Lysosomes degrade macromolecules and recycle metabolites as well as being involved in diverse processes that regulate cellular homeostasis. The lysosome is limited by a single phospholipid bilayer that forms a barrier to separate the potent luminal hydrolases from other cellular constituents, thus protecting the latter from unwanted degradation. The mechanisms that maintain lysosomal membrane integrity remain unknown. Here, we identified SCAV-3, the Caenorhabditis elegans homologue of human LIMP-2, as a key regulator of lysosome integrity, motility, and dynamics. Loss of scav-3 caused rupture of lysosome membranes and significantly shortened lifespan. Both of these phenotypes were suppressed by reinforced expression of LMP-1 or LMP-2, the C. elegans LAMPs, indicating that longevity requires maintenance of lysosome integrity. Remarkably, reduction in insulin/insulin-like growth factor 1 (IGF-1) signaling suppressed lysosomal damage and extended the lifespan in scav-3(lf) animals in a DAF-16-dependent manner. Our data reveal that SCAV-3 is essential for preserving lysosomal membrane stability and that modulation of lysosome integrity by the insulin/IGF-1 signaling pathway affects longevity.

The Six1 transcription factor is a homeodomain protein involved in controlling gene expression during embryonic development. Six1 establishes gene expression profiles that enable skeletal myogenesis and nephrogenesis, among others. While several homeodomain factors have been extensively characterized with regards to their DNA-binding properties, relatively little is known of the properties of Six1. We have used the genomic binding profile of Six1 during the myogenic differentiation of myoblasts to obtain a better understanding of its preferences for recognizing certain DNA sequences. DNA sequence analyses on our genomic binding dataset, combined with biochemical characterization using binding assays, reveal that Six1 has a much broader DNA-binding sequence spectrum than had been previously determined. Moreover, using a position weight matrix optimization algorithm, we generated a highly sensitive and specific matrix that can be used to predict novel Six1-binding sites with highest accuracy. Furthermore, our results support the idea of a mode of DNA recognition by this factor where Six1 itself is sufficient for sequence discrimination, and where Six1 domains outside of its homeodomain contribute to binding site selection. Together, our results provide new light on the properties of this important transcription factor, and will enable more accurate modeling of Six1 function in bioinformatic studies.

Phagocytic removal of apoptotic cells involves formation, maturation, and digestion of cell corpse-containing phagosomes. The retrieval of lysosomal components following phagolysosomal digestion of cell corpses remains poorly understood. Here we reveal that the amino acid transporter SLC-36.1 is essential for lysosome reformation during cell corpse clearance in Caenorhabditis elegans embryos. Loss of slc-36.1 leads to formation of phagolysosomal vacuoles arising from cell corpse-containing phagosomes. In the absence of slc-36.1, phagosome maturation is not affected, but the retrieval of lysosomal components is inhibited. Moreover, loss of PPK-3, the C. elegans homologue of the PtdIns3P 5-kinase PIKfyve, similarly causes accumulation of phagolysosomal vacuoles that are defective in phagocytic lysosome reformation. SLC-36.1 and PPK-3 function in the same genetic pathway, and they directly interact with one another. In addition, loss of slc-36.1 and ppk-3 causes strong defects in autophagic lysosome reformation in adult animals. Our findings thus suggest that the PPK-3-SLC-36.1 axis plays a central role in both phagocytic and autophagic lysosome formation.

During spermatogenesis, interconnected haploid spermatids segregate undesired cellular contents into residual bodies (RBs) before detaching from RBs. It is unclear how this differentiation process is controlled to produce individual spermatids or motile spermatozoa. Here, we developed a live imaging system to visualize and investigate this process in C. elegans. We found that non-muscle myosin 2 (NMY-2)/myosin II drives incomplete cytokinesis to generate connected haploid spermatids, which are then polarized to segregate undesired cellular contents into RBs under the control of myosin II and myosin VI. NMY-2/myosin II extends from the pseudo-cleavage furrow formed between two haploid spermatids to the spermatid poles, thus promoting RB expansion. In the meantime, defective spermatogenesis 15 (SPE-15)/myosin VI migrates from spermatids towards the expanding RB to promote spermatid budding. Loss of myosin II or myosin VI causes distinct cytoplasm segregation defects, while loss of both myosins completely blocks RB formation. We found that the final separation of spermatids from RBs is achieved through myosin VI-mediated cytokinesis, while myosin II is dispensable at this step. SPE-15/myosin VI and F-actin form a detergent-resistant actomyosin VI ring that undergoes continuous contraction to promote membrane constriction between spermatid and RB. We further identified that RGS-GAIP-interacting protein C terminus (GIPC)-1 and GIPC-2 cooperate with myosin VI to regulate contractile ring formation and spermatid release. Our study reveals distinct roles of myosin II and myosin VI in spermatid differentiation and uncovers a novel myosin VI-mediated cytokinesis process that controls spermatid release.

While the signaling pathways and transcription factors active in adult slow- and fast-type muscles begin to be characterized, genesis of muscle fiber-type diversity during mammalian development remains unexplained. We provide evidence showing that Six homeoproteins are required to activate the fast-type muscle program in the mouse primary myotome. Affymetrix transcriptomal analysis of Six1(-/-)Six4(-/-) E10.5 somites revealed the specific down-regulation of many genes of the fast-type muscle program. This data was confirmed by in situ hybridization performed on Six1(-/-)Six4(-/-) embryos. The first mouse myocytes express both fast-type and slow-type muscle genes. In these fibers, Six1 and Six4 expression is required to specifically activate fast-type muscle genes. Chromatin immunoprecipitation experiments confirm the binding of Six1 and Six4 on the regulatory regions of these muscle genes, and transfection experiments show the ability of these homeoproteins to activate specifically identified fast-type muscle genes. This in vivo wide transcriptomal analysis of the function of the master myogenic determinants, Six, identifies them as novel markers for the differential activation of a specific muscle program during mammalian somitic myogenesis.

Reaumuria soongorica (Pall.) Maxim., a resurrection semi-shrub, is a typical constructive and dominant species in desert ecosystems in northwestern China. However, the gene expression characteristics of R. soongorica under drought stress have not been elucidated. Digital gene expression analysis was performed using Illumina technique to investigate differentially expressed genes (DEGs) between control and PEG-treated samples of R. soongorica. A total of 212,338 and 211,052 distinct tags were detected in the control and PEG-treated libraries, respectively. A total of 1,325 genes were identified as DEGs, 379 (28.6%) of which were up-regulated and 946 (71.4%) were down-regulated in response to drought stress. Functional annotation analysis identified numerous drought-inducible genes with various functions in response to drought stress. A number of regulatory proteins, functional proteins, and proteins induced by other stress factors in R. soongorica were identified. Alteration in the regulatory proteins (transcription factors and protein kinase) may be involved in signal transduction. Functional proteins, including flavonoid biosynthetic proteins, late embryogenesis abundant (LEA) proteins, small heat shock proteins (sHSP), and aquaporin and proline transporter may play protective roles in response to drought stress. Flavonoids, LEA proteins and sHSP function as reactive oxygen species scavenger or molecular chaperone. Aquaporin and proline transporters regulate the distribution of water and proline throughout the whole plant. The tolerance ability of R. soongorica may be gained through effective signal transduction and enhanced protection of functional proteins to reestablish cellular homeostasis. DEGs obtained in this study may provide useful insights to help further understand the drought-tolerant mechanism of R. soongorica.

What are the differences in gene expression of cumulus cells (CCs) between young women with diminished ovarian reserve (DOR) and those of similar age with normal ovarian reserve (NOR)?

Ribosome degradation through the autophagy-lysosome pathway is crucial for cell survival during nutrient starvation, but whether it occurs under normal growth conditions and contributes to animal physiology remains unaddressed. In this study, we identified RNST-2, a C. elegans T2 family endoribonuclease, as the key enzyme that degrades ribosomal RNA in lysosomes. We found that loss of rnst-2 causes accumulation of rRNA and ribosomal proteins in enlarged lysosomes and both phenotypes are suppressed by blocking autophagy, which indicates that RNST-2 mediates autophagic degradation of ribosomal RNA in lysosomes. rnst-2(lf) mutants are defective in embryonic and larval development and are short-lived. Remarkably, simultaneous loss of RNST-2 and de novo synthesis of pyrimidine nucleotides leads to complete embryonic lethality, which is suppressed by supplements of uridine or cytidine. Our study reveals an essential role of autophagy-dependent degradation of ribosomal RNA in maintaining nucleotide homeostasis during animal development.

Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However, further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still, the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study, we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming βIII-TUB/MAP2+ neurons, GFAP+ astrocytes, and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors, piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons, astrocytes, and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans.

Quiescent satellite cells are myogenic progenitors that enable regeneration of skeletal muscle. One of the early events of satellite cell activation following myotrauma is the induction of the myogenic regulatory factor MyoD, which eventually induces terminal differentiation and muscle function gene expression. The purpose of this study was to elucidate the mechanism by which MyoD is induced during activation of satellite cells in mouse muscle undergoing regeneration. We show that Six1, a transcription factor essential for embryonic myogenesis, also regulates MyoD expression in muscle progenitor cells. Six1 knock-down by RNA interference leads to decreased expression of MyoD in myoblasts. Chromatin immunoprecipitation assays reveal that Six1 binds the Core Enhancer Region of MyoD. Further, transcriptional reporter assays demonstrate that Core Enhancer Region reporter gene activity in myoblasts and in regenerating muscle depends on the expression of Six1 and on Six1 binding sites. Finally, we provide evidence indicating that Six1 is required for the proper chromatin structure at the Core Enhancer Region, as well as for MyoD binding at its own enhancer. Together, our results reveal that MyoD expression in satellite cells depends on Six1, supporting the idea that Six1 plays an important role in adult myogenesis, in addition to its role in embryonic muscle formation.

Precise regulation of gene expression is crucial to myogenesis and is thought to require the cooperation of various transcription factors. On the basis of a bioinformatic analysis of gene regulatory sequences, we hypothesized that myogenic regulatory factors (MRFs), key regulators of skeletal myogenesis, cooperate with members of the SIX family of transcription factors, known to play important roles during embryonic skeletal myogenesis. To this day little is known regarding the exact molecular mechanism by which SIX factors regulate muscle development. We have conducted a functional genomic study of the role played by SIX1 and SIX4 during the differentiation of skeletal myoblasts, a model of adult muscle regeneration. We report that SIX factors cooperate with the members of the MRF family to activate gene expression during myogenic differentiation, and that their function is essential to this process. Our findings also support a model where SIX factors function not only 'upstream' of the MRFs during embryogenesis, but also 'in parallel' to them during myoblast differentiation. We have identified new essential nodes that depend on SIX factor function, in the myogenesis regulatory network, and have uncovered a novel way by which MRF function is modulated during differentiation.

The Six1 transcription factor is implicated in controlling the development of several tissue types, notably skeletal muscle. Six1 also contributes to muscle metabolism and its activity is associated with the fast-twitch, glycolytic phenotype. Six1 regulates the expression of certain genes of the fast muscle program by directly stimulating their transcription or indirectly acting through a long non-coding RNA. We hypothesized that additional mechanisms of action of Six1 might be at play.

Reaumuria soongorica is one of the typical desert plants that present excellent tolerance to adverse environments. However, its molecular response to UV-B radiation remains poorly understood. To test the response and tolerance mechanisms of R. soongorica to the increasing UV-B radiation, the differentially expressed genes (DEGs) were investigated between the control and UV-B radiation groups. A total of 2150 DEGs were detected between the two groups, of which 561 were up-regulated and 1589 were down-regulated. For functional analysis, DEGs were divided into three groups: (i) Chloroplast-localized proteins, including photosynthesis-associated proteins, ribulose-phosphate-3-epimerase, and ATP-dependent Clp protease. Their transcripts were inhibited, implying that the normal function of chloroplast was affected by UV-B radiation. (ii) Proteins involved in signaling transduction, such as phototropins and GTP-binding proteins. The transcriptional alternation of phototropins may reduce the penetration of UV-B radiation by regulating phototropism, stomatal opening, and chloroplast relocation. The down regulation of GTP-binding proteins may inhibit replication of potentially damaged DNA through preventing cell division; and (iii) proteins for lipid transfer and flavonoids biosynthesis. The up-regulation of these genes suggested that lipid transfer and flavonoids may have a protective function in response to UV-B radiation. Thus, UV-B radiation may lead to the disruption of chloroplasts function. The induction of genes for signal transduction and protective proteins may be a strategy for responding to UV-B radiation in R. soongorica.

The importance of soil microbial flora in agro-ecosystems is well known, but there is limited understanding of the effects of long-term fertilization on soil microbial community succession in different farming management practices. Here, we report the responses of soil microbial community structure, abundance and activity to chemical (CF) and organic fertilization (OF) treatments in a sandy agricultural system of wheat-maize rotation over a 17-year period. Illumina MiSeq sequencing showed that the microbial community diversity and richness showed no significant changes in bacteria but decreased in fungi under both CF and OF treatments. The dominant species showing significant differences between fertilization regimes were Actinobacteria, Acidobacteria and Ascomycota at the phylum level, as well as some unclassified genera of other phyla at the genus level. As expected, soil organic matter content, nutrient element concentrations and bacterial abundance were enhanced by both types of fertilization, especially in OF, but fungal abundance was inhibited by OF. Redundancy analysis revealed that soil enzyme activities were closely related to both bacterial and fungal communities, and the soil nutrient, texture and pH value together determined the community structures. Bacterial abundance might be the primary driver of crop yield, and soil enzyme activities may reflect crop yield. Our results suggest a relatively permanent response of soil microbial communities to the long-term fertilization regimes in a reclaimed sandy agro-ecosystem from a mobile dune, and indicate that the appropriate dosage of chemical fertilizers is beneficial to sandy soil sustainability.