A drug-resistant mutation in plant target of rapamycin validates the specificity of ATP-competitive TOR inhibitors in vivo.

Kinases are major components of cellular signaling pathways, regulating key cellular activities through phosphorylation. Kinase inhibitors are efficient tools for studying kinase targets and functions, however assessing their kinase specificity in vivo is essential. The identification of resistant kinase mutants has been proposed to be the most convincing approach to achieve this goal. Here, we address this issue in plants via a pharmacogenetic screen for mutants resistant to the ATP-competitive TOR inhibitor AZD-8055. The eukaryotic TOR (Target of Rapamycin) kinase is emerging as a major hub controlling growth responses in plants largely thanks to the use of ATP-competitive inhibitors. We identified a dominant mutation in the DFG motif of the Arabidopsis TOR kinase domain that leads to very strong resistance to AZD-8055. This resistance was characterized by measuring root growth, photosystem II (PSII) activity in leaves and phosphorylation of YAK1 (Yet Another Kinase 1) and RPS6 (Ribosomal protein S6), a direct and an indirect target of TOR respectively. Using other ATP-competitive TOR inhibitors, we also show that the dominant mutation is particularly efficient for resistance to drugs structurally related to AZD-8055. Altogether, this proof-of-concept study demonstrates that a pharmacogenetic screen in Arabidopsis can be used to successfully identify the target of a kinase inhibitor in vivo and therefore to demonstrate inhibitor specificity. Thanks to the conservation of kinase families in eukaryotes, and the possibility of creating amino acid substitutions by genome editing, this work has great potential for extending studies on the evolution of signaling pathways in eukaryotes.

mTORC1/rpS6 and p-FAK-Y407 signaling regulate spermatogenesis: Insights from studies of the adjudin pharmaceutical/toxicant model.

In rodents and humans, the major cellular events at spermatogenesis include self-renewal of spermatogonial stem cells and undifferentiated spermatogonia via mitosis, commitment of spermatogonia to differentiation and transformation to spermatocytes, meiosis, spermiogenesis, and the release of spermatozoa at spermiation. While details of the morphological changes during these cellular events have been delineated, knowledge gap exists between the morphological changes in the seminiferous epithelium and the underlying molecular mechanism(s) that regulate these cellular events. Even though many of the regulatory proteins and biomolecules that modulate spermatogenesis are known based on studies using genetic models, the underlying regulatory mechanism(s), in particular signaling pathways/proteins, remain unexplored since much of the information regarding the signaling regulation is unknown. Studies in the past decade, however, have unequivocally demonstrated that the testis is using several signaling proteins and/or pathways to regulate multiple cellular events to modulate spermatogenesis. These include mTORC1/rpS6/Akt1/2 and p-FAK-Y407. While selective inhibitors and/or agonists and antagonists are available to examine some of these signaling proteins, their use have limitations due to their specificities and also potential systemic cytotoxicity. On the other hand, the use of genetic models has had profound implications for our understanding of the molecular regulation of spermatogenesis, and these knockout (null) models have also revealed the factors that are critical for spermatogenesis. Nonetheless, additional studies using in vitro and in vivo models are necessary to unravel the signaling pathways involved in regulating seminiferous epithelial cycle. Emerging data from studies, such as the use of the adjudin pharmaceutical/toxicant model, have illustrated that this non-hormonal male contraceptive drug is utilizing specific signaling pathways/proteins to induce specific defects in spermatogenesis, yielding mechanistic insights on the regulation of spermatogenesis. We sought to review these recent data in this article, highlighting an interesting approach that can be considered for future studies.

The Neurobeachin-like 2 protein (NBEAL2) controls the homeostatic level of the ribosomal protein RPS6 in mast cells.

The Beige and Chediak-Higashi (BEACH) domain-containing, Neurobeachin-like 2 (NBEAL2) protein is a molecule with a molecular weight of 300 kDa. Inactivation of NBEAL2 by loss-of-function mutations in humans as well as deletion of the Nbeal2 gene in mice results in functional defects in cells of the innate immune system such as neutrophils, NK-cells, megakaryocytes, platelets and of mast cells (MCs). To investigate the detailed function of NBEAL2 in murine MCs we generated MCs from wild type (wt) and Nbeal2-/- mice, and deleted Nbeal2 by CRISPR/Cas9 technology in the murine mast cell line MC/9. We also predicted the structure of NBEAL2 to infer its function and to examine potential mechanisms for its association with interaction partners by using the deep learning-based method RoseTTAFold and the Pymol© software. The function of NBEAL2 was analysed by molecular and immunological techniques such as co-immunoprecipitation (co-IP) experiments, western blotting, enzyme-linked immunosorbent assay and flow cytometry. We identified RPS6 as an interaction partner of NBEAL2. Thereby, the NBEAL2/RPS6 complex formation is probably required to control the protein homeostasis of RPS6 in MCs. Consequently, inactivation of NBEAL2 leads to accumulation of strongly p90RSK-phosphorylated RPS6 molecules which results in the development of an abnormal MC phenotype characterised by prolonged growth factor-independent survival and in a pro-inflammatory MC-phenotype.

