Microscopic image recognition of diatoms based on deep learning.

Diatoms are a crucial component in the study of aquatic ecosystems and ancient environmental records. However, traditional methods for identifying diatoms, such as morphological taxonomy and molecular detection, are costly, are time consuming, and have limitations. To address these issues, we developed an extensive collection of diatom images, consisting of 7983 images from 160 genera and 1042 species, which we expanded to 49,843 through preprocessing, segmentation, and data augmentation. Our study compared the performance of different algorithms, including backbones, batch sizes, dynamic data augmentation, and static data augmentation on experimental results. We determined that the ResNet152 network outperformed other networks, producing the most accurate results with top-1 and top-5 accuracies of 85.97% and 95.26%, respectively, in identifying 1042 diatom species. Additionally, we propose a method that combines model prediction and cosine similarity to enhance the model's performance in low-probability predictions, achieving an 86.07% accuracy rate in diatom identification. Our research contributes significantly to the recognition and classification of diatom images and has potential applications in water quality assessment, ecological monitoring, and detecting changes in aquatic biodiversity.

CDK1 serves as a therapeutic target of adrenocortical carcinoma via regulating epithelial-mesenchymal transition, G2/M phase transition, and PANoptosis.

BACKGROUND: Adrenocortical carcinoma (ACC) is an extremely rare, aggressive tumor with few effective therapeutic options or drugs. Mitotane (Mtn), which is the only authorized therapeutic drug, came out in 1970 and is still the only first-line treatment for ACC in spite of serious adverse reaction and a high recurrence rate. METHODS: By in silico analysis of the ACC dataset in the cancer genome atlas (TCGA), we determined that high expression levels of cyclin-dependent kinase-1 (CDK1) were significantly related to the adverse clinical outcomes of ACC. In vitro and in vivo experiments were performed to evaluate the role of CDK1 in ACC progression through gain and loss of function assays in ACC cells. CDK1 inhibitors were screened to identify potential candidates for the treatment of ACC. RNA sequencing, co-immunoprecipitation, and immunofluorescence assays were used to elucidate the mechanism. RESULTS: Overexpression of CDK1 in ACC cell lines promoted proliferation and induced the epithelial-to-mesenchymal transition (EMT), whereas knockdown of CDK1 expression inhibited growth of ACC cell lines. The CDK1 inhibitor, cucurbitacin E (CurE), had the best inhibitory effect with good time-and dose-dependent activity both in vitro and in vivo. CurE had a greater inhibitory effect on ACC xenografts in nude mice than mitotane, without obvious adverse effects. Most importantly, combined treatment with CurE and mitotane almost totally eliminated ACC tumors. With respect to mechanism, CDK1 facilitated the EMT of ACC cells via Slug and Twist and locked ACC cells into the G2/M checkpoint through interaction with UBE2C and AURKA/B. CDK1 also regulated pyroptosis, apoptosis, and necroptosis (PANoptosis) of ACC cells through binding with the PANoptosome in a ZBP1-dependent way. CONCLUSIONS: CDK1 could be exploited as an essential therapeutic target of ACC via regulating the EMT, the G2/M checkpoint, and PANoptosis. Thus, CurE may be a potential candidate drug for ACC therapy with good safety and efficacy, which will meet the great need of patients with ACC.

Systematic pan-cancer analysis identifies APOC1 as an immunological biomarker which regulates macrophage polarization and promotes tumor metastasis.

Apolipoprotein C1 (APOC1) has been found to play an essential part in proliferation and metastasis of numerous cancers, but related mechanism has not been elucidated, especially its function and role in tumor immunity. Through systematic pan-cancer analysis, we identified that APOC1 was closely associated with the infiltration of various immune cells in multiple cancers. Besides, APOC1 was significantly co-expressed with the immune checkpoints, major histocompatibility complex (MHC) molecules, chemokines and other immune-related genes. Furthermore, single-cell sequencing analysis suggested that the vast majority of APOC1 was expressed in macrophages or tumor-associated macrophages (TAMs). Additionally, the expression of APOC1 was significantly related to the prognosis of different cancers. Since APOC1 was most significantly abnormally expressed in renal cell cancer (RCC), subsequent experiments were carried out in RCC to explore the role of APOC1 in tumor immunity. The expression of APOC1 was significantly elevated in the tumor and serum of RCC patients. Besides, APOC1 was mainly expressed in the macrophage and it was closely related to the immune cell infiltration of RCC. Co-culture with RCC cells could induce the generation of TAMs with M2 phenotype which be blocked by silencing APOC1. The expression of APOC1 was elevated in the M2 or TAMs and APOC1 promoted M2 polarization of macrophages through interacting with CD163 and CD206. Furthermore, macrophages overexpressing APOC1 promoted the metastasis of RCC cells via secreting CCL5. Together, these data indicate that APOC1 is an immunological biomarker which regulates macrophage polarization and promotes tumor metastasis.

ADH1C inhibits progression of colorectal cancer through the ADH1C/PHGDH /PSAT1/serine metabolic pathway.

Colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women worldwide. CRC is the second leading cause of cancer-related deaths. Although some progress in the treatment of CRC has been achieved, the molecular mechanism of CRC is still unclear. In this study, alcohol dehydrogenase 1C(ADH1C) was first identified as a target gene closely associated with the development of CRC by the comprehensive application of transcriptomics, proteomics, metabonomics and in silico analysis. The ADH1C mRNA and protein expression in CRC cell lines and tumor tissues was lower than that in normal intestinal epithelial cell lines and healthy tissues. Overexpression of ADH1C inhibited the growth, migration, invasion and colony formation of CRC cell lines and prevented the growth of xenograft tumors in nude mice. The inhibitory effects of ADH1C on CRC cells in vitro were exerted by reducing the expression of PHGDH/PSAT1 and the serine level. This inhibition could be partially reversed by adding serine to the culture medium. These results showed that ADH1C is a potential drug target in CRC.

Benzimidazoles induce concurrent apoptosis and pyroptosis of human glioblastoma cells via arresting cell cycle.

Glioblastoma multiforme (GBM) is the most malignant and lethal primary brain tumor in adults accounting for about 50% of all gliomas. The only treatment available for GBM is the drug temozolomide, which unfortunately has frequent drug resistance issue. By analyzing the hub genes of GBM via weighted gene co-expression network analysis (WGCNA) of the cancer genome atlas (TCGA) dataset, and using the connectivity map (CMAP) platform for drug repurposing, we found that multiple azole compounds had potential anti-GBM activity. When their anti-GBM activity was examined, however, only three benzimidazole compounds, i.e. flubendazole, mebendazole and fenbendazole, potently and dose-dependently inhibited proliferation of U87 and U251 cells with IC50 values below 0.26 µM. Benzimidazoles (0.125-0.5 µM) dose-dependently suppressed DNA synthesis, cell migration and invasion, and regulated the expression of key epithelial-mesenchymal transition (EMT) markers in U87 and U251 cells. Benzimidazoles treatment also dose-dependently induced the GBM cell cycle arrest at the G2/M phase via the P53/P21/cyclin B1 pathway. Furthermore, the drugs triggered pyroptosis of GBM cells through the NF-κB/NLRP3/GSDMD pathway, and might also concurrently induced mitochondria-dependent apoptosis. In a nude mouse U87 cell xenograft model, administration of flubendazole (12.5, 25, and 50 mg · kg-1 · d-1, i.p, for 3 weeks) dose-dependently suppressed the tumor growth without obvious adverse effects. Taken together, our results demonstrated that benzimidazoles might be promising candidates for the treatment of GBM.

Apolipoprotein C1 promotes glioblastoma tumorigenesis by reducing KEAP1/NRF2 and CBS-regulated ferroptosis.

Glioblastoma (GBM), a malignant brain tumor, is a world-wide health problem because of its poor prognosis and high rates of recurrence and mortality. Apolipoprotein C1 (APOC1) is the smallest of apolipoproteins, implicated in many diseases. Recent studies have shown that APOC1 promotes tumorigenesis and development of several types of cancer. In this study we investigated the role of APOC1 in GBM tumorigenesis. Using in silico assays we showed that APOC1 was highly expressed in GBM tissues and its expression was closely related to GBM progression. We showed that APOC1 protein expression was markedly increased in four GBM cell lines (U251, U138, A172 and U87) compared to the normal brain glia cell lines (HEB, HA1800). In U251 cells, overexpression of APOC1 promoted cell proliferation, migration, invasion and colony information, which was reversed by APOC1 knockdown. APOC1 knockdown also markedly inhibited the growth of GBM xenografts in the ventricle of nude mice. We further demonstrated that APOC1 reduced ferroptosis by inhibiting KEAP1, promoting nuclear translocation of NRF2 and increasing expression of HO-1 and NQO1 in GBM cells. APOC1 also induced ferroptosis resistance by increasing cystathionine beta-synthase (CBS) expression, which promoted trans-sulfuration and increased GSH synthesis, ultimately leading to an increase in glutathione peroxidase-4 (GPX4). Thus, APOC1 plays a key role in GBM tumorigenesis, conferring resistance to ferroptosis, and may be a promising therapeutic target for GBM.

Avasimibe exerts anticancer effects on human glioblastoma cells via inducing cell apoptosis and cell cycle arrest.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, but there is no effective drug available for GBM. Avasimibe is a potent inhibitor of acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1), which was used to treat atherosclerosis. Experimental evidence and bioinformatics have shown that avasimibe has anticancer activity. In this study we investigated the anticancer effects of avasimibe on human glioblastoma cells and the underlying mechanisms. Our results showed that avasimibe dose-dependently inhibited the proliferation of U251 and U87 human glioblastoma cells with IC50 values of 20.29 and 28.27 µM, respectively, at 48 h. Avasimibe (7.5, 15, 30 µM) decreased the DNA synthesis, and inhibited the colony formation of the tumor cells. Treatment of avasimibe also dose-dependently increased the apoptotic rate of tumor cells, decreased the mitochondrial membrane potential, induced the activity of caspase-3/7, and increased the protein expression of cleaved caspase-9, cleaved PARP and Bax in U251 and U87 cells. RNA-sequencing analyses revealed that avasimibe suppressed the expression of CDK2, cyclin E1, CDK4, cyclin D, CDK1, cyclin B1, Aurora A, and PLK1, while induced the expression of p53, p21, p27, and GADD45A, which was validated by Western blot analysis. These results demonstrated that avasimibe induced mitochondria-dependent apoptosis in glioblastoma cells, which was associated with arresting the cell cycle at G0/G1 phase and G2/M phase by regulating the p53/p21 pathway, p53/GADD45A and Aurora A/PLK1 signaling pathways. In U87 xenograft nude mice model, administration of avasimibe (15, 30 mg·kg-1·d-1, ip, for 18 days) dose-dependently inhibit the tumor growth. Taken together, our results demonstrated that avasimibe might be a promising chemotherapy drug in the treatment of GBM.

Combined artificial high-silicate medium and LED illumination promote carotenoid accumulation in the marine diatom Phaeodactylum tricornutum.

BACKGROUND: Diatoms, which can accumulate large amounts of carotenoids, are a major group of microalgae and the dominant primary producer in marine environments. Phaeodactylum tricornutum, a model diatom species, acquires little silicon for its growth although silicon is known to contribute to gene regulation and play an important role in diatom intracellular metabolism. In this study, we explored the effects of artificial high-silicate medium (i.e. 3.0 mM sodium metasilicate) and LED illumination conditions on the growth rate and pigment accumulation in P. tricornutum, which is the only known species so far that can grow without silicate. It's well known that light-emitting diodes (LEDs) as novel illuminants are emerging to be superior monochromatic light sources for algal cultivation with defined and efficient red and blue lights. RESULTS: Firstly, we cultivated P. tricornutum in a synthetic medium supplemented with either 0.3 mM or 3.0 mM silicate. The morphology and size of diatom cells were examined: the proportion of the oval and triradiate cells decreased while the fusiform cells increased with more silicate addition in high-silicate medium; the average length of fusiform cells also slightly changed from 14.33 µm in 0.3 mM silicate medium to 12.20 µm in 3.0 mM silicate medium. Then we cultivated P. tricornutum under various intensities of red light in combination with the two different levels of silicate in the medium. Higher biomass productivity also achieved in 3.0 mM silicate medium than in 0.3 mM silicate medium under red LED light irradiation at 128 µmol/m2/s or higher light intensity. Increasing silicate reversed the down-regulation of fucoxanthin and chlorophyll a under high red-light illumination (i.e. 255 µmol/m2/s). When doubling the light intensity, fucoxanthin content decreased under red light but increased under combined red and blue (50:50) lights while chlorophyll a content reduced under both conditions. Fucoxanthin accumulation and biomass productivity increased with enhanced red and blue (50:50) lights. CONCLUSION: High-silicate medium and blue light increased biomass and fucoxanthin production in P. tricornutum under high light conditions and this strategy may be beneficial for large-scale production of fucoxanthin in diatoms.

Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum.

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

Structural Basis for Substrate Preference of SMYD3, a SET Domain-containing Protein Lysine Methyltransferase.

SMYD3 is a SET domain-containing N-lysine methyltransferase associated with multiple cancers. Its reported substrates include histones (H3K4 and H4K5), vascular endothelial growth factor receptor 1 (VEGFR1 Lys(831)) and MAP3 kinase kinase (MAP3K2 Lys(260)). To reveal the structural basis for substrate preference and the catalytic mechanism of SMYD3, we have solved its co-crystal structures with VEGFR1 and MAP3K2 peptides. Our structural and biochemical analyses show that MAP3K2 serves as a robust substrate of SMYD3 because of the presence of a phenylalanine residue at the -2 position. A shallow hydrophobic pocket on SMYD3 accommodates the binding of the phenylalanine and promotes efficient catalytic activities of SMYD3. By contrast, SMYD3 displayed a weak activity toward a VEGFR1 peptide, and the location of the acceptor lysine in the folded kinase domain of VEGFR1 requires drastic conformational rearrangements for juxtaposition of the acceptor lysine with the enzymatic active site. Our results clearly revealed structural determinants for the substrate preference of SMYD3 and provided mechanistic insights into lysine methylation of MAP3K2. The knowledge should be useful for the development of SMYD3 inhibitors in the fight against MAP3K2 and Ras-driven cancer.

The in vitro selection world.

Through iterative cycles of selection, amplification, and mutagenesis, in vitro selection provides the ability to isolate molecules of desired properties and function from large pools (libraries) of random molecules with as many as 10(16) distinct species. This review, in recognition of a quarter of century of scientific discoveries made through in vitro selection, starts with a brief overview of the method and its history. It further covers recent developments in in vitro selection with a focus on tools that enhance the capabilities of in vitro selection and its expansion from being purely a nucleic acids selection to that of polypeptides and proteins. In addition, we cover how next generation sequencing and modern biological computational tools are being used to complement in vitro selection experiments. On the very least, sequencing and computational tools can translate the large volume of information associated with in vitro selection experiments to manageable, analyzable, and exploitable information. Finally, in vivo selection is briefly compared and contrasted to in vitro selection to highlight the unique capabilities of each method.

Algal Cell Factories: Approaches, Applications, and Potentials.

With the advent of modern biotechnology, microorganisms from diverse lineages have been used to produce bio-based feedstocks and bioactive compounds. Many of these compounds are currently commodities of interest, in a variety of markets and their utility warrants investigation into improving their production through strain development. In this review, we address the issue of strain improvement in a group of organisms with strong potential to be productive "cell factories": the photosynthetic microalgae. Microalgae are a diverse group of phytoplankton, involving polyphyletic lineage such as green algae and diatoms that are commonly used in the industry. The photosynthetic microalgae have been under intense investigation recently for their ability to produce commercial compounds using only light, CO2, and basic nutrients. However, their strain improvement is still a relatively recent area of work that is under development. Importantly, it is only through appropriate engineering methods that we may see the full biotechnological potential of microalgae come to fruition. Thus, in this review, we address past and present endeavors towards the aim of creating productive algal cell factories and describe possible advantageous future directions for the field.

Multidimensional analytical approach based on UHPLC-UV-ion mobility-MS for the screening of natural pigments.

Here, we propose a novel strategy that combines a typical ultra high performance liquid chromatography (UHPLC), data-independent mass spectrometry (MS(E)) workflow with traveling wave ion mobility (TWIM) and UV detection, to improve the characterization of carotenoids and chlorophylls in complex biological matrices. UV detection selectively highlighted pigments absorbing at specific wavelengths, while TWIM coupled to MS was used to maximize the peak capacity. We applied this approach for the analysis of pigments in different microalgae samples, including Chlorella vulgaris, Dunaliella salina, and Phaeodactylum tricornutum. Using UHPLC-UV-MS(E) information (retention time, absorbance at 450 nm, and accurate masses of precursors and product ions), we tentatively identified 26 different pigments (carotenes, chlorophylls, and xanthophylls). By adding TWIM information (collision cross sections), we further resolved 5 isobaric pigments, not resolved by UHPLC-UV-MS(E) alone. The characterization of the molecular phenotypes allowed us to differentiate the microalgae species. Our results demonstrate that a combination of TWIM and UV detection with traditional analytical approaches increases the selectivity and specificity of analysis, providing a new tool to characterize pigments in biological samples. We anticipate that such an analytical approach will be extended to other lipidomics and metabolomics applications.

Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation.

Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.

Effects of abiotic stressors on lutein production in the green microalga Dunaliella salina.

BACKGROUND: Recent years have witnessed a rising trend in exploring microalgae for valuable carotenoid products as the demand for lutein and many other carotenoids in global markets has increased significantly. In green microalgae lutein is a major carotenoid protecting cellular components from damage incurred by reactive oxygen species under stress conditions. In this study, we investigated the effects of abiotic stressors on lutein accumulation in a strain of the marine microalga D. salina which had been selected for growth under stress conditions of combined blue and red lights by adaptive laboratory evolution. RESULTS: Nitrate concentration, salinity and light quality were selected as three representative influencing factors and their impact on lutein production in batch cultures of D. salina was evaluated using response surface analysis. D. salina was found to be more tolerant to hyper-osmotic stress than to hypo-osmotic stress which caused serious cell damage and death in a high proportion of cells while hyper-osmotic stress increased the average cell size of D. salina only slightly. Two models were developed to explain how lutein productivity depends on the stress factors and for predicting the optimal conditions for lutein productivity. Among the three stress variables for lutein production, stronger interactions were found between nitrate concentration and salinity than between light quality and the other two. The predicted optimal conditions for lutein production were close to the original conditions used for adaptive evolution of D. salina. This suggests that the conditions imposed during adaptive evolution may have selected for the growth optima arrived at. CONCLUSIONS: This study shows that systematic evaluation of the relationship between abiotic environmental stresses and lutein biosynthesis can help to decipher the key parameters in obtaining high levels of lutein productivity in D. salina. This study may benefit future stress-driven adaptive laboratory evolution experiments and a strategy of applying stress in a step-wise manner can be suggested for a rational design of experiments.