The mitochondrial ribosomal protein mRpL4 regulates Notch signaling.

Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. Here, we report a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. We find that mRpL4 physically and genetically interacts with the WD40 repeat protein wap and activates the transcription of Notch signaling targets. We show that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, we have discovered a previously unknown function of mRpL4 during animal development.

Molecular Engineering of Perylene Diimide Polymers with a Robust Built-in Electric Field for Enhanced Solar-Driven Water Splitting.

The built-in electric field of the polymer semiconductors could be regulated by the dipole moment of its building blocks, thereby promoting the separation of photogenerated carriers and achieving efficient solar-driven water splitting. Herein, three perylene diimide (PDI) polymers, namely oPDI, mPDI and pPDI, are synthesized with different phenylenediamine linkers. Notably, the energy level structure, light-harvesting efficiency, and photogenerated carrier separation and migration of polymers are regulated by the orientation of PDI unit. Among them, oPDI enables a large dipole moment and robust built-in electric field, resulting in enhanced solar-driven water splitting performance. Under simulated sunlight irradiation, oPDI exhibits the highest photocurrent of 115.1 µA cm-2 for photoelectrochemical oxygen evolution, which is 11.5 times that of mPDI, 26.8 times that of pPDI and 104.6 times that of its counterparts PDI monomer at the same conditions. This work provides a strategy for designing polymers by regulating the orientation of structural units to construct efficient solar energy conversion systems.

JAK/STAT signaling regulates the Harmonia axyridis leg regeneration by coordinating cell proliferation.

Harmonia axyridis presents remarkable appendage regeneration capacity and can therefore be considered as an emerging regeneration research model. Amino acid sequences of the Janus kinase Hopscotch (Hahop) and the transcription factor STAT (HaStat), the main components of the JAK/STAT signaling pathway, conserved with their homologs in other models. The expression levels of these two genes were continuously up-regulated during the appendage regeneration process. To identify the functions of JAK/STAT signaling, we performed RNAi experiments of Hahop and HaStat in H. axyridis, and found regeneration defects following in HahopRNAi and HaStatRNAi treatments at different regeneration stages. Additionally, we confirmed that regeneration defects caused by the low-level of JAK/STAT activity were due to the inhibition of cell proliferation. The results of the current study suggest that JAK/STAT signaling regulates the entire regeneration process by coordinating cell proliferation of regenerating appendages.

Hippo signaling promotes Ets21c-dependent apical cell extrusion in the Drosophila wing disc.

Cell extrusion is a crucial regulator of epithelial tissue development and homeostasis. Epithelial cells undergoing apoptosis, bearing pathological mutations or possessing developmental defects are actively extruded toward elimination. However, the molecular mechanisms of Drosophila epithelial cell extrusion are not fully understood. Here, we report that activation of the conserved Hippo (Hpo) signaling pathway induces both apical and basal cell extrusion in the Drosophila wing disc epithelia. We show that canonical Yorkie targets Diap1, Myc and Cyclin E are not required for either apical or basal cell extrusion induced by activation of this pathway. Another target gene, bantam, is only involved in basal cell extrusion, suggesting novel Hpo-regulated apical cell extrusion mechanisms. Using RNA-seq analysis, we found that JNK signaling is activated in the extruding cells. We provide genetic evidence that JNK signaling activation is both sufficient and necessary for Hpo-regulated cell extrusion. Furthermore, we demonstrate that the ETS-domain transcription factor Ets21c, an ortholog of proto-oncogenes FLI1 and ERG, acts downstream of JNK signaling to mediate apical cell extrusion. Our findings reveal a novel molecular link between Hpo signaling and cell extrusion.

Notch Signaling in Insect Development: A Simple Pathway with Diverse Functions.

Notch signaling is an evolutionarily conserved pathway which functions between adjacent cells to establish their distinct identities. Despite operating in a simple mechanism, Notch signaling plays remarkably diverse roles in development to regulate cell fate determination, organ growth and tissue patterning. While initially discovered and characterized in the model insect Drosophila melanogaster, recent studies across various insect species have revealed the broad involvement of Notch signaling in shaping insect tissues. This review focuses on providing a comprehensive picture regarding the roles of the Notch pathway in insect development. The roles of Notch in the formation and patterning of the insect embryo, wing, leg, ovary and several specific structures, as well as in physiological responses, are summarized. These results are discussed within the developmental context, aiming to deepen our understanding of the diversified functions of the Notch signaling pathway in different insect species.

Chronic exposure to the star polycation (SPc) nanocarrier in the larval stage adversely impairs life history traits in Drosophila melanogaster.

BACKGROUND: Nanomaterials are widely used as pesticide adjuvants to increase pesticide efficiency and minimize environmental pollution. But it is increasingly recognized that nanocarrier is a double-edged sword, as nanoparticles are emerging as new environmental pollutants. This study aimed to determine the biotoxicity of a widely applied star polycation (SPc) nanocarrier using Drosophila melanogaster, the fruit fly, as an in vivo model. RESULTS: The lethal concentration 50 (LC50) value of SPc was identified as 2.14 g/L toward third-instar larvae and 26.33 g/L for adults. Chronic exposure to a sub lethal concentration of SPc (1 g/L) in the larval stage showed long-lasting adverse effects on key life history traits. Exposure to SPc at larval stage adversely impacted the lifespan, fertility, climbing ability as well as stresses resistance of emerged adults. RNA-sequencing analysis found that SPc resulted in aberrant expression of genes involved in metabolism, innate immunity, stress response and hormone production in the larvae. Orally administrated SPc nanoparticles were mainly accumulated in intestine cells, while systemic responses were observed. CONCLUSIONS: These findings indicate that SPc nanoparticles are hazardous to fruit flies at multiple levels, which could help us to develop guidelines for further large-scale application.

A novel expression vector for Corynebacterium glutamicum with an auxotrophy complementation system.

Corynebacterium glutamicum is an important industrial strain used for the production of amino acids and vitamins. Most tools developed for overexpression of genes in C. glutamicum are based on the inducible promoter regulated by the lacIq gene or contain an antibiotic resistance gene as a selection marker. These vectors are essential for rapid identification of recombinant strains and detailed study of gene functions, but, as a considerable disadvantage, these vectors are not suitable for large-scale industrial production due to the need for the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) and antibiotics. In this study, the novel Escherichia coli-C. glutamicum shuttle expression vector pLY-4, derived from the expression vector pXMJ19, was constructed. The constitutive vector pLY-4 contains a large multiple cloning site, the strong promoter tacM and two selective markers: the original chloramphenicol resistance gene cat is used for molecular cloning operations, and the auxotrophy complementation marker alr, which can be stably replicated in the auxotrophic host strain without antibiotic selection pressure, is used for industrial fermentation. Heterologous expression of the gapC gene using the vector pLY-4 in C. glutamicum for L-methionine production indicated the potential application of pLY-4 in the development of C. glutamicum strain engineering and industrial fermentation.

Visible-Light-Promoted C2 Selective Arylation of Quinoline and Pyridine N-Oxides with Diaryliodonium Tetrafluoroborate.

A protocol of visible-light-promoted C2 selective arylation of quinoline and pyridine N-oxides, with diaryliodonium tetrafluoroborate as an arylation reagent, using eosin Y as a photocatalyst for the construction of N-heterobiaryls was presented. This methodology provided an efficient way for the synthesis of 2-aryl-substituted quinoline and pyridine N-oxides. This strategy has the following advantages: specific regioselectivity, simple operation, good functional group tolerance, and high to moderate yields under mild conditions.

Three Melanin Pathway Genes, TH, yellow, and aaNAT, Regulate Pigmentation in the Twin-Spotted Assassin Bug, Platymeris biguttatus (Linnaeus).

Pigmentation plays a vital role in insect survival and reproduction. Many melanin pathway genes have been studied in holometabolous insects; however, they have only been studied in two hemimetabolous insect genera, Oncopeltus and Periplaneta. Here we analyzed three melanin pathway genes (TH, yellow, and aaNAT) using RNA interference (RNAi) in another hemimetabolous insect, namely the twin-spotted assassin bug, Platymeris biguttatus. TH was highly expressed in freshly molted nymphs and adults. TH RNAi resulted in a complete loss of black pigment, with yellow coloration maintained. Therefore, black pigment in this assassin bug is solely generated from the melanin pathway, whereas yellow pigment is generated from other unknown pigmentation pathways. yellow and aaNAT were highly expressed in the white spot of the hemelytra. Downregulation of yellow caused a brown phenotype with high mortality, indicating an important role of yellow functions in cuticle formation and in the process of converting melanin from brown to black. Interestingly, aaNAT RNAi caused not only loss of white pigment, but also loss of yellow and red pigments. This phenotype of aaNAT has not been reported in other insects. Our results provide new information for understanding the melanin pathway in which aaNAT is essential for the formation of colorless patterns.

Ubpy controls the stability of the ESCRT-0 subunit Hrs in development.

Ubiquitylated developmental membrane signaling proteins are often internalized for endocytic trafficking, through which endosomal sorting complexes required for transport (ESCRT) act sequentially to deliver internalized cargos to lysosomes. The ESCRT function in endocytic sorting is well established; however, it is not fully understood how the sorting machinery itself is regulated. Here, we show that Ubiquitin isopeptidase Y (Ubpy) plays a conserved role in vivo in the homeostasis of an essential ESCRT-0 complex component Hrs. We find that, in the absence of Drosophila Ubpy, multiple membrane proteins that are essential components of important signaling pathways accumulate in enlarged, aberrant endosomes. We further demonstrate that this phenotype results from endocytic pathway defects. We provide evidence that Ubpy interacts with and deubiquitylates Hrs. In Ubpy-null cells, Hrs becomes ubiquitylated and degraded in lysosomes, thus disrupting the integrity of ESCRT sorting machinery. Lastly, we find that signaling proteins are enriched in enlarged endosomes when Hrs activity is abolished. Together, our data support a model in which Ubpy plays a dual role in both cargo deubiquitylation and the ESCRT-0 stability during development.

Identification and characterization of the major antifungal substance against Fusarium Sporotrichioides from Chaetomium globosum.

Fusarium sporotrichioides, is a common soil-borne plant pathogen causing dry rot of potato in Northeast China. The objective of this study was to identify the main antifungal substances from Chaetomium globosum W7 against F. sporotrichioides. Strain W7 can significantly inhibit F. sporotrichioides without direct contact, suggesting that its antifungal substance was extracellular, and the solubility of this antifungal substance in ethyl acetate was superior to that in water. Acetone was selected as the optimum solvent for the extraction of the metabolites of C. globosum. Metabolites were then separated with thin-layer chromatography. Following antifungal tests on bands, a dark brown band with Rf value of 0.20 was determined as the antifungal substance, and identified as chaetoglobosin A. The antifungal activity test showed that the minimum inhibitory concentration of chaetoglobosin A to F. sporotrichioides was 9.45-10.50 µg/mL, IC50 being 4.344 µg/mL. Chaetoglobosin A also proved to have an excellent preventive effect on potato dry rot caused by F. sporotrichioides. To summarize, chaetoglobosin A was identified as the main active substance of C. globosum to inhibit F. sporotrichioides for the first time, and demonstrated a potential application value in agriculture.

Metabolic engineering of Corynebacterium glutamicum for methionine production by removing feedback inhibition and increasing NADPH level.

Relieving the feedback inhibition of key enzymes in a metabolic pathway is frequently the first step of producer-strain construction by genetic engineering. However, the strict feedback regulation exercised by microorganisms in methionine biosynthesis often makes it difficult to produce methionine at a high level. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for methionine production. First, the metD gene encoding the methionine uptake system was deleted to achieve extracellular accumulation of methionine. Then, random mutagenesis was performed to remove feedback inhibition by metabolic end-products. The resulting strain C. glutamicum ENM-16 was further engineered to block or decrease competitive branch pathways by deleting the thrB gene and changing the start codon of the dapA gene, followed by point mutations of lysC (C932T) and pyc (G1A, C1372T) to increase methionine precursor supply. To enrich the NADPH pool, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the pentose phosphate pathway were mutated to reduce their sensitivity to inhibition by intracellular metabolites. The resultant strain C. glutamicum LY-5 produced 6.85 ± 0.23 g methionine l(-1) with substrate-specific yield (Y P/S) of 0.08 mol per mol of glucose after 72 h fed-batch fermentation. The strategies described here will be useful for construction of methionine engineering strains.

Heat Shock Protein Genes Affect the Rapid Cold Hardening Ability of Two Invasive Tephritids.

Bactrocera dorsalis and Bactrocera correcta are two invasive species that can cause major economic damage to orchards and the fruit import and export industries. Their distribution is advancing northward due to climate change, which is threatening greater impacts on fruit production. This study tested the rapid cold-hardening ability of the two species and identified the temperature associated with the highest survival rate. Transcriptome data and survival data from the two Bactrocera species' larvae were obtained after rapid cold-hardening experiments. Based on the sequencing of transcripts, four Hsp genes were found to be affected: Hsp68 and Hsp70, which play more important roles in the rapid cold hardening of B. dorsalis, and Hsp23 and Hsp70, which play more important roles in the rapid cold hardening of B. correcta. This study explored the adaptability of the two species to cold, demonstrated the expression and function of four Hsps in response to rapid cold hardening, and explained the occurrence and expansion of these two species of tephritids, offering information for further studies.

Computational identification of long non-coding RNAs associated with graphene therapy in glioblastoma multiforme.

Glioblastoma multiforme represents the most prevalent primary malignant brain tumour, while long non-coding RNA assumes a pivotal role in the pathogenesis and progression of glioblastoma multiforme. Nonetheless, the successful delivery of long non-coding RNA-based therapeutics to the tumour site has encountered significant obstacles attributable to inadequate biocompatibility and inefficient drug delivery systems. In this context, the use of a biofunctional surface modification of graphene oxide has emerged as a promising strategy to surmount these challenges. By changing the surface of graphene oxide, enhanced biocompatibility can be achieved, facilitating efficient transport of long non-coding RNA-based therapeutics specifically to the tumour site. This innovative approach presents the opportunity to exploit the therapeutic potential inherent in long non-coding RNA biology for treating glioblastoma multiforme patients. This study aimed to extract relevant genes from The Cancer Genome Atlas database and associate them with long non-coding RNAs to identify graphene therapy-related long non-coding RNA. We conducted a series of analyses to achieve this goal, including univariate Cox regression, least absolute shrinkage and selection operator regression and multivariate Cox regression. The resulting graphene therapy-related long non-coding RNAs were utilized to develop a risk score model. Subsequently, we conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses on the identified graphene therapy-related long non-coding RNAs. Additionally, we employed the risk model to construct the tumour microenvironment model and analyse drug sensitivity. To validate our findings, we referenced the IMvigor210 immunotherapy model. Finally, we investigated differences in the tumour stemness index. Through our investigation, we identified four promising graphene therapy-related long non-coding RNAs (AC011405.1, HOXC13-AS, LINC01127 and LINC01574) that could be utilized for treating glioblastoma multiforme patients. Furthermore, we identified 16 compounds that could be utilized in graphene therapy. Our study offers novel insights into the treatment of glioblastoma multiforme, and the identified graphene therapy-related long non-coding RNAs and compounds hold promise for further research in this field. Furthermore, additional biological experiments will be essential to validate the clinical significance of our model. These experiments can help confirm the potential therapeutic value and efficacy of the identified graphene therapy-related long non-coding RNAs and compounds in treating glioblastoma multiforme.

Follicular fluid exosome-derived miR-339-5p enhances in vitro maturation of porcine oocytes via targeting SFPQ, a regulator of the ERK1/2 pathway.

In this study, we aimed to investigate cytoplasmic maturation and miRNA expression of mature oocytes cultured in porcine follicular fluid exosomes. We also examined the effect of miR-339-5p on oocyte maturation. Twenty eight differentially expressed miRNAs were detected using miRNA-seq. We then transfected cumulus oocyte complexes with miR-339-5p mimics and inhibitor during culture. The results showed that exosomes increased endoplasmic reticulum levels and the amount of lipid droplets, and decreased ROS levels, lipid droplet size, and percentage of oocytes with abnormal cortical granule distribution. Overexpressing miR-339-5p significantly decreased cumulus expansion genes, oocyte maturation-related genes, target gene proline/glutamine-rich splicing factor (SFPQ), ERK1/2 phosphorylation levels, oocyte maturation rate, blastocyst rate, and lipid droplet number, but increased lipid droplet size and the ratio of oocytes with abnormal cortical granule distribution. Inhibiting miR-339-5p reversed the decrease observed during overexpression. Mitochondrial membrane potential and ROS levels did not differ significantly between groups. In summary, exosomes promote oocyte cytoplasmic maturation and miR-339-5p regulating ERK1/2 activity through SFPQ expression, thereby elevating oocyte maturation and blastocyst formation rate in vitro.

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.

[Diagnosis of nitrogen content in upper and lower corn leaves based on hyperspectral data].

Based on the spectral characters of corn leaf nitrogen content in the space, the spectral models for rapid estimating crop nitrogen content were set up, which is practically meaningful to effectively providing the guidance in fertilization. Spectral technology was applied to explore corn leaves nitrogen content distribution regularity and the relationship between the nitrogen content and plant index was analysed and then the estimation models were built. The results showed N content in upper leaves is higher than that in lower leaves in four growing stages; lower leaves at tassel emerge stage are sensitive to nitrogen losses, which could be used in guiding fertilization in grain production; optimum estimation models were built atjointing stage, the full-grown stage and tasseling stage, The research results provided the proof of crop nutrient analysis and rational fertilization.

The Wnt pathway regulates wing morph determination in Acyrthosiphon pisum.

Wing dimorphism occurs in insects as a survival strategy to adapt to environmental changes. In response to environmental cues, mother aphids transmit signals to their offspring, and the offspring either emerge as winged adults or develop as wingless adults with degeneration of the wing primordia in the early instar stage. However, how the wing morph is determined in the early instar stage is still unclear. Here, we established a surgical sampling method to obtain precise wing primordium tissues for transcriptome analysis. We identified Wnt as a regulator of wing determination in the early second instar stage in the pea aphid. Inhibiting Wnt signaling via knockdown of Wnt2, Wnt11b, the Wnt receptor-encoding gene fz2 or the downstream targets vg and omb resulted in a decreased proportion of winged aphids. Activation of Wnt signaling via knockdown of miR-8, an inhibitor of the Wnt/Wg pathway, led to an increased proportion of winged aphids. Furthermore, the wing primordia of wingless nymphs underwent apoptosis in the early second instar, and cell death was activated by knockdown of fz2 under the wing-inducing condition. These results indicate that the developmental plasticity of aphid wings is modulated by the intrinsic Wnt pathway in response to environmental challenges.

Insulin-like peptide 8 (Ilp8) regulates female fecundity in flies.

Introduction: Insulin-like peptides (Ilps) play crucial roles in nearly all life stages of insects. Ilp8 is involved in developmental stability, stress resistance and female fecundity in several insect species, but the underlying mechanisms are not fully understood. Here we report the functional characterization of Ilp8s in three fly species, including Bactrocera dorsalis, Drosophila mercatorum and Drosophila melanogaster. Methods: Phylogenetic analyses were performed to identify and characterize insect Ilp8s. The amino acid sequences of fly Ilp8s were aligned and the three-dimensional structures of fly Ilp8s were constructed and compared. The tissue specific expression pattern of fly Ilp8s were examined by qRT-PCR. In Bactrocera dorsalis and Drosophila mercatorum, dsRNAs were injected into virgin females to inhibit the expression of Ilp8 and the impacts on female fecundity were examined. In Drosophila melanogaster, the female fecundity of Ilp8 loss-of-function mutant was compared with wild type control flies. The mutant fruit fly strain was also used for sexual behavioral analysis and transcriptomic analysis. Results: Orthologs of Ilp8s are found in major groups of insects except for the lepidopterans and coleopterans, and Ilp8s are found to be well separated from other Ilps in three fly species. The key motif and the predicted three-dimensional structure of fly Ilp8s are well conserved. Ilp8 are specifically expressed in the ovary and are essential for female fecundity in three fly species. Behavior analysis demonstrates that Ilp8 mutation impairs female sexual attractiveness in fruit fly, which results in decreased mating success and is likely the cause of fecundity reduction. Further transcriptomic analysis indicates that Ilp8 might influence metabolism, immune activity, oocyte development as well as hormone homeostasis to collectively regulate female fecundity in the fruit fly. Discussion: Our findings support a universal role of insect Ilp8 in female fecundity, and also provide novel clues for understanding the modes of action of Ilp8.

Comprehensive assessment of base excision repair (BER)-related lncRNAs as prognostic and functional biomarkers in lung adenocarcinoma: implications for personalized therapeutics and immunomodulation.

BACKGROUND: Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer, and comprehending its molecular mechanisms is pivotal for advancing treatment efficacy. This study aims to explore the prognostic and functional significance of base excision repair (BER)-related long non-coding RNAs (BERLncs) in LUAD. METHODS: A risk score model for BERLncs was developed using the least absolute shrinkage and selection operator regression and Cox regression analysis. Model validation and prognostic evaluation were performed using Kaplan-Meier and receiver-operating characteristic curve analyses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to elucidate the potential biological functions of BERLncs. Comparative analyses were carried out to investigate disparities in tumor mutation burden (TMB), immune infiltration, tumor immune dysfunction and exclusion (TIDE) score, chemosensitivity, and immune checkpoint gene expression between the two risk groups. RESULTS: A predictive risk score model comprising 19 BERLncs was successfully developed. Patients were divided into high-risk and low-risk groups according to the median risk score. The high-risk subgroup exhibited significantly inferior overall survival. Functional enrichment analysis revealed pathways associated with lung cancer development, notably the neuroactive ligand-receptor interaction pathway. High-risk patients demonstrated elevated TMB, diminished TIDE scores, and an immunosuppressive tumor microenvironment, while low-risk patients displayed potential benefits from immunotherapy. Additionally, the risk model identified potential anticancer agents. CONCLUSION: The risk score model based on BERLncs shows promise as a prognostic biomarker for LUAD patients, providing valuable insights for clinical decision-making, therapeutic strategies, and understanding of underlying biological mechanisms.