Ribosomal Protein Dynamics and Its Association with Actin Filaments and Local Translation in Axonal Growth Cones of Dorsal Root Ganglia Neurons.

Local translation in growth cones plays a critical role in responses to extracellular stimuli, such as axon guidance cues. We previously showed that brain-derived neurotrophic factor activates translation and enhances novel protein synthesis through the activation of mammalian target of rapamycin complex 1 signaling in growth cones of dorsal root ganglion neurons. In this study, we focused on 40S ribosomal protein S6 (RPS6), 60S ribosomal protein P0/1/2 (RPP0/1/2), and actin filaments to determine how localization of ribosomal proteins changes with overall protein synthesis induced by neurotrophins. Our quantitative analysis using immunocytochemistry and super-resolution microscopy indicated that RPS6, RPP0/1/2, and actin tend to colocalize in the absence of stimulation, and that these ribosomal proteins tend to dissociate from actin and associate with each other when local protein synthesis is enhanced. We propose that this is because stimulation causes ribosomal subunits to associate with each other to form actively translating ribosomes (polysomes). This study further clarifies the role of cytoskeletal components in local translation in growth cones.

Ribosomal protein S6 kinase 2 (RPS6KB2) is a potential immunotherapeutic target for cancer that upregulates proinflammatory cytokines.

BACKGROUND: Cancer is still a leading cause of mortality. Over the years, cancer therapy has undergone significant advances driven by advancements in science and technology. A promising area of drug discovery in this field involves the development of therapeutic targets for cancer treatment. The urgent need to identify new pharmacological targets arises from the impact of tumor resistance on the effectiveness of current medications. Specifically, the RPS6KB2 gene on chromosome 11 has been implicated in cell cycle regulation and exhibits higher expression levels in tumor tissue. Given this association, there is a potential for this gene to serve as a target for cancer treatment. METHODS: We conducted an analysis using the GTEx, TCGA, and CCLE databases to explore the relationship between RPS6KB2 and immune infiltration, the tumor microenvironment (TME), microsatellite instability (MSI), and more. Cell proliferation was assessed using EDU detection, while cell invasion and migration were evaluated via wound healing and Transwell assays. Additionally, western blot analysis was employed to measure expression of Bax, Bcl-2, MMP2, MMP9, PCNA, and proinflammatory factors. RESULTS: Through data analysis and molecular biology methods, our study carefully examined the potential role of RPS6KB2 in cancer therapy. The data revealed that RPS6KB2 is aberrantly expressed in most cancers and is associated with poor prognosis. Further analysis indicated its involvement in cancer cell apoptosis and migration, as well as its role in cancer immune processes. We validated the significance of RPS6KB2 in hepatocellular carcinoma (HCC), highlighting its capacity to upregulate proinflammatory cytokines. CONCLUSION: Our research indicates that RPS6KB2 is a prognostic biomarker associated with immune infiltration in cancer that can affect antitumor immunity by increasing secretion of proinflammatory factors, providing a potential drug target for cancer treatment.

MiR-200c-3p as a novel genetic marker and therapeutic tool for alopecia areata.

