrane and traveled to the cholesterol-rich domains soon afterward. Both DSPE@Chol-Pc and Chol-Pc effectively mediated photodynamic therapy to kill the breast cancer cells. After light irradiation, we found that the organizations of clustered cholesterol-rich domains in cells were destroyed, presumably leading to the death of cells for photodynamic therapy. It should be noted that DSPE@Chol-Pc is highly soluble in aqueous solution and has strong red fluorescence under two-photon excitation. Thus, it could be an excellent probe for detecting cholesterol-rich domains and studying transport processes of cholesterol in living cells.

The Roles of Protein Tyrosine Phosphatases in Hepatocellular Carcinoma.

The protein tyrosine phosphatase (PTP) family is involved in multiple cellular functions and plays an important role in various pathological and physiological processes. In many chronic diseases, for example cancer, PTP is a potential therapeutic target for cancer treatment. In the last two decades, dozens of PTP inhibitors which specifically target individual PTP molecules were developed as therapeutic agents. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and is the second most lethal cancer worldwide due to a lack of effective therapies. Recent studies have unveiled both oncogenic and tumor suppressive functions of PTP in HCC. Here, we review the current knowledge on the involvement of PTP in HCC and further discuss the possibility of targeting PTP in HCC.

Probing the biophysical properties of tumor cells during mitosis by atomic force microscopy.

Mitosis is an important physiological event accompanying with dramatic changes of cellar biophysical properties. Failure of mitosis results in cell death or chromosome aneuploidy. In this study, we used atomic force microscopy to probe and compare the biophysical properties of tumor cells at different stages during mitosis. The rounding forces of MCF-7 cells oscillated during mitosis. At anaphase, the average elasticity of cells was higher than that at other phases. Cholesterol depletion with M[Formula: see text]CD led to an increase in the average elasticity, whereas the average roughness of membrane surface decreased at the absence of cholesterol. Our study indicated that the distribution of actin filaments could affect the biophysical properties of tumor cells and cellular morphology during mitosis. Furthermore, the biophysical properties of tumor cells were also regulated by membrane cholesterol during mitosis. This work provides a new detection approach for monitoring tumor cell development at single cell level.

Evaluation of the prognostic and physiological functions of death associated protein kinase 1 in breast cancer.

Death associated protein kinase 1 (DAPK1) is a notable serine/threonine kinase involved in the regulation of multiple cellular pathways, including apoptosis and autophagy. Although DAPK1 is usually considered to be a tumor suppressor, it was previously reported to promote the viability of p53 mutant cancer cell lines and possess physiological oncogenic functions in breast cancer. However, the ability of endogenous DAPK1 to suppress breast cancer cell mobility has not been assessed. In the present study, the prognostic function of DAPK1 in a Chinese patient cohort was evaluated, and no significant association was observed between DAPK1 expression and patient survival or lymph node metastasis. In order to investigate the physiological function of endogenous DAPK1, stable inducible DAPK1 knockdown MCF7 and MDA-MB-231 cell lines were established. Consistent with previous studies, endogenous DAPK1 only regulated cell viability in p53 mutant MDA-MB-231 cells. However, knockdown of DAPK1 did not significantly affect cell motility of either MCF7 or MDA-MB-231 cells. Altogether, these results further explored the function of endogenous DAPK1 in breast cancer and may shed light in understanding the molecular signaling pathways regulating the physiological function of DAPK1.