Spatiotemporal association of DNAJB13 with the annulus during mouse sperm flagellum development

<p>Abstract</p> <p>Background</p> <p>The sperm annulus is a septin-based fibrous ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Although ultrastructural abnormalities and functional importance of the annulus have been addressed in <it>Sept4</it>-null mutant mice and a subset of human patients with asthenospermia syndrome, little is known about how the structure is assembled and positioned to the midpiece-principal piece junction during mammalian sperm flagellum development.</p> <p>Results</p> <p>By performing immunofluorescence and biochemical approaches with antibodies against DNAJB13 and an annulus constituent SEPT4, we report here a spatiotemporal association of DNAJB13 with sperm annulus during mouse sperm flagellum development. DNAJB13 co-localized with SEPT4 to the annulus, and both were first able to be detected in step 9 spermatids. As spermiogenesis proceeded, the annular DNAJB13 immunosignal increased until the annulus reached the midpiece-principal piece junction, and then gradually disappeared from it in late spermiogenesis. In contrast, the SEPT4 immunosignal was relatively unaltered, and still present on annulus of mature spermatozoa. In <it>Sept4</it>-null mouse spermatids lacking the annulus structure, the annulus-like DNAJB13 immunosignal was still able to be detected, albeit weaker, at the neck region of the flagella. In vitro DNAJB13 was co-localized and interacted with SEPT4 directly.</p> <p>Conclusion</p> <p>The direct interaction of DNAJB13 with SEPT4 in vitro and its spatiotemporal association with the annulus during sperm flagellum development, and even its annulus-like appearance in the annulus-deficient spermatids, suggest that DNAJB13 may be involved in assembling the annulus structure and positioning it towards the midpiece-principal piece junction.</p

Integrating scRNA and bulk-RNA sequencing develops a cell senescence signature for analyzing tumor heterogeneity in clear cell renal cell carcinoma

IntroductionCellular senescence (CS) plays a critical role in cancer development, including clear cell renal cell carcinoma (ccRCC). Traditional RNA sequencing cannot detect precise molecular composition changes within tumors. This study aimed to analyze cellular senescence’s biochemical characteristics in ccRCC using single RNA sequencing (ScRNA-seq) and traditional RNA sequencing (Bulk RNA-seq).MethodsResearchers analyzed the biochemical characteristics of cellular senescence in ccRCC using ScRNA-seq and Bulk RNA-seq. They combined these approaches to identify differences between malignant and non-malignant phenotypes in ccRCC across three senescence-related pathways. Genes from these pathways were used to identify molecular subtypes associated with senescence, and a new risk model was constructed. The function of the gene DUSP1 in ccRCC was validated through biological experiments.ResultsThe combined analysis of ScRNA-seq and Bulk RNA-seq revealed significant differences between malignant and non-malignant phenotypes in ccRCC across three senescence-related pathways. Researchers identified genes from these pathways to identify molecular subtypes associated with senescence, constructing a new risk model. Different subgroups showed significant differences in prognosis level, clinical stage and grade, immune infiltration, immunotherapy, and drug sensitivity.DiscussionSenescence signature markers are practical biomarkers and predictors of molecular typing in ccRCC. Differences in prognosis level, clinical stage and grade, immune infiltration, immunotherapy, and drug sensitivity between different subgroups indicate that this approach could provide valuable insights into senescence-related treatment options and prognostic assessment for patients with ccRCC. The function of the gene DUSP1 in ccRCC was validated through biological experiments, confirming its feasibility as a novel biomarker for ccRCC. These findings suggest that targeted therapies based on senescence-related mechanisms could be an effective treatment option for ccRCC

Expanding the portfolio of tribo-positive materials: aniline formaldehyde condensates for high charge density triboelectric nanogenerators

The rapid uptake of energy harvesting triboelectric nanogenerators (TENGs) for self-powered electronics requires the development of high-performance tribo-materials capable of providing large power outputs. This work reports on the synthesis and use of aniline formaldehyde resin (AFR) for energy-harvesting applications. The facile, acidic-medium reaction between aniline and formaldehyde produces the aniline-formaldehyde condensate, which upon an in-vacuo high temperature curing step provides smooth AFR films with abundant nitrogen and oxygen surface functional groups which can acquire a tribo-positive charge and thus endow AFR with a significantly higher positive tribo-polarity than the existing state-of-art polyamide-6 (PA6). A TENG comprising of optimized thin-layered AFR against a polytetrafluoroethylene (PTFE) film produced a peak-to-peak voltage of up to ~1,000 V, a current density of ~65 mA m⁻², a transferred charge density of ~200 μC m⁻² and an instantaneous power output (energy pulse) of ~11 W m⁻² (28.1 μJ cycle⁻¹), respectively. The suitability of AFR was further supported through the Kelvin probe force microscopy (KPFM) measurements, which reveal a significantly higher average surface potential value of 1.147 V for AFR as compared to 0.87 V for PA6 and a step-by-step increase of the surface potential with the increase of energy generation cycles. The work not only proposes a novel and scalable mouldable AFR synthesis process but also expands with excellent prospects, the current portfolio of tribo-positive materials for triboelectric energy harvesting applications

Chromosome Imbalances in Neuroblastoma&mdash;Recent Molecular Insight into Chromosome 1p-deletion, 2p-gain, and 11q-deletion Identifies New Friends and Foes for the Future

Neuroblastoma is the most common extracranial solid pediatric tumor, with around 15% childhood cancer-related mortality. High-risk neuroblastomas exhibit a range of genetic, morphological, and clinical heterogeneities, which add complexity to diagnosis and treatment with existing modalities. Identification of novel therapies is a high priority in high-risk neuroblastoma, and the combination of genetic analysis with increased mechanistic understanding&mdash;including identification of key signaling and developmental events&mdash;provides optimism for the future. This focused review highlights several recent findings concerning chromosomes 1p, 2p, and 11q, which link genetic aberrations with aberrant molecular signaling output. These novel molecular insights contribute important knowledge towards more effective treatment strategies for neuroblastoma

Mammalian sperm flagella and cilia

In mammals, both sperm flagella and cilia are axoneme-containing organelles. The sperm flagella are major parts of the spermatozoa that are male gametes produced in the testis from progenitor spermatogonia through a process called spermatogenesis. During last stage of spermatogenesis, also called spermiogenesis, an axoneme grows out from one of the two centrioles to form the flagellum. With the growth of the axoneme, accessory components including mitochondria, outer dense fibers, fibrous sheath and annulus are added to the flagellum to form its middle, principal and end pieces. The sperm flagellum is responsible for the sperm motility, and defects in it often cause male infertility. Unlike sperm flagella, cilia contain only axonemes covered with the plasma membrane. Generally two types of cilia exist in mammals: multiple motile cilia and single primary cilia. All cilia extend from basal bodies that are derived from centrioles. Cilia play important physiological roles in the body. Defects in them cause a large number of genetic diseases, such as polycystic kidney disease, retinal degeneration, hydrocephalus, laterality defects, chronic respiratory problems, and even obesity and diabetes. In this thesis, I characterized two proteins related to mammalian sperm flagellum development and ciliogenesis: DNAJB13 and SMC1. Dnajb13 is a type II Hsp40 gene once reported to be highly expressed in testis. By multi-tissue RT-PCR, I found that it was also expressed in several ciliated tissues. In mouse testis, Dnajb13 mRNA was detectable at postnatal week 1 while DNAJB13 was undetectable until postnatal week 4, indicating a translational control for this gene. Although being an Hsp40, DNAJB13 was not heat-inducible. In the cross-sections of seminiferous epithelium, DNAJB13 was first detectable in step2-3 spermatids, peaked at step9-10 and then gradually decreased afterwards. In a mature spermatozoon, DNAJB13 was present in the flagellum throughout the length. Its presence in normal sperm flagellum but not in SDS-resistant sperm tail indicates that it is axoneme-associated, which is further confirmed by its presence in motile cilia of airway and oviduct epithelia. By immunoelectron microscopy DNAJB13 was defined as a radial spoke protein. DNAJB13 is also associated with the annulus spatiotemporally during mouse sperm flagellum development. This association started from the formation of annulus. The annular DNAJB13 increased with the maturation of annulus, and peaked when the annulus reaches the midpieceprincipal piece junction of the flagellum, and then gradually disappeared during late spermiogenesis. In annulus-deficient spermatids, DNAJB13 still formed an annulus-like ring in the neck region. In vitro DNAJB13 was colocalized and interacted with an annulus constituent SEPT4. All the data suggest that DNAJB13 may be involved in the assembly and positioning of the annulus during mouse sperm flagellum development. Cohesin protein SMC1 is well known for its roles in sister chromatid cohesion and DNA repair. It was also reported to be present in primary cilia. By immunofluorescence, I found that SMC1 also localized to centrosomes throughout the cell cycle in a microtubule-independent manner. In addition, SMC1 was associated with both centrioles of a centrosome. Biochemically, SMC1 was cofractionated with the centrosomal marker gamma-tubulin in centrosomal preparation. In vivo SMC1 localized to the basal bodies of motile cilia. These data suggest that SMC1 is also a centrosome/basal body protein and may play a role in ciliogenesis

DataSheet_1_ALK F1174S mutation impairs ALK kinase activity in EML4-ALK variant 1 and sensitizes EML4-ALK variant 3 to crizotinib.docx

ObjectiveTo assess the influence of F1174S mutation on kinase activity and drug sensitivity of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion (EML4-ALK) variants 1 and 3.MethodsWe constructed mammalian expression plasmids of both wildtype and F1174 mutant EML4-ALK variants 1 and 3, and then characterized them with cell models by performing immunoblotting, neurite outgrowth assay, focus formation assay as well as protein stability assay. Drug sensitivity to ALK tyrosine kinase inhibitors was also compared between wildtype and F1174 mutant EML4-ALK fusions. In addition, we characterized the effect of different F1174 kinase domain mutations in the context of EML4-ALK fusions.ResultsIn contrast to the oncogenic ALK-F1174S mutation that has been reported to be activating in the context of full-length ALK in neuroblastoma, EML4-ALK (F1174S) variant 1 exhibits impaired kinase activity leading to loss of oncogenicity. Furthermore, unlike the previously reported F1174C/L/V mutations, mutation of F1174 to S sensitizes EML4-ALK variants 3a and 3b to crizotinib.ConclusionThese findings highlight the complexity of drug selection when treating patients harboring resistance mutations and suggest that the F1174S mutation in EML4-ALK variant 1 is likely not a potent oncogenic driver. Additional oncogenic driver or other resistance mechanisms should be considered in the case of EML4-ALK variant 1 with F1174S mutation.</p

Optical second-harmonic generation imaging for identifying gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors arising in the digest tract. It brings a challenge to diagnosis because it is asymptomatic clinically. It is well known that tumor development is often accompanied by the changes in the morphology of collagen fibers. Nowadays, an emerging optical imaging technique, second-harmonic generation (SHG), can directly identify collagen fibers without staining due to its noncentrosymmetric properties. Therefore, in this study, we attempt to assess the feasibility of SHG imaging for detecting GISTs by monitoring the morphological changes of collagen fibers in tumor microenvironment. We found that collagen alterations occurred obviously in the GISTs by comparing with normal tissues, and furthermore, two morphological features from SHG images were extracted to quantitatively assess the morphological difference of collagen fibers between normal muscular layer and GISTs by means of automated image analysis. Quantitative analyses show a significant difference in the two collagen features. This study demonstrates the potential of SHG imaging as an adjunctive diagnostic tool for label-free identification of GISTs

Novel mechanisms of ALK activation revealed by analysis of the Y1278S neuroblastoma mutation

Numerous mutations have been observed in the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) in both germline and sporadic neuroblastoma. Here, we have investigated the Y1278S mutation, observed in four patient cases, and its potential importance in the activation of the full length ALK receptor. Y1278S is located in the 1278-YRASYY-1283 motif of the ALK activation loop, which has previously been reported to be important in the activation of the ALK kinase domain. In this study, we have characterized activation loop mutations within the context of the full length ALK employing cell culture and Drosophila melanogaster model systems. Our results show that the Y1278S mutant observed in patients with neuroblastoma harbors gain-of-function activity. Secondly, we show that the suggested interaction between Y1278 and other amino acids might be of less importance in the activation process of the ALK kinase than previously proposed. Thirdly, of the three individual tyrosines in the 1278-YRASYY-1283 activation loop, we find that Y1283 is the critical tyrosine in the activation process. Taken together, our observations employing different model systems reveal new mechanistic insights on how the full length ALK receptor is activated and highlight differences with earlier described activation mechanisms observed in the NPM-ALK fusion protein, supporting a mechanism of activation more in line with those observed for the Insulin Receptor (InR).Originally included in thesis in manuscript form.</p

Loss of RET Promotes Mesenchymal Identity in Neuroblastoma Cells

International audienceAberrant activation of anaplastic lymphoma kinase (ALK) drives neuroblastoma (NB). Previous work identified the RET receptor tyrosine kinase (RTK) as a downstream target of ALK activity in NB models. We show here that ALK activation in response to ALKAL2 ligand results in the rapid phosphorylation of RET in NB cells, providing additional insight into the contribution of RET to the ALK-driven gene signature in NB. To further address the role of RET in NB, RET knockout (KO) SK-N-AS cells were generated by CRISPR/Cas9 genome engineering. Gene expression analysis of RET KO NB cells identified a reprogramming of NB cells to a mesenchymal (MES) phenotype that was characterized by increased migration and upregulation of the AXL and MNNG HOS transforming gene (MET) RTKs, as well as integrins and extracellular matrix components. Strikingly, the upregulation of AXL in the absence of RET reflects the development timeline observed in the neural crest as progenitor cells undergo differentiation during embryonic development. Together, these findings suggest that a MES phenotype is promoted in mesenchymal NB cells in the absence of RET, reflective of a less differentiated developmental status