BACKGROUND: MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression in diverse biological processes. They hold promise as therapeutic candidates for targeting human disease pathways, although our understanding of their gene regulatory mechanism remains incomplete. Alopecia areata (AA) is a prevalent inflammatory ailment distinguished by the infiltration of T cells targeting the anagen-stage hair follicles. The scarcity of effective remedies for AA may stem from limited understanding regarding its precise cellular mechanism. AIM: To investigate and examine the importance and role of the miR-200c-3p as a genetic indicator for AA, and its possible impact on disease progression. SUBJECTS AND METHODS: Case-control study included 65 patients with AA and 65 matched healthy controls. A real-time PCR technique was used to measure the expression of miR-200c-3p for both groups. Bioinformatic tools were used for prediction with genes and gene-gene interaction, and protein-protein interaction. RESULTS: The expression levels of miR-200c-3p were significantly higher in AA patients than in healthy controls. We predicted that miR-200c-3p plays a markable role in the development of AA by its effect on the EGFR tyrosine kinase inhibitor resistance pathway. CONCLUSION: We were able to identify the influence of miR-200c-3p on both PLCG1 and RPS6KP1 genes which in turn regulate the EGFR tyrosine kinases resistance pathway that displayed the most substantial increase in activity. Our outcomes shed light on the era of the potential theranostic role of this innovative miRNA in AA.

Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice.

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.

Upregulation of dihydropyrimidinase-like 3 (DPYSL3) protein predicts poor prognosis in urothelial carcinoma.

BACKGROUND: Dihydropyrimidinase-like 3 (DPYSL3) is a cytosolic phosphoprotein expressed in the nervous system and is crucial for neurogenesis. A previous study showed that increased DPYSL3 expression promotes tumour aggressiveness in pancreatic ductal adenocarcinoma, gastric cancer, and colon cancer. However, the role of DPYSL3 in affecting the biological behaviour of urothelial carcinoma (UC) is not yet understood. METHODS: A UC transcriptomic dataset from the Gene Expression Omnibus and the Urothelial Bladder Cancer (BLCA) dataset from The Cancer Genome Atlas were used for the in silico study. We collected 340 upper urinary tract urothelial carcinoma (UTUC) and 295 urinary bladder urothelial carcinoma (UBUC) samples for the immunohistochemical study. Fresh tumour tissue from 50 patients was used to examine the DPYSL3 mRNA level. In addition, urothelial cell lines with and without DPYSL3 knockdown were used for the functional study. RESULTS: The in silico study revealed that DPYSL3 correlated with advanced tumour stage and metastasis development while functioning primarily in the nucleobase-containing compound metabolic process (GO:0006139). DPYSL3 mRNA expression is significantly upregulated in advanced UC. Furthermore, overexpression of the DPYSL3 protein is significantly associated with the aggressive behaviour of UTUC and UBUC. DPYSL3 expression independently predicts disease-specific survival (DSS) and metastatic-free survival (MFS) in patients with UC. In non-muscle-invasive UBUC, DPYSL3 expression predicts local recurrence-free survival. UC cell lines with DPYSL3 knockdown exhibited decreased proliferation, migration, invasion, and human umbilical vein endothelial cells (HUVECs) tube formation but increased apoptosis and G1 arrest. Gene ontology enrichment analysis revealed that the enriched processes related to DPYSL3 overexpression in UC were tissue morphogenesis, cell mesenchyme migration, smooth muscle regulation, metabolic processes, and RNA processing. In vivo study revealed DPYSL3 knockdown in UC tumours significantly suppressed the growth of tumours and decreased MYC and GLUT1 protein expression. CONCLUSIONS: DPYSL3 promotes the aggressiveness of UC cells by changing their biological behaviours and is likely associated with cytoskeletal and metabolic process modifications. Furthermore, DPYSL3 protein overexpression in UC was associated with aggressive clinicopathological characteristics and independently predicted poor clinical outcomes. Therefore, DPYSL3 can be used as a novel therapeutic target for UC.

Blocking ribosomal protein S6 phosphorylation inhibits podocyte hypertrophy and focal segmental glomerulosclerosis.

Ribosomal protein S6 (rpS6) phosphorylation mediates the hypertrophic growth of kidney proximal tubule cells. However, the role of rpS6 phosphorylation in podocyte hypertrophy and podocyte loss during the pathogenesis of focal segmental glomerulosclerosis (FSGS) remains undefined. Here, we examined rpS6 phosphorylation levels in kidney biopsy specimens from patients with FSGS and in podocytes from mouse kidneys with Adriamycin-induced FSGS. Using genetic and pharmacologic approaches in the mouse model of FSGS, we investigated the role of rpS6 phosphorylation in podocyte hypertrophy and loss during development and progression of FSGS. Phosphorylated rpS6 was found to be markedly increased in the podocytes of patients with FSGS and Adriamycin-induced FSGS mice. Genetic deletion of the Tuberous sclerosis 1 gene in kidney glomerular podocytes activated mammalian target of rapamycin complex 1 signaling to rpS6 phosphorylation, resulting in podocyte hypertrophy and pathologic features similar to those of patients with FSGS including podocyte loss, leading to segmental glomerulosclerosis. Since protein phosphatase 1 is known to negatively regulate rpS6 phosphorylation, treatment with an inhibitor increased phospho-rpS6 levels, promoted podocyte hypertrophy and exacerbated formation of FSGS lesions. Importantly, blocking rpS6 phosphorylation (either by generating congenic rpS6 knock-in mice expressing non-phosphorylatable rpS6 or by inhibiting ribosomal protein S6 kinase 1-mediated rpS6 phosphorylation with an inhibitor) significantly blunted podocyte hypertrophy, inhibited podocyte loss, and attenuated formation of FSGS lesions. Thus, our study provides genetic and pharmacologic evidence indicating that specifically targeting rpS6 phosphorylation can attenuate the development of FSGS lesions by inhibiting podocyte hypertrophy and associated podocyte depletion.

Differential Expression of RSK4 Transcript Isoforms in Cancer and Its Clinical Relevance.

While we previously revealed RSK4 as a therapeutic target in lung and bladder cancers, the wider role of this kinase in other cancers remains controversial. Indeed, other reports instead proposed RSK4 as a tumour suppressor in colorectal and gastric cancers and are contradictory in breast malignancies. One explanation for these discrepancies may be the expression of different RSK4 isoforms across cancers. Four RNAs are produced from the RSK4 gene, with two being protein-coding. Here, we analysed the expression of the latter across 30 normal and 33 cancer tissue types from the combined GTEx/TCGA dataset and correlated it with clinical features. This revealed the expression of RSK4 isoforms 1 and 2 to be independent prognostic factors for patient survival, pathological stage, cancer metastasis, recurrence, and immune infiltration in brain, stomach, cervical, and kidney cancers. However, we found that upregulation of either isoform can equally be associated with good or bad prognosis depending on the cancer type, and changes in the expression ratio of isoforms fail to predict clinical outcome. Hence, differential isoform expression alone cannot explain the contradictory roles of RSK4 in cancers, and further research is needed to highlight the underlying mechanisms for the context-dependent function of this kinase.

Coffin-Lowry Syndrome Induced by RPS6KA3 Gene Variation in China: A Case Report in Twins.

Background and objectives: Coffin-Lowry Syndrome (CLS), a rare neurodegenerative disorder, is mainly diagnosed based on clinical manifestations and molecular analyses. In total, about 20 cases of CLS have been reported in China. Here, we report two cases of CLS in identical twin brothers and examine their potential causative mutations. Methods: The Trio mode was used in this analysis, i.e., DNA from the proband and his parents was sequenced. Furthermore, DNA from the proband's twin brother was used for confirmation. Results: A hemizygous variation was detected in the 11th exon of the RPS6KA3 gene, c.898C>T (p.R300*) of the proband, and the same site variation was detected in his identical twin brother; however, the mutation was not detected in his parents. Conclusions: The RPS6KA3 gene mutation c.898C>T (p.R300*) is the causative factor of familial CLS. The variant detected was reported for the first time in the Chinese population. Additionally, by analyzing the previous literature, we were able to summarize the phenotypic and genetic characteristics of GLS in China.

Transcriptome analysis of the transdifferentiation of canine BMSCs into insulin producing cells.

BACKGROUND: Bone marrow mesenchymal stem cells are a potential resource for the clinical therapy of certain diseases. Canine, as a companion animal, living in the same space with human, is an ideal new model for human diseases research. Because of the high prevalence of diabetes, alternative transplantation islets resource (i.e. insulin producing cells) for diabetes treatment will be in urgent need, which makes our research on the transdifferentiation of Bone marrow mesenchymal stem cells into insulin producing cells become more important. RESULT: In this study, we completed the transdifferentiation process and achieved the transcriptome profiling of five samples with two biological duplicates, namely, "BMSCs", "islets", "stage 1", "stage 2" and "stage 3", and the latter three samples were achieved on the second, fifth and eighth day of induction. A total of 11,530 differentially expressed transcripts were revealed in the profiling data. The enrichment analysis of differentially expressed genes revealed several signaling pathways that are essential for regulating proliferation and transdifferentiation, including focal adhesion, ECM-receptor interaction, tight junction, protein digestion and absorption, and the Rap1 signaling pathway. Meanwhile, the obtained protein-protein interaction network and functional identification indicating involvement of three genes, SSTR2, RPS6KA6, and VIP could act as a foundation for further research. CONCLUSION: In conclusion, to the best of our knowledge, this is the first survey of the transdifferentiation of canine BMSCs into insulin-producing cells according with the timeline using next-generation sequencing technology. The three key genes we pick out may regulate decisive genes during the development of transdifferentiation of insulin producing cells.

Regulation of Mumps Virus Replication and Transcription by Kinase RPS6KB1.

Mumps virus (MuV) caused the most viral meningitis before mass immunization. Unfortunately, MuV has reemerged in the United States in the past several years. MuV is a member of the genus Rubulavirus, in the family Paramyxoviridae, and has a nonsegmented negative-strand RNA genome. The viral RNA-dependent RNA polymerase (vRdRp) of MuV consists of the large protein (L) and the phosphoprotein (P), while the nucleocapsid protein (NP) encapsulates the viral RNA genome. These proteins make up the replication and transcription machinery of MuV. The P protein is phosphorylated by host kinases, and its phosphorylation is important for its function. In this study, we performed a large-scale small interfering RNA (siRNA) screen targeting host kinases that regulated MuV replication. The human kinase ribosomal protein S6 kinase beta-1 (RPS6KB1) was shown to play a role in MuV replication and transcription. We have validated the role of RPS6KB1 in regulating MuV using siRNA knockdown, an inhibitor, and RPS6KB1 knockout cells. We found that MuV grows better in cells lacking RPS6KB1, indicating that it downregulates viral growth. Furthermore, we detected an interaction between the MuV P protein and RPS6KB1, suggesting that RPS6KB1 directly regulates MuV replication and transcription.IMPORTANCE Mumps virus is an important human pathogen. In recent years, MuV has reemerged in the United State, with outbreaks occurring in young adults who have been vaccinated. Our work provides insight into a previously unknown mumps virus-host interaction. RPS6KB1 negatively regulates MuV replication, likely through its interaction with the P protein. Understanding virus-host interactions can lead to novel antiviral drugs and enhanced vaccine production.

p-Coumaric acid prevents obesity via activating thermogenesis in brown adipose tissue mediated by mTORC1-RPS6.

Brown adipose tissue (BAT) has long been recognized as an energy-consuming organ and a possible target for combating metabolism disorder. Although numerous studies have demonstrated the ability of phytochemical phenolic acids to improve obesity by activating BAT, the underlying mechanism or mechanism therein remain obscure. In this study, diet-induced obese mice, genetically obese mice, and C3H10T1/2 cells were used to examine the effects of p-Coumaric acid (CA) on metabolism profiles. The results showed that CA prevented metabolic syndromes in the two mice models through the activation of BAT. This phenomenon was closely linked to the upregulation of uncoupling protein 1 (UCP1) and the accelerated burning of fatty acids and glucose, which consequently enhanced the energy expenditure and thermogenesis. Similar results were also obtained in vitro. Importantly, these effects were mediated by the mammalian target of rapamycin complex 1 (mTORC1)-RPS6 pathway. These findings reveal, to the best of our knowledge for the first time, the close correlation between mTORC1-RPS6 and BAT-mediated thermogenesis, and, in addition, the key role played by mTORC1-RPS6 in mediating phenolic acids-induced activation of BAT, thus preventing obesity.

14q32.11 microdeletion including CALM1, TTC7B, PSMC1, and RPS6KA5: A new potential cause of developmental and language delay in three unrelated patients.

Three unrelated patients with similar microdeletions of chromosome 14q32.11 with shared phenotypes including language and developmental delay, and four overlapping genes -CALM1, TTC7B, PSMC1, and RPS6KA5 have been presented. All four genes are expressed in the brain and have haploinsufficiency scores, which reflect low tolerance to loss of function variation. An insight on the genes in the overlapping region, which may influence the resulting phenotype has been provided. Given the three patients' similar phenotypes and lack of normal variation in this region, it was suggested that this microdeletion may be associated with developmental and language delay.

TOR and RPS6 transmit light signals to enhance protein translation in deetiolating Arabidopsis seedlings.

Deetiolation is an essential developmental process transforming young plant seedlings into the vegetative phase with photosynthetic activities. Light signals initiate this important developmental process by triggering massive reprogramming of the transcriptome and translatome. Compared with the wealth of knowledge of transcriptional regulation, the molecular mechanism underlying this light-triggered translational enhancement remains unclear. Here we show that light-enhanced translation is orchestrated by a light perception and signaling pathway composed of photoreceptors, CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), the phytohormone auxin, target of rapamycin (TOR), and ribosomal protein S6 (RPS6). In deetiolating Arabidopsis seedlings, photoreceptors, including phytochrome A and cryptochromes, perceive far-red and blue light to inactivate the negative regulator COP1, which leads to activation of the auxin pathway for TOR-dependent phosphorylation of RPS6. Arabidopsis mutants defective in TOR, RPS6A, or RPS6B exhibited delayed cotyledon opening, a characteristic of the deetiolating process to ensure timely vegetative development of a young seedling. This study provides a mechanistic view of light-triggered translational enhancement in deetiolating Arabidopsis.

The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast-epithelial crosstalk.

Healthy kidney structure and environment rely on epithelial integrity and interactions between epithelial cells and other kidney cells. The Ser/Thr kinase 90 kDa ribosomal protein S6 kinase 1 (p90RSK) belongs to a protein family that regulates many cellular processes, including cell motility and survival. p90RSK is predominantly expressed in the kidney, but its possible role in chronic kidney disease (CKD) remains largely unknown. Here, we found that p90RSK expression is dramatically activated in a classic mouse obstructive chronic kidney disease model, largely in the interstitial FSP-1-positive fibroblasts. We generated FSP-1-specific p90RSK transgenic mouse (RSK-Tg) and discovered that these mice, after obstructive injury, display significantly increased fibrosis and enhanced tubular epithelial damage compared with their wt littermates (RSK-wt), indicating a role of p90RSK in fibroblast-epithelial communication. We established an in vitro fibroblast-epithelial coculture system with primary kidney fibroblasts from RSK-Tg and RSK-wt mice and found that RSK-Tg fibroblasts consistently produce excessive H2O2 causing epithelial oxidative stress and inducing nuclear translocation of the signaling protein ß-catenin. Epithelial accumulation of ß-catenin, in turn, promoted epithelial apoptosis by activating the transcription factor forkhead box class O1 (FOXO1). Of note, blockade of reactive oxygen species (ROS) or ß-catenin or FOXO1 activity abolished fibroblast p90RSK-mediated epithelial apoptosis. These results make it clear that p90RSK promotes kidney fibrosis by inducing fibroblast-mediated epithelial apoptosis through ROS-mediated activation of ß-catenin/FOXO1 signaling pathway.

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.

Silicon influences the localization and expression of Phytophthora sojae effectors in interaction with soybean.

In plant-pathogen interactions, expression and localization of effectors in the aqueous apoplastic region play a crucial role in the establishment or suppression of pathogen development. Silicon (Si) has been shown to protect plants in several host-pathogen interactions, but its mode of action remains a source of debate. Its deposition in the apoplastic area of plant cells suggests that it might interfere with receptor-effector recognition. In this study, soybean plants treated or not with Si were inoculated with Phytophthora sojae and differences in the ensuing infection process were assessed through different microscopy techniques, transcript analysis of effector and defense genes, and effector (Avr6) localization through immunolocalization and fluorescence labeling. In plants grown without Si, the results showed the rapid (4 d post-inoculation) host recognition by P. sojae through the development of haustorium-like bodies, followed by expression and release of effectors into the apoplastic region. In contrast, Si treatment resulted in limited pathogen development, and significantly lower expression and presence of Avr6 in the apoplastic region. Based on immunolocalization and quantification of Avr6 through fluorescence labeling, our results suggest that the presence of Si in the apoplast interferes with host recognition and/or limits receptor-effector interactions, which leads to an incompatible interaction.

Genome-wide association analysis for lethal brachycephalic-like facial dysmorphia in Labrador Retrievers.

A GWAS was performed for inborn X-linked facial dysmorphia with severe growth retardation in Labrador Retrievers. This lethal condition was mapped on the X chromosome at 17-21 Mb and supported by eight SNPs in complete LD. Dams of affected male puppies were heterozygous for the significantly associated SNPs and male affected puppies carried the associated alleles hemizygously. In the near vicinity to the associated region, RPS6KA3 was identified as a candidate gene causing facial dysmorphia in humans and mice known as Coffin-Lowry syndrome. Haplotype analysis showed significant association with the phenotypes of all 18 animals under study. This haplotype was validated through normal male progeny from a dam with the not-associated haplotype on both X chromosomes but male affected full-sibs with the associated haplotype.