Molecular Basis Research Articles

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Procyanidin B2 alleviates oxidized low-density lipoprotein-induced cell injury, inflammation, monocyte chemotaxis, and oxidative stress by inhibiting the nuclear factor kappa-B pathway in human umbilical vein endothelial cells

BackgroundOxidized low-density lipoprotein (ox-LDL) can initiate and affect almost all atherosclerotic events including endothelial dysfunction. In this text, the role and underlying molecular basis of procyanidin B2 (PCB2) with potential anti-oxidant and anti-inflammatory activities in ox-LDL-induced HUVEC injury were examined.MethodsHUVECs were treated with ox-LDL in the presence or absence of PCB2. Cell viability and apoptotic rate were examined by CCK-8 assay and flow cytometry, respectively. The mRNA and protein levels of genes were tested by RT-qPCR and western blot assays, respectively. Potential downstream targets and pathways of apple procyanidin oligomers were examined by bioinformatics analysis for the GSE9647 dataset. The effect of PCB2 on THP-1 cell migration was examined by recruitment assay. The effect of PCB2 on oxidative stress was assessed by reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and mitochondrial membrane potential (MMP).Resultsox-LDL reduced cell viability, induced cell apoptosis, and facilitated the expression of oxidized low-density lipoprotein receptor 1 (LOX-1), C-C motif chemokine ligand 2 (MCP-1), vascular cell adhesion protein 1 (VCAM-1) in HUVECs. PCB2 alleviated ox-LDL-induced cell injury in HUVECs. Apple procyanidin oligomers triggered the differential expression of 592 genes in HUVECs (|log2fold-change| > 0.58 and adjusted p-value < 0.05). These dysregulated genes might be implicated in apoptosis, endothelial cell proliferation, inflammation, and monocyte chemotaxis. PCB2 inhibited C-X-C motif chemokine ligand 1/8 (CXCL1/8) expression and THP-1 cell recruitment in ox-LDL-stimulated HUVECs. PCB2 inhibited ox-LDL-induced oxidative stress and nuclear factor kappa-B (NF-κB) activation in HUVECs.ConclusionPCB2 weakened ox-LDL-induced cell injury, inflammation, monocyte recruitment, and oxidative stress by inhibiting the NF-κB pathway in HUVECs.

Molecular regulation of calcium‐sensing receptor (CaSR)‐mediated signaling

AbstractCalcium‐sensing receptor (CaSR), a family C G‐protein‐coupled receptor, plays a crucial role in regulating calcium homeostasis by sensing small concentration changes of extracellular Ca2+, Mg2+, amino acids (e.g., L‐Trp and L‐Phe), small peptides, anions (e.g., HCO3− and PO43−), and pH. CaSR‐mediated intracellular Ca2+ signaling regulates a diverse set of cellular processes including gene transcription, cell proliferation, differentiation, apoptosis, muscle contraction, and neuronal transmission. Dysfunction of CaSR with mutations results in diseases such as autosomal dominant hypocalcemia, familial hypocalciuric hypercalcemia, and neonatal severe hyperparathyroidism. CaSR also influences calciotropic disorders, such as osteoporosis, and noncalciotropic disorders, such as cancer, Alzheimer's disease, and pulmonary arterial hypertension. This study first reviews recent advances in biochemical and structural determination of the framework of CaSR and its interaction sites with natural ligands, as well as exogenous positive allosteric modulators and negative allosteric modulators. The establishment of the first CaSR protein–protein interactome network revealed 94 novel players involved in protein processing in endoplasmic reticulum, trafficking, cell surface expression, endocytosis, degradation, and signaling pathways. The roles of these proteins in Ca2+‐dependent cellular physiological processes and in CaSR‐dependent cellular signaling provide new insights into the molecular basis of diseases caused by CaSR mutations and dysregulated CaSR activity caused by its protein interactors and facilitate the design of therapeutic agents that target CaSR and other family C G‐protein‐coupled receptors.

Genome-wide analysis of UDP-glycosyltransferases family and identification of UGT genes involved in drought stress of Platycodon grandiflorus.

The uridine diphosphate (UDP)-glycosyltransferase (UGT) family is the largest glycosyltransferase family, which is involved in the biosynthesis of natural plant products and response to abiotic stress. UGT has been studied in many medicinal plants, but there are few reports on Platycodon grandiflorus. This study is devoted to genome-wide analysis of UGT family and identification of UGT genes involved in drought stress of Platycodon grandiflorus (PgUGTs). The genome data of Platycodon grandiflorus was used for genome-wide identification of PgUGTs, online website and bioinformatics analysis software was used to conduct bioinformatics analysis of PgUGT genes and the genes highly responsive to drought stress were screened out by qRT-PCR, these genes were cloned and conducted bioinformatics analysis. A total of 75 PgUGT genes were identified in P.grandiflorus genome and clustered into 14 subgroups. The PgUGTs were distributed on nine chromosomes, containing multiple cis-acting elements and 22 pairs of duplicate genes were identified. Protein-protein interaction analysis was performed to predict the interaction between PgUGT proteins. Additionally, six genes were upregulated after 3d under drought stress and three genes (PGrchr09G0563, PGrchr06G0523, PGrchr06G1266) responded significantly to drought stress, as confirmed by qRT-PCR. This was especially true for PGrchr06G1266, the expression of which increased 16.21-fold after 3d of treatment. We cloned and conducted bioinformatics analysis of three candidate genes, both of which contained conserved motifs and several cis-acting elements related to stress response, PGrchr06G1266 contained the most elements. PgGT1 was confirmed to catalyze the C-3 position of platycodin D and only eight amino acids showed differences between gene PGr008G1527 and PgGT1, which means PGr008G1527 may be able to catalyze the C-3 position of platycodin D in the same manner as PgGT1. Seven genes were highly expressed in the roots, stems, and leaves, these genes may play important roles in the development of the roots, stems, and leaves of P. grandiflorus. Three genes were highly responsive to drought stress, among which the expression of PGrchr06G1266 was increased 16.21-fold after 3d of drought stress treatment, indicating that PGrchr06G1266 plays an important role in drought stress tolerance. To summarize, this study laied the foundation to better understand the molecular bases of responses to drought stress and the biosynthesis of platycodin.

Effect of Pressure on the Conformational Landscape of Human γD-Crystallin from Replica Exchange Molecular Dynamics Simulations.

Human γD-crystallin belongs to a crucial family of proteins known as crystallins located in the fiber cells of the human lens. Since crystallins do not undergo any turnover after birth, they need to possess remarkable thermodynamic stability. However, their sporadic misfolding and aggregation, triggered by environmental perturbations or genetic mutations, constitute the molecular basis of cataracts, which is the primary cause of blindness in the globe according to the World Health Organization. Here, we investigate the impact of high pressure on the conformational landscape of wild-type HγD-crystallin using replica exchange molecular dynamics simulations augmented with principal component analysis. We find pressure to have a modest impact on global measures of protein stability, such as root-mean-square displacement and radius of gyration. Upon projecting our trajectories along the first two principal components from principal component analysis, however, we observe the emergence of distinct free energy basins at high pressures. By screening local order parameters previously shown or hypothesized as markers of HγD-crystallin stability, we establish correlations between a tyrosine-tyrosine aromatic contact within the N-terminal domain and the protein's end-to-end distance with projections along the first and second principal components, respectively. Furthermore, we observe the simultaneous contraction of the hydrophobic core and its intrusion by water molecules. This exploration sheds light on the intricate responses of HγD-crystallin to elevated pressures, offering insights into potential mechanisms underlying its stability and susceptibility to environmental perturbations, crucial for understanding cataract formation.

Identification of mRNA expression profiles and their characterization in age-related hearing loss.

Age-related hearing loss (ARHL), is a pervasive health problem worldwide. ARHL seriously affects the quality of life and reportedly leads to social isolation and dementia in the elderly. ARHL is caused by the degeneration or disorders of cochlear hair cells and auditory neurons. Numerous studies have verified that genetic factors contributed to this impairment, however, the mechanism behind remains unclear. In this study, we analyzed an mRNA expression dataset (GSE49543) from the GEO database. Differentially expressed genes (DEGs) between young control mice and presbycusis mice were analyzed using limma in R and weighted gene co-expression network analysis (WGCNA) methods. Functional enrichment analyses of the DEGs were conducted with the clusterProfiler R package and the results were visualized using ggplot2 R package. The STRING database was used for the construction of the protein-protein interaction (PPI) network of the screened DEGs. Two machine learning algorithms LASSO and SVM-RFE were used to screen the hub genes. We identified 54 DEGs in presbycusis using limma and WGCNA. DEGs were associated with the synaptic vesicle cycle, distal axon, neurotransmitter transmembrane transporter activity in GO analysis, and alcoholic liver disease, pertussis, lysosome pathway according to KEGG analyses. PPI network analysis identified three significant modules. Five hub genes (CLEC4D, MS4A7, CTSS, LAPTM5, ALOX5AP) were screened by LASSO and SVM-RFE. These hub genes were highly expressed in presbycusis mice compared with young control mice. We screened DEGs and identified hub genes involved in ARHL development, which might provide novel clues to understanding the molecular basis of ARHL.

Widely Targeted Metabolomic Analysis Revealed the Diversity in Milk from Goats, Sheep, Cows, and Buffaloes and Its Association with Flavor Profiles.

The milk flavor can be attributed to the presence of numerous flavor molecules and precursors. In this study, we employed widely targeted metabolomic analysis techniques to analyze the metabolic profiles of various milk samples obtained from goats, sheep, dairy cows, and buffaloes. A total of 631 metabolites were identified in the milk samples, which were further categorized into 16 distinct classes. Principal component analysis (PCA) suggested that the metabolite profiles of samples from the same species exhibit clustering, while separated patterns of metabolite profiles are observed across goat, sheep, cow, and buffalo species. The differential metabolites between the groups of each species were screened based on fold change and variable importance in projection (VIP) values. Five core differential metabolites were subsequently identified, including 3-(3-hydroxyphenyl)-3-hydroxypropanoic acid, inosine 5'-triphosphate, methylcysteine, N-cinnamylglycine, and small peptide (L-tyrosine-L-aspartate). Through multiple comparisons, we also screened biomarkers of each type of milk. Our metabolomic data showed significant inter-species differences in the composition and concentration of some compounds, such as organic acids, amino acids, sugars, nucleotides, and their derivatives, which may affect the overall flavor properties of the milk sample. These findings provided insights into the molecular basis underlying inter-species variations in milk flavor.

Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl-Alkenes Catalyzed by Laboratory Evolved P450s.

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C-H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-β-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C-H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation

BackgroundPaired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing.MethodsThe repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1’s role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.ResultsHere, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.ConclusionsOur study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

The Genetic Diversity of Natural Ilex chinensis Sims (Aquifoliaceae) Populations as Revealed by SSR Markers

Ilex chinensis Sims. is an evergreen tree species native to China and mainly distributed in the region south of the Qinling Mountains and the Huai River. This species has important ornamental, medicinal, ecological, and economic values, and plays a positive role in improving the environment and people’s lives. To reveal the genetic diversity and genetic structure of 401 individuals from 14 populations in the major distribution area of I. chinensis, 11 pairs of SSR primers were selected for PCR amplification. The products were then subjected to capillary electrophoresis, and the genetic diversity of Ilex individuals was analyzed using relevant software. The results showed that the genetic diversity of I. chinensis was at a moderate-to-high level. A total of 54 alleles were detected at 11 SSR loci in the 14 Ilex populations, with an average of 4.831 alleles per locus. AMOVA analysis indicated that the genetic variation of I. chinensis populations mainly originated within populations. A STRUCTURE analysis divided the 401 I. chinensis individuals into four different genetic clusters. The unweighted pair group methods using arithmetic averages (UPGMA) clustering based on Nei’s genetic distance revealed that the population from Xinping of Yuxi, Yunnan Province (XP), and the population from Longan of Qianxinan, Guizhou Province (LoA) were located in the outermost layer of the phylogenetic tree, indicating the furthest genetic relationship between these two population and other populations. The remaining populations could be roughly divided into two groups. Principal coordinate analysis (PCoA) demonstrated that the 401 individuals were clearly divided into three groups, which was consistent with the results of the STRUCTURE analysis and UPGMA clustering. This study identified the hotspots of genetic diversity of I. chinensis, as well as units for the conservation of individuals. It also revealed the patterns of genetic variation and population distribution of I. chinensis in different regions, providing a molecular basis for the geographical zoning and formulation of breeding programs for I. chinensis, as well as germplasm resource management.

Transcriptome profiles reveal response mechanisms and key role of PsNAC1 in Pinus sylvestris var. mongolica to drought stress

BackgroundDrought stress severely impedes plant growth, and only a limited number of species exhibit long-term resistance to such conditions. Pinus sylvestris var. mongolica, a dominant tree species in arid and semi-arid regions of China, exhibits strong drought resistance and plays a crucial role in the local ecosystem. However, the molecular mechanisms underlying this resistance remain poorly understood.ResultsHere, we conducted transcriptome sequence and physiological indicators analysis of needle samples during drought treatment and rehydration stages. De-novo assembly yielded approximately 114,152 unigenes with an N50 length of 1,363 bp. We identified 6,506 differentially expressed genes (DEGs), with the majority being concentrated in the heavy drought stage (4,529 DEGs). Functional annotation revealed enrichment of drought-related GO terms such as response to water (GO:0009415: enriched 108 genes) and response to water deprivation (GO:0009414: enriched 106 genes), as well as KEGG categories including MAPK signaling pathway (K04733: enriched 35 genes) and monoterpenoid biosynthesis (K21374: enriched 27 genes). Multiple transcription factor families and functional protein families were differentially expressed during drought treatment. Co-expression network analysis identified a potential drought regulatory network between cytochrome P450 genes (Unigene4122_c1_g1) and a core regulatory transcription factor Unigene9098_c3_g1 (PsNAC1) with highly significant expression differences. We validated PsNAC1 overexpression in Arabidopsis and demonstrated enhanced drought resistance.ConclusionsThese findings provide insight into the molecular basis of drought resistance in P. sylvestris var. mongolica and lay the foundation for further exploration of its regulatory network.

GHCU, a Molecular Chaperone, Regulates Leaf Curling by Modulating the Distribution of KNGH1 in Cotton.

Leaf shape is considered to be one of the most significant agronomic traits in crop breeding. However, the molecular basis underlying leaf morphogenesis in cotton is still largely unknown. In this study, through genetic mapping and molecular investigation using a natural cotton mutant cu with leaves curling upward, the causal gene GHCU is successfully identified as the key regulator of leaf flattening. Knockout of GHCU or its homolog in cotton and tobacco using CRISPR results in abnormal leaf shape. It is further discovered that GHCU facilitates the transport of the HD protein KNOTTED1-like (KNGH1) from the adaxial to the abaxial domain. Loss of GHCU function restricts KNGH1 to the adaxial epidermal region, leading to lower auxin response levels in the adaxial boundary compared to the abaxial. This spatial asymmetry in auxin distribution produces the upward-curled leaf phenotype of the cu mutant. By analysis of single-cell RNA sequencing and spatiotemporal transcriptomic data, auxin biosynthesis genes are confirmed to be expressed asymmetrically in the adaxial-abaxial epidermal cells. Overall, these findings suggest that GHCU plays a crucial role in the regulation of leaf flattening through facilitating cell-to-cell trafficking of KNGH1 and hence influencing the auxin response level.

Reversible strain-promoted DNA polymerization.

Molecular strain can be introduced to influence the outcome of chemical reactions. Once a thermodynamic product is formed, however, reversing the course of a strain-promoted reaction is challenging. Here, a reversible, strain-promoted polymerization in cyclic DNA is reported. The use of nonhybridizing, single-stranded spacers as short as a single nucleotide in length can promote DNA cyclization. Molecular strain is generated by duplexing the spacers, leading to ring opening and subsequent polymerization. Then, removal of the strain-generating duplexers triggers depolymerization and cyclic dimer recovery via enthalpy-driven cyclization and entropy-mediated ring contraction. This reversibility is retained even when a protein is conjugated to the DNA strands, and the architecture of the protein assemblies can be modulated between bivalent and polyvalent states. This work underscores the utility of using DNA not only as a programmable ligand for assembly but also as a route to access restorable bonds, thus providing a molecular basis for DNA-based materials with shape-memory, self-healing, and stimuli-responsive properties.

Molecular mechanisms underlying structural plasticity of electroconvulsive therapy in major depressive disorder.

Although previous studies reported structural changes associated with electroconvulsive therapy (ECT) in major depressive disorder (MDD), the underlying molecular basis of ECT remains largely unknown. Here, we combined two independent structural MRI datasets of MDD patients receiving ECT and transcriptomic gene expression data from Allen Human Brain Atlas to reveal the molecular basis of ECT for MDD. We performed partial least square regression to explore whether/how gray matter volume (GMV) alterations were associated with gene expression level. Functional enrichment analysis was conducted using Metascape to explore ontological pathways of the associated genes. Finally, these genes were further assigned to seven cell types to determine which cell types contribute most to the structural changes in MDD patients after ECT. We found significantly increased GMV in bilateral hippocampus in MDD patients after ECT. Transcriptome-neuroimaging association analyses showed that expression levels of 726 genes were positively correlated with the increased GMV in MDD after ECT. These genes were mainly involved in synaptic signaling, calcium ion binding and cell-cell signaling, and mostly belonged to excitatory and inhibitory neurons. Moreover, we found that the MDD risk genes of CNR1, HTR1A, MAOA, PDE1A, and SST as well as ECT related genes of BDNF, DRD2, APOE, P2RX7, and TBC1D14 showed significantly positive associations with increased GMV. Overall, our findings provide biological and molecular mechanisms underlying structural plasticity induced by ECT in MDD and the identified genes may facilitate future therapy for MDD.

Osteopontin-driven partial epithelial-mesenchymal transition governs the development of middle ear cholesteatoma

ABSTRACT Cholesteatoma is a common disease of the middle ear. Currently, surgical removal is the only treatment option and patients face a high risk of relapse. The molecular basis of cholesteatoma remains largely unknown. Here, we show that Osteopontin (OPN), a predominantly secreted protein, plays a crucial role in the development of middle ear cholesteatoma. Global transcriptome analysis revealed the loss of epithelial features and an enhanced immune response in human cholesteatoma tissues. Quantitative RT-PCR and immunohistochemical staining of middle ear cholesteatoma validated the reduced expression of epithelial markers, as well as the elevated expression of mesenchymal markers including Vimentin and Fibronectin, but not N-Cadherin, α-smooth muscle actin (α-SMA) or ferroptosis suppressor protein 1 (FSP1), indicating a partial epithelial-mesenchymal transition (EMT) state. Besides, the expression of OPN was significantly elevated in human cholesteatoma tissues. Treatment with OPN promoted cell proliferation, survival and migration and led to a partial EMT in immortalized human keratinocyte cells. Importantly, blockade of OPN signaling could remarkably improve the cholesteatoma-like symptoms in SD rats. Our mechanistic study demonstrated that the AKT-zinc finger E-box binding homeobox 2 (ZEB2) axis mediated the effects of OPN. Overall, these findings suggest that targeting the OPN signaling represents a promising strategy for the treatment of middle ear cholesteatoma.

The MdMYB44-MdTPR1 repressive complex inhibits MdCCD4 and MdCYP97A3 expression through histone deacetylation to regulate carotenoid biosynthesis in apple.

Carotenoids are photosynthetic pigments and antioxidants that contribute to different plant colors. However, the involvement of TOPLESS (TPL/TPR)-mediated histone deacetylation in the modulation of carotenoid biosynthesis through ethylene-responsive element-binding factor-associated amphiphilic repression (EAR)-containing transcription factors (TFs) in apple (Malus domestica Borkh.) is poorly understood. MdMYB44 is a transcriptional repressor that contains an EAR repression motif. In the present study, we used functional analyses and molecular assays to elucidate the molecular mechanisms through which MdMYB44-MdTPR1-mediated histone deacetylation influences carotenoid biosynthesis in apples. We identified two carotenoid biosynthetic genes, MdCCD4 and MdCYP97A3, that were confirmed to be involved in MdMYB44-mediated carotenoid biosynthesis. MdMYB44 enhanced β-branch carotenoid biosynthesis by repressing MdCCD4 expression, whereas MdMYB44 suppressed lutein level by repressing MdCYP97A3 expression. Moreover, MdMYB44 partially influences carotenoid biosynthesis by interacting with the co-repressor TPR1 through the EAR motif to inhibit MdCCD4 and MdCYP97A3 expression via histone deacetylation. Our findings indicate that the MdTPR1-MdMYB44 repressive cascade regulates carotenoid biosynthesis, providing profound insights into the molecular basis of histone deacetylation-mediated carotenoid biosynthesis in plants. These results also provide evidence that the EAR-harboring TF/TPL repressive complex plays a universal role in histone deacetylation-mediated inhibition of gene expression in various plants.

Frequent Amplification and Overexpression of PSMA in Basallike Breast Cancer from Analysis of The Cancer Genome Atlas.

Prostate-specific membrane antigen (PSMA) is frequently overexpressed in nonprostate malignancies. This preclinical study investigated the molecular basis of the application of PMSA-targeting radiopharmaceuticals in breast cancer subtypes. Methods: The somatic copy number status and the transcriptomic and protein expressions of FOLH1 (gene name of PSMA) were analyzed across breast cancer subtypes in 998 patients from The Cancer Genome Atlas dataset. Results: FOLH1 was frequently amplified in basallike breast cancer (BLBC) (32%) compared with luminal and human epidermal growth factor receptor 2-positive subtypes (16% and 17%, respectively; P < 0.01). FOLH1 expression was higher in BLBC (P < 0.001) and was negatively correlated with estrogen-receptor and progesterone-receptor expressions. Consistently, the PSMA protein level was higher in BLBC (P < 0.05). Interestingly, FOLH1 expression was associated with relapse-free and distant metastasis-free survival in patients with BLBC. Conclusion: The BLBC subtype exhibited frequent amplification and overexpression of PSMA, supporting the exploration of PSMA-targeting radiopharmaceuticals in this aggressive breast cancer subtype.

Shared Features Underlying Compact Genomes and Extreme Habitat Use in Chironomid Midges.

Nonbiting midges (family Chironomidae) are found throughout the world in a diverse array of aquatic and terrestrial habitats, can often tolerate harsh conditions such as hypoxia or desiccation, and have consistently compact genomes. Yet we know little about the shared molecular basis for these attributes and how they have evolved across the family. Here, we address these questions by first creating high-quality, annotated reference assemblies for Tanytarsus gracilentus (subfamily Chironominae, tribe Tanytarsini) and Parochlus steinenii (subfamily Podonominae). Using these and other publicly available assemblies, we created a time-calibrated phylogenomic tree for family Chironomidae with outgroups from order Diptera. We used this phylogeny to test for features associated with compact genomes, as well as examining patterns of gene family evolution and positive selection that may underlie chironomid habitat tolerances. Our results suggest that compact genomes evolved in the common ancestor of Chironomidae and Ceratopogonidae and that this occurred mainly through reductions in noncoding regions (introns, intergenic sequences, and repeat elements). Significantly expanded gene families in Chironomidae included biological processes that may relate to tolerance of stressful environments, such as temperature homeostasis, carbohydrate transport, melanization defense response, and trehalose transport. We identified several positively selected genes in Chironomidae, notably sulfonylurea receptor, CREB-binding protein, and protein kinase D. Our results improve our understanding of the evolution of small genomes and extreme habitat use in this widely distributed group.

Mammalian PIWI-piRNA-target complexes reveal features for broad and efficient target silencing.

The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host-pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate-which enforces seed-target fidelity in microRNA RISC-adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed-target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA-target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species.

Unraveling the molecular basis of cannabidiolic acid methyl Ester's anti-depressive effects in a rat model of treatment-resistant depression

Major depressive disorder (MDD) stands as a significant cause of disability globally. Cannabidiolic Acid-Methyl Ester (CBDA-ME) (EPM-301, HU-580), a derivative of Cannabidiol, demonstrates immediate antidepressant-like effects, yet it has undergone only minimal evaluation in psychopharmacology. Our goal was to investigate the behavioral and potential molecular mechanisms associated with the chronic oral administration of this compound in the Wistar Kyoto (WKY) genetic model of treatment-resistant depression. Male WKY rats were subjected to behavioral assessments before and after receiving chronic (14-day) oral doses of CBDA-ME (0.5 mg/kg), 15 mg/kg of imipramine or vehicle. At the end of the study, plasma corticosterone levels and mRNA expression of various genes in the medial Prefrontal Cortex and Hippocampus were measured. Behavioral outcomes from CBDA-ME treatment indicated an antidepressant-like effect similar to imipramine, as oral ingestion reduced immobility and increased swimming duration in the Forced Swim Test. Neither treatment influenced locomotion in the Open Field Test nor preference in the Saccharin Preference Test. The behavioral impact in WKY rats coincided with reduced corticosterone serum levels, upregulated mRNA expression of Cannabinoid receptor 1, Fatty Acid Amide Hydrolase, and Corticotropin-Releasing Hormone Receptor 1, alongside downregulation of the Serotonin Transporter in the hippocampus. Additionally, there was an upregulation of CB1 mRNA expression and downregulation of Brain-Derived Neurotrophic Factor in the mPFC. These findings contribute to our limited understanding of the antidepressant effects of CBDA-ME and shed light on its potential psychopharmacological mechanisms. This discovery opens up possibilities for utilizing cannabinoids in the treatment of major depressive disorder and related conditions.