An in silico scheme for optimizing the enzymatic acquisition of natural biologically active peptides based on machine learning and virtual digestion.

BACKGROUND: As a potential natural active substance, natural biologically active peptides (NBAPs) are recently attracting increasing attention. The traditional proteolysis methods of obtaining effective NBAPs are considerably vexing, especially since multiple proteases can be used, which blocks the exploration of available NBAPs. Although the development of virtual digesting brings some degree of convenience, the activity of the obtained peptides remains unclear, which would still not allow efficient access to the NBAPs. It is necessary to develop an efficient and accurate strategy for acquiring NBAPs. RESULTS: A new in silico scheme named SSA-LSTM-VD, which combines a sparrow search algorithm-long short-term memory (SSA-LSTM) deep learning and virtually digested, was presented to optimize the proteolysis acquisition of NBAPs. Therein, SSA-LSTM reached the highest Efficiency value reached 98.00 % compared to traditional machine learning algorithms, and basic LSTM algorithm. SSA-LSTM was trained to predict the activity of peptides in the proteins virtually digested results, obtain the percentage of target active peptide, and select the appropriate protease for the actual experiment. As an application, SSA-LSTM was employed to predict the percentage of neuroprotective peptides in the virtual digested result of walnut protein, and trypsin was ultimately found to possess the highest value (85.29 %). The walnut protein was digested by trypsin (WPTrH) and the peptide sequence obtained was analyzed closely matches the theoretical neuroprotective peptide. More importantly, the neuroprotective effects of WPTrH had been demonstrated in nerve damage mouse models. SIGNIFICANCE: The proposed SSA-LSTM-VD in this paper makes the acquisition of NBAPs efficient and accurate. The approach combines deep learning and virtually digested skillfully. Utilizing the SSA-LSTM-VD based strategy holds promise for discovering and developing peptides with neuroprotective properties or other desired biological activities.

Observation on efficacy and underlying mechanism of cheek acupuncture on ovulation induction for infertile women with PCOS: Case series.

RATIONALE: Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disorder among women of childbearing age and is the primary cause of anovulatory infertility, accounting for 70% to 80% of cases. Ovulation induction is the main treatment approach for infertile patients with PCOS. Commonly utilized medications for this purpose are clomiphene citrate (CC) and letrozole (LE). Clomiphene citrate administration results in an ovulation rate ranging from 60% to 85%, while the pregnancy rate is limited to 35% to 40%, and a further reduction is observed in live birth rates. Letrozole demonstrates a slightly higher pregnancy rate and live birth rate compared to clomiphene citrate, although challenges persist in terms of longer stimulation cycles, multiple pregnancies, and the risk of ovarian hyperstimulation syndrome (OHSS). Clinical reports indicate that acupuncture therapy shows promising efficacy in treating patients with PCOS-related infertility, despite a partially unclear understanding of its underlying mechanisms. PATIENT CONCERNS: In this study, one patient did not achieve pregnancy despite more than a year of ovulation induction using clomiphene citrate and letrozole. However, after 3 months of receiving cheek acupuncture therapy, she successfully conceived and gave birth to a liveborn baby. Another patient achieved natural conception and live birth after 2 months of exclusive cheek acupuncture therapy. DIAGNOSIS: PCOS. INTERVENTIONS: Cheek acupuncture therapy. OUTCOMES: Both of them successfully conceived and gave birth to a liveborn baby. LESSONS: These findings suggest that cheek acupuncture therapy can effectively stimulate follicle development and ovulation, potentially improving endometrial receptivity. According to holographic theory, there is a biologically holographic model within the cheek region that shares a homology with the human body structure. This model provides an explanation for the regulatory effects of cheek acupuncture point stimulation on the Hypothalamic-Pituitary-Ovarian axis (HPO), which subsequently influences follicle development and ovulation in patients. Consequently, when cheek acupuncture therapy is applied alone or in combination with ovulation induction medication, patients have the ability to achieve successful pregnancy and experience a smooth delivery.

Effects of an Akt-activating peptide obtained from walnut protein degradation on the prevention of memory impairment in mice.

Akt acts as a central protein influencing multiple pathologies in neurodegenerative diseases including AD and PD, and using Akt activators is a promising management strategy. The current study characterized the effects of an Akt-activating peptide (Glu-Pro-Glu-Val-Leu-Pro, EPEVLR) obtained from walnut protein degradation on D-gal-induced memory impairment in mice. EPEVLR was obtained by hydrolysis of walnut proteins, identification of peptide sequences, and screening for molecular docking sequentially. The MWM test in mice indicated that the oral administration of EPEVLR (80, 200 and 400 mg per kg per day) significantly (p < 0.05) reversed D-gal-induced memory impairment. WB tests of the mouse hippocampus confirmed that EPEVLR could activate Akt by promoting its phosphorylation. In addition, further characterization (including TEM, ELISA, and immunohistochemistry) related to Akt phosphorylation showed lower Aß and p-tau levels, as well as more autophagosomes than those in the model group. Moreover, the EPEVLR treatment significantly increased Lactobacillus abundance and reduced Helicobacter abundance in the gut microbiome and caused up-regulation of SCFAs and down-regulation of LPS of serum metabolites. Therefore, EPEVLR ingestion reversed cognitive impairment symptoms, possibly related to the activation of Akt and regulation of the intestinal flora pathway. Consumption of an EPEVLR-containing diet is beneficial for treating cognitive dysfunction.

Pharmacological profiling of a berbamine derivative for lymphoma treatment.

ABSTRACT: Ca2+/calmodulin-dependent protein kinase II γ (CAMKIIγ) has been identified as a potential target for treating cancer. Based on our previous study of berbamine (BBM) as a CAMKIIγ inhibitor, we have synthesized a new BBM derivative termed PA4. Compared with BBM, PA4 showed improved potency and specificity and was more cytotoxic against lymphoma and leukemia than against other types of cancer. In addition to indirectly targeting c-Myc protein stability, we demonstrated that its cytotoxic effects were also mediated via increased reactive oxygen species production in lymphoma cells. PA4 significantly impeded tumor growth in vivo in a xenograft T-cell lymphoma mouse model. Pharmacokinetics studies demonstrated quick absorption into plasma after oral administration, with a maximum concentration of 1680 ± 479 ng/mL at 5.33 ± 2.31 hours. The calculated oral absolute bioavailability was 34.1%. Toxicity assessment of PA4 showed that the therapeutic window used in our experiments was safe for future development. Given its efficacy, safety, and favorable pharmacokinetic profile, PA4 is a potential lead candidate for treating lymphoma.

CYP8B1 downregulation mediates the metabolic effects of vertical sleeve gastrectomy in mice.

BACKGROUND AND AIMS: Although the benefits of vertical sleeve gastrectomy (VSG) surgery are well known, the molecular mechanisms by which VSG alleviates obesity and its complications remain unclear. We aim to determine the role of CYP8B1 (cytochrome P450, family 8, subfamily B, polypeptide 1) in mediating the metabolic benefits of VSG. APPROACH AND RESULTS: We found that expression of CYP8B1, a key enzyme in controlling the 12α-hydroxylated (12α-OH) bile acid (BA) to non-12α-OH BA ratio, was strongly downregulated after VSG. Using genetic mouse models of CYP8B1 overexpression, knockdown, and knockout, we demonstrated that overexpression of CYP8B1 dampened the metabolic improvements associated with VSG. In contrast, short hairpin RNA-mediated CYP8B1 knockdown improved metabolism similar to those observed after VSG. Cyp8b1 deficiency diminished the metabolic effects of VSG. Further, VSG-induced alterations to the 12α-OH/non-12α-OH BA ratio in the BA pool depended on CYP8B1 expression level. Consequently, intestinal lipid absorption was restricted, and the gut microbiota (GM) profile was altered. Fecal microbiota transplantation from wild type-VSG mice (vs. fecal microbiota transplantation from wild-type-sham mice) improved metabolism in recipient mice, while there were no differences between mice that received fecal microbiota transplantation from knockout-sham and knockout-VSG mice. CONCLUSIONS: CYP8B1 is a critical downstream target of VSG. Modulation of BA composition and gut microbiota profile by targeting CYP8B1 may provide novel insight into the development of therapies that noninvasively mimic bariatric surgery to treat obesity and its complications.

Clinical efficacy of Gualou Xiaoyong Decoction and painless lactation manipulation in treating lactation acute mastitis and breast abscess: An observational study.

Information on the effects of Chinese medicine in the treatment of lactational acute mastitis and breast abscess is limited; thus, we conducted an observational study to analyze the clinical efficacy of Gualou Xiaoyong Decoction combined with painless lactation manipulation in the treatment of lactational acute mastitis and breast abscess. A total of 41 patients with lactational acute mastitis and breast abscess who were treated with Gualou Xiaoyong Decoction and painless lactation manipulation from October 2021 to October 2022 were included in this study. The age, fetal times(primiparous/multiparous), delivery mode (cesarean section/vaginal delivery), onset time, breast lump diameter, skin rash diameter, body temperature, visual analogue score, blood routine, C-reactive protein, procalcitonin, bacterial culture in milk, B ultrasound and other data of these patients were statistically analyzed. After treatment, the breast lump diameter of these patients decreased significantly, the skin rash diameter was reduced or disappeared, the body temperature decreased or returned to a normal range, and the visual analogue score also decreased. Besides, these patients had a decreased total number of white blood cells and a reduced percentage of neutrophils, C-reactive protein, and procalcitonin after treatment. In addition, bacteria in the milk of most patients disappeared, and there was no abnormality in B ultrasonic imaging. Except for 2 patients with breast abscess who stopped breastfeeding on the affected side for 1 day and 3 days respectively, all other patients continued to provide breast milk for their infants, and no adverse reactions were observed in these infants. The combination of Gualou Xiaoyong Decoction and painless lactation manipulation can achieve favorable clinical effects in the treatment of lactational acute mastitis and breast abscess. This combined therapy has good efficacy, short course of treatment, low costs, and great convenience with the avoidance of pain, hospitalization, influence on lactation, breast scar and other adverse outcomes.

Multi-dimensional deep learning drives efficient discovery of novel neuroprotective peptides from walnut protein isolates.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, are multi-factor induced neurological disorders that require management from multiple pathologies. The peptides from natural proteins with diverse physiological activity can be candidates as multifunctional neuroprotective agents. However, traditional methods for screening neuroprotective peptides are not only time-consuming and laborious but also poorly accurate, which makes it difficult to effectively obtain the needed peptides. In this case, a multi-dimensional deep learning model called MiCNN-LSTM was proposed to screen for multifunctional neuroprotective peptides. Compared to other multi-dimensional algorithms, MiCNN-LSTM reached a higher accuracy value of 0.850. The MiCNN-LSTM was used to acquire candidate peptides from walnut protein hydrolysis. Following molecular docking, behavioral and biochemical index experimental validation eventually found 4 hexapeptides (EYVTLK, VFPTER, EPEVLR and ELEWER) demonstrating excellent multifunctional neuroprotective properties. Therein, EPEVLR performed the best and can be investigated in depth as a multifunctional neuroprotective agent. This strategy will greatly improve the efficiency of screening multifunctional bioactive peptides, and it will be beneficial for the development of food functional peptides.

Gell: A GPU-powered 3D hybrid simulator for large-scale multicellular system.

As a powerful but computationally intensive method, hybrid computational models study the dynamics of multicellular systems by evolving discrete cells in reacting and diffusing extracellular microenvironments. As the scale and complexity of studied biological systems continuously increase, the exploding computational cost starts to limit large-scale cell-based simulations. To facilitate the large-scale hybrid computational simulation and make it feasible on easily accessible computational devices, we develop Gell (GPU Cell), a fast and memory-efficient open-source GPU-based hybrid computational modeling platform for large-scale system modeling. We fully parallelize the simulations on GPU for high computational efficiency and propose a novel voxel sorting method to further accelerate the modeling of massive cell-cell mechanical interaction with negligible additional memory footprint. As a result, Gell efficiently handles simulations involving tens of millions of cells on a personal computer. We compare the performance of Gell with a state-of-the-art paralleled CPU-based simulator on a hanging droplet spheroid growth task and further demonstrate Gell with a ductal carcinoma in situ (DCIS) simulation. Gell affords ~150X acceleration over the paralleled CPU method with one-tenth of the memory requirement.

Bile acids, gut microbiota and metabolic surgery.

Metabolic surgery, or bariatric surgery, is currently the most effective approach for treating obesity and its complications. Vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass (RYGB) are the top two types of commonly performed metabolic surgery now. The precise mechanisms of how the surgeries work are still unclear, therefore much research has been conducted in this area. Gut hormones such as GLP-1 and PYY have been studied extensively in the context of metabolic surgery because they both participate in satiety and glucose homeostasis. Bile acids, whose functions cover intestinal lipid absorption and various aspects of metabolic regulation via the action of FXR, TGR5, and other bile acid receptors, have also been actively investigated as potential mediators of metabolic surgery. Additionally, gut microbiota and their metabolites have also been studied because they can affect metabolic health. The current review summarizes and compares the recent scientific progress made on identifying the mechanisms of RYGB and VSG. One of the long-term goals of metabolic/bariatric surgery research is to develop new pharmacotherapeutic options for the treatment of obesity and diabetes. Because obesity is a growing health concern worldwide, there is a dire need in developing novel non-invasive treatment options.

In situ generated Fe3C embedded Fe-N-doped carbon nanozymes with enhanced oxidase mimic activity for total antioxidant capacity assessment.

In this work, we reported new Fe3C embedded Fe-N-doped carbon nanomaterials (Fe3C@Fe-N-CMs) generated in situ by the facile pyrolysis of Fe-Zn ZIF precursors. The resulting Fe3C@Fe-N-CMs were equipped with several desirable nanozyme features, including multiple efficient intrinsic active sites (i.e. Fe-Nx, Fe3C@C, and C-N moieties), large specific surface area and abundant mesoporous structures. As a result, these Fe3C@Fe-N-CMs displayed exceptional ability to mimic three enzymes: peroxidase, catalase and oxidase, while the Fe3C@Fe-N-CMs pyrolyzed at 800 °C, named CMs-800, showed the best enzyme-like properties. After systematically investigating the catalytic mechanism, we further explored the application of the oxidase-like properties of CMs-800 in the detection of the total antioxidant capacity (TAC) in beverages and tablets. This study not only provided a new approach to construct multifunctional carbon-based nanozymes, but also expanded the application of carbon nanozymes in the field of food quality and safety.

New chemical constituents from the fruits of Tetradium ruticarpum.

Two new γ-lactone derivatives, evodinoids A (1) and B (2), along with a new essential oil (3) were isolated from the nearly ripe fruits of Tetradium ruticarpum. The structures of these isolations were determined by 1D and 2D NMR, HR-ESI-MS and ECD data analysis. In addition, the cytotoxic effect of compounds 1-3 was evaluated against human cancer cells A498, A549, HepG-2, MCF-7 and SHSY-5Y, which displayed no significant cytotoxicity (IC50 > 100 µM).

Vertical sleeve gastrectomy confers metabolic improvements by reducing intestinal bile acids and lipid absorption in mice.

Vertical sleeve gastrectomy (VSG) is one of the most effective and durable therapies for morbid obesity and its related complications. Although bile acids (BAs) have been implicated as downstream mediators of VSG, the specific mechanisms through which BA changes contribute to the metabolic effects of VSG remain poorly understood. Here, we confirm that high fat diet-fed global farnesoid X receptor (Fxr) knockout mice are resistant to the beneficial metabolic effects of VSG. However, the beneficial effects of VSG were retained in high fat diet-fed intestine- or liver-specific Fxr knockouts, and VSG did not result in Fxr activation in the liver or intestine of control mice. Instead, VSG decreased expression of positive hepatic Fxr target genes, including the bile salt export pump (Bsep) that delivers BAs to the biliary pathway. This reduced small intestine BA levels in mice, leading to lower intestinal fat absorption. These findings were verified in sterol 27-hydroxylase (Cyp27a1) knockout mice, which exhibited low intestinal BAs and fat absorption and did not show metabolic improvements following VSG. In addition, restoring small intestinal BA levels by dietary supplementation with taurocholic acid (TCA) partially blocked the beneficial effects of VSG. Altogether, these findings suggest that reductions in intestinal BAs and lipid absorption contribute to the metabolic benefits of VSG.

Structural Insights into the Specificity of Ligand Binding and Coactivator Assembly by Estrogen-Related Receptor ß.

Estrogen-related receptor ß (ERRß) is a nuclear receptor critical for many biological processes. Despite the biological and pharmaceutical importance of ERRß, deciphering the structure of ERRß has been hampered by the difficulties in obtaining a pure and stable protein for structural studies. In fact, the ERRß ligand-binding domain remains the last unsolved ERR structure and also one of only a few unknown nuclear receptor structures. Here, we report the identification of a critical single-residue mutation resulted in robust solubility and stability of an active ERRß ligand-binding domain, thereby providing a protein tool enabling the first probe into the biochemical and structural studies of this important receptor. The crystal structure reveals key structural features that have enabled the integration of the molecular determinants of signals transduced across the ligand binding and coregulator recruitment by all three ERR subtypes, which also provides a framework for the rational design of selective and potent ligands for the treatment of various ERR-mediated diseases.

UPLC-ESI-MS/MS based identification and antioxidant, antibacterial, cytotoxic activities of aqueous extracts from storey onion (Allium cepa L. var. proliferum Regel).

Storey onion (Allium cepa L. var. proliferum Regel) is a variety of onion commonly grown in northern China that has not been researched in detail. This study aimed to identify the chemical compositions of storey onion aqueous extracts by UPLC-ESI-MS/MS, as well as characterize the antioxidant, antibacterial and cytotoxic activities, compared with welsh onion and onion. A total of 42 compounds were identified, among which the contents of organosulfur compounds (962.20 ± 34.55 µg/g), polyphenols (100.40 ± 12.55 µg/g) and organic acids (54.04 ± 2.69 µg/g) in storey onion were higher than those in welsh onion and onion. Additionally, the contents of cycloalliin (551.74 ± 8.12 µg/g), ajoene (159.31 ± 5.30 µg/g) and (E)-1-propene-1-sulfenic acid (72.12 ± 2.98 µg/g) in storey onion were the highest. Storey onion had pronounced DPPH• (IC50 = 1.24 ± 0.52 mg/mL) and OH• scavenging activities (IC50 = 14.45 ± 1.29 mg/mL) as well as ferric ion reducing power (absorbance from 0.32 to 2.21). Onion had the highest ABTS•+ scavenging activity (IC50 = 1.64 ± 0.64 mg/mL), while welsh onion had the lowest antioxidant activity. Storey onion had the strongest inhibitory effect on all the tested strains (MIC 31.3-125 mg/mL), and cell viability assays against human liver (HepG2) cancer cell lines also illustrated that aqueous extracts from storey onion significantly inhibited cell proliferation (when incubated for 24 h, IC50 = 33.21 ± 1.12 mg/mL) and induced cell apoptosis. Welsh onion and onion also had weaker antibacterial and anticancer activites, with those of onion being the weakest. The results showed that storey onion with excellent biological activity may benefit to human health and can be developed into functional foods.

Real-Time Quantification of Cell Internalization Kinetics by Functionalized Bioluminescent Nanoprobes.

Cells transport mass dynamically, crossing cell membranes to maintain metabolism and systemic homeostasis, through which biomolecules are also delivered to cells for gene editing, cell reprograming, therapy, and other purposes. Quantifying the translocation kinetics is fundamentally and clinically essential, but remains limited by fluorescence-based technologies, which are semi-quantitative and only provide kinetics information at cellular level or in discrete time. Herein, a real-time method of quantifying cell internalization kinetics is reported using functionalized firefly-luciferase nanocapsules as the probe. This quantitative assay will facilitate the rational design of delivery vectors and enable high-throughput screening of peptides and other functional molecules, constituting an effective tool for broad applications, including drug development and cancer therapy.

Bile-ology: from bench to bedside.

Bile acids (BAs) are originally known as detergents essential for the digestion and absorption of lipids. In recent years, extensive research has unveiled new functions of BAs as gut hormones that modulate physiological and pathological processes, including glucose and lipid metabolism, energy expenditure, inflammation, tumorigenesis, cardiovascular disease, and even the central nervous system in addition to cholesterol homeostasis, enterohepatic protection and liver regeneration. BAs are closely linked with gut microbiota which might explain some of their crucial roles in organs. The signaling actions of BAs can also be mediated through specific nuclear receptors and membrane-bound G protein-coupled receptors. Several pharmacological agents or bariatric surgeries have demonstrated efficacious therapeutic effects on metabolic diseases through targeting BA signaling. In this mini-review, we summarize recent advances in bile-ology, focusing on its translational studies.

Coassembly Kinetics of Graphene Oxide and Block Copolymers at the Water/Oil Interface.

The coassembly kinetics of graphene oxide (GO) nanosheets and diblock copolymers at the water/toluene interface is probed by tracking the dynamic interfacial tension using pendant drop tensiometry. The diblock copolymer significantly enhances the surfactancy of the GO nanosheets at the interface. It is found that diblock copolymers rapidly adsorb to the water/toluene interface and enhance the adsorption affinity of GO nanosheets to the interface. The continuous adsorption of GO at the interface leads to a random loose packing state, at which the adsorbed GO and diblock copolymers start to form an elastic film. After this transition, GO continues to adsorb to the interface, however, at a much slower speed, yielding a more solidlike elastic film in the long time equilibrium limit.

Targeting hexokinase 2 inhibition promotes radiosensitization in HPV16 E7-induced cervical cancer and suppresses tumor growth.

In order to improve the sensitivity of cervical cancer cells to irradiation therapy, we targeted hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis, and explore its role in cervical cancer cells. We suppressed HK2 expression and/or function by shRNA and/or metformin and found HK2 inhibition enhanced cells apoptosis with accelerating expression of cleaved PARP and caspase-3. HK2 inhibition also induced much inferior proliferation of cervical cancer cells both in vitro and in vivo with diminishing expression of mTOR, MIB and MGMT. Moreover, HK2 inhibition altered the metabolic profile of cervical cancer cells to one less dependent on glycolysis with a reinforcement of mitochondrial function and an ablation of lactification ability. Importantly, cervical cancer cells contained HK2 inhibition displayed more sensitivity to irradiation. Further results indicated that HPV16 E7 oncoprotein altered the glucose homeostasis of cervical cancer cells into glycolysis by coordinately promoting HK2 expression and its downregulation of glycolysis. Taken together, our findings supported a mechanism whereby targeting HK2 inhibition contributed to suppress HPV16 E7-induced tumor glycolysis metabolism phenotype, inhibiting tumor growth, and induced apoptosis, blocking the cancer cell energy sources and ultimately enhanced the sensitivity of HPV(+) cervical cancer cells to irradiation therapy.

A Novel Class of Natural FXR Modulators with a Unique Mode of Selective Co-regulator Assembly.

The farnesoid X receptor (FXR) is an important target for drug discovery. Small molecules induce a conformational change in FXR that modulates its binding to co-regulators, thus resulting in distinct FXR functional profiles. However, the mechanisms for selectively recruiting co-regulators by FXR remain elusive, partly because of the lack of FXR-selective modulators. We report the identification of two natural terpenoids, tschimgine and feroline, as novel FXR modulators. Remarkably, their crystal structures uncovered a secondary binding pocket important for ligand binding. Further, tschimgine or feroline induced dynamic conformational changes in the activation function 2 (AF-2) surface, thus leading to differential co-regulator recruiting profiles, modulated by both hydrophobic and selective hydrogen-bond interactions unique to specific co-regulators. Our findings thus provide a novel structure template for optimization for FXR-selective modulators of clinical value.

MOF Acetylates the Histone Demethylase LSD1 to Suppress Epithelial-to-Mesenchymal Transition.

The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression.