Experimental verification of a conserved intronic microRNA located in the human <Emphasis Type="Italic">TrkC</Emphasis> gene with a cell type-dependent apoptotic function

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75^NTR receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.     

<Emphasis Type="Italic">C</Emphasis>-<Emphasis Type="Italic">Raf</Emphasis> deficiency leads to hearing loss and increased noise susceptibility

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K⁺ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf ^+/? mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.     

Sorting nexin 3 mutation impairs development and neuronal function in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

The sorting nexins family of proteins (SNXs) plays pleiotropic functions in protein trafficking and intracellