AI-powered microscopy image analysis for parasitology: integrating human expertise.

Microscopy image analysis plays a pivotal role in parasitology research. Deep learning (DL), a subset of artificial intelligence (AI), has garnered significant attention. However, traditional DL-based methods for general purposes are data-driven, often lacking explainability due to their black-box nature and sparse instructional resources. To address these challenges, this article presents a comprehensive review of recent advancements in knowledge-integrated DL models tailored for microscopy image analysis in parasitology. The massive amounts of human expert knowledge from parasitologists can enhance the accuracy and explainability of AI-driven decisions. It is expected that the adoption of knowledge-integrated DL models will open up a wide range of applications in the field of parasitology.

A knowledge-integrated deep learning framework for cellular image analysis in parasite microbiology.

Cellular image analysis is an important method for microbiologists to identify and study microbes. Here, we present a knowledge-integrated deep learning framework for cellular image analysis, using three tasks as examples: classification, detection, and reconstruction. Alongside thorough descriptions of different models and datasets, we describe steps for computing environment setup, knowledge representation, data pre-processing, and training and tuning. We then detail evaluation and visualization. For complete details on the use and execution of this protocol, please refer to Li et al. (2021),1 Jiang et al. (2020),2 and Zhang et al. (2022).3.

Tumor-derived exosomal miR-148b-3p mediates M2 macrophage polarization via TSC2/mTORC1 to promote breast cancer migration and invasion.

BACKGROUND: Emerging evidence has revealed that tumor-associated macrophages (TAMs) and exosomes play a crucial role in the microenvironment for tumor growth. However, the mechanisms through which exosomal miRNAs modulate TAMs and tumor development in breast cancer are not fully understood. METHODS: We constructed a macrophage model and an indirect coculture system consist of breast cancer cells and macrophages. Exosomes were isolated from BC cells culture supernatant and identified by transmission electron microscopy, Western blot and Nanosight LM10 system. The expression of miR-148b-3p in exosomes was determined by qRT-PCR and the effect of exosomal miR-148b-3p on macrophage polarization was measured using qRT-PCR and ELISA. The proliferation, migration and invasion of BC cells were estimated by EdU, wound healing assay and transwell assay. We employed bioinformatics, luciferase reporter assay and Western blot to identify the target gene of miR-148b-3p. Western blot was used to clarify the mechanism of exosomal miR-148b-3p mediated the crosstalk between BC cells and M2 macrophages. RESULTS: Cancer-derived exosomes could induce M2 polarization of macrophages, which promoted the migration and invasion of breast cancer cells. We found that exosomal miR-148b-3p was overexpressed in breast cancer cell-derived exosomes and correlated with lymph node metastasis, late tumor stage and worse prognosis. Upregulated miR-148b-3p expression in exosomes modulated macrophage polarization by targeting TSC2, which promoted the proliferation and might affect migration and invasion of breast cancer cells. Interestingly, we found that exosomal miR-148b-3p could induce M2 macrophage polarization via the TSC2/mTORC1 signaling pathway in breast cancer. CONCLUSION: Overall, our study elucidated that miR-148b-3p could be transported by exosomes from breast cancer cells to surrounding macrophages and induced M2 polarization by targeting TSC2, providing novel insights for breast cancer therapy.

Cancer-associated fibroblast-derived exosomal miR-18b promotes breast cancer invasion and metastasis by regulating TCEAL7.

Studies have shown that cancer-associated fibroblasts (CAFs) play an irreplaceable role in the occurrence and development of tumors. Therefore, exploring the action and mechanism of CAFs on tumor cells is particularly important. In this study, we compared the effects of CAFs-derived exosomes and normal fibroblasts (NFs)-derived exosomes on breast cancer cells migration and invasion. The results showed that exosomes from both CAFs and NFs could enter into breast cancer cells and CAFs-derived exosomes had a more enhancing effect on breast cancer cells migration and invasion than NFs-derived exosomes. Furthermore, microRNA (miR)-18b was upregulated in CAFs-derived exosomes, and CAFs-derived exosomes miR-18b can promote breast cancer cell migration and metastasis by specifically binding to the 3'UTR of Transcription Elongation Factor A Like 7 (TCEAL7). The miR-18b-TCEAL7 pathway promotes nuclear Snail ectopic activation by activating nuclear factor-kappa B (NF-κB), thereby inducing epithelial-mesenchymal transition (EMT) and promoting cell invasion and metastasis. Moreover, CAFs-derived exosomes miR-18b could promote mouse xenograft model tumor metastasis. Overall, our findings suggest that CAFs-derived exosomes miR-18b promote nuclear Snail ectopic by targeting TCEAL7 to activate the NF-κB pathway, thereby inducing EMT, invasion, and metastasis of breast cancer. Targeting CAFs-derived exosome miR-18b may be a potential treatment option to overcome breast cancer progression.

Alternative Pathway Is Involved in Hydrogen Peroxide-Enhanced Cadmium Tolerance in Hulless Barley Roots.

Hulless barley, grown in the Qinghai Tibet Plateau, has a wide range of environmental stress tolerance. Alternative pathway (AP) and hydrogen peroxide (H2O2) are involved in enhancing plant tolerance to environmental stresses. However, the relationship between H2O2 and AP in hulless barley tolerance to cadmium (Cd) stress remains unclear. In the study, the role and relationship of AP and H2O2 under Cd stress were investigated in hulless barley (Kunlun14) and common barley (Ganpi6). Results showed that the expression level of alternative oxidase (AOX) genes (mainly AOX1a), AP capacity (Valt), and AOX protein were clearly induced more in Kunlun14 than in Ganpi 6 under Cd stress; moreover, these parameters were further enhanced by applying H2O2. Malondialdehyde (MDA) content, electrolyte leakage (EL) and NAD(P)H to NAD(P) ratio also increased in Cd-treated roots, especially in Kunlun 14, which can be markedly alleviated by exogenous H2O2. However, this mitigating effect was aggravated by salicylhydroxamic acid (SHAM, an AOX inhibitor), suggesting AP contributes to the H2O2-enhanced Cd tolerance. Further study demonstrated that the effect of SHAM on the antioxidant enzymes and antioxidants was minimal. Taken together, hulless barley has higher tolerance to Cd than common barley; and in the process, AP exerts an indispensable function in the H2O2-enhanced Cd tolerance. AP is mainly responsible for the decrease of ROS levels by dissipating excess reducing equivalents.

[CCL18 Promotes the Invasion of Lung Adenocarcinoma through ANXA2].

BACKGROUND: ANXA2 plays a very important role in cancer progression. chemokine ligand 18 (CCL18) is associated with the invasion, migration, metastasis and poor prognosis of lung adenocarcinoma (LUAD). In this study, we aimed to explore whether CCL18 promotes LUAD invasion through ANXA2, and its role and molecular mechanism in LUAD invasion. METHODS: Western blot was used to detect ANXA2 expression in LUAD tissues and adjacent non-tumor tissues, the transfection efficiency of SiANXA2#2 in cells and the role of ANXA2 as an upstream regulator in the AKT/cofilin signaling pathway. In vitro cytological experiments such as chemotaxis experiment and transwell invasion test was used to explore the mechanism of ANXA2 on LUAD metastasis. F-actin polymerization experiment and Western blot were used to detect whether invasion ability alteration of SiANXA2#2 A549 cells are related to F-actin. RESULTS: Western blot analysis showed that compared with adjacent non-tumor tissues, the protein expression level of ANXA2 in cancer tissues increased (P<0.05). In the chemotaxis experiment and invasion experiment, the chemotaxis and invasion ability induced by CCL18 decreased when ANXA2 knockdowned (P<0.05). Compared with the control group, F-actin polymerization was significantly lower in ANXA2 knockdown group, while phosphorylation of AKT at Ser473 and Thr308 and phosphorylation of Cofilin and LIMK were reduced in ANXA2 knockdown group (P<0.05). CONCLUSIONS: ANXA2 knockdown can reduce the invasive effect of CCL18 on LUAD cells by reducing phosphorylation of AKT and downstream pathways.

Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley.

G6PDH provides intermediate metabolites and reducing power (nicotinamide adenine dinucleotide phosphate, NADPH) for plant metabolism, and plays a pivotal role in the cellular redox homeostasis. In this study, we cloned five G6PDH genes (HvG6PDH1 to HvG6PDH5) from highland barley and characterized their encoded proteins. Functional analysis of HvG6PDHs in E. coli showed that HvG6PDH1 to HvG6PDH5 encode the functional G6PDH proteins. Subcellular localization and phylogenetic analysis indicated that HvG6PDH2 and HvG6PDH5 are localized in the cytoplasm, while HvG6PDH1, HvG6PDH3, and HvG6PDH4 are plastidic isoforms. Analysis of enzymatic activities and gene expression showed that HvG6PDH1 to HvG6PDH4 are involved in responses to salt and drought stresses. The cytosolic HvG6PDH2 is the major isoform against oxidative stress. HvG6PDH5 may be a house-keeping gene. In addition, HvG6PDH1 to HvG6PDH4 and their encoded enzymes responded to jasmonic acid (JA) and abscisic acid (ABA) treatments, implying that JA and ABA are probably critical regulators of HvG6PDHs (except for HvG6PDH5). Reactive oxygen species analysis showed that inhibition of cytosolic and plastidic G6PDH activities leads to increased H2O2 and O2- contents in highland barley under salt and drought stresses. These results suggest that G6PDH can maintain cellular redox homeostasis and that cytosolic HvG6PDH2 is an irreplaceable isoform against oxidative stress in highland barley.

Alternative Pathway is Involved in Nitric Oxide-Enhanced Tolerance to Cadmium Stress in Barley Roots.

Alternative pathway (AP) has been widely accepted to be involved in enhancing tolerance to various environmental stresses. In this study, the role of AP in response to cadmium (Cd) stress in two barley varieties, highland barley (Kunlun14) and barley (Ganpi6), was investigated. Results showed that the malondialdehyde (MDA) content and electrolyte leakage (EL) level under Cd stress increased in two barley varieties. The expressions of alternative oxidase (AOX) genes (mainly AOX1a), AP capacity (Valt), and AOX protein amount were clearly induced more in Kunlun14 under Cd stress, and these parameters were further enhanced by applying sodium nitroprussid (SNP, a NO donor). Moreover, H2O2 and O2- contents were raised in the Cd-treated roots of two barley varieties, but they were markedly relieved by exogenous SNP. However, this mitigating effect was aggravated by salicylhydroxamic acid (SHAM, an AOX inhibitor), suggesting that AP contributes to NO-enhanced Cd stress tolerance. Further study demonstrated that the effect of SHAM application on reactive oxygen species (ROS)-related scavenging enzymes and antioxidants was minimal. These observations showed that AP exerts an indispensable function in NO-enhanced Cd stress tolerance in two barley varieties. AP was mainly responsible for regulating the ROS accumulation to maintain the homeostasis of redox state.

iTRAQ-Based Comparative Proteomic Analysis Provides Insights into Molecular Mechanisms of Salt Tolerance in Sugar Beet (Beta vulgaris L.).

Salinity is one of the major abiotic stress factors that limit plant growth and crop yield worldwide. To understand the molecular mechanisms and screen the key proteins in response of sugar beet (Beta vulgaris L.) to salt, in the present study, the proteomics of roots and shoots in three-week-old sugar beet plants exposed to 50 mM NaCl for 72 h was investigated by isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology. The results showed that 105 and 30 differentially expressed proteins (DEPs) were identified in roots and shoots of salt-treated plants compared with untreated plants, respectively. There were 46 proteins up-regulated and 59 proteins down-regulated in roots; and 13 up-regulated proteins and 17 down-regulated proteins found in shoots, respectively. These DEPs were mainly involved in carbohydrate metabolism, energy metabolism, lipid metabolism, biosynthesis of secondary metabolites, transcription, translation, protein folding, sorting, and degradation as well as transport. It is worth emphasizing that some novel salt-responsive proteins were identified, such as PFK5, MDH, KAT2, ACAD10, CYP51, F3H, TAL, SRPR, ZOG, V-H⁺-ATPase, V-H⁺-PPase, PIPs, TIPs, and tubulin α-2/ß-1 chain. qRT-PCR analysis showed that six of the selected proteins, including BvPIP1-4, BvVP and BvVAP in root and BvTAL, BvURO-D1, and BvZOG in shoot, displayed good correlation between the expression levels of protein and mRNA. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings should significantly improve the understanding of the molecular mechanisms involved in salt tolerance of sugar beet plants.

Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.).

Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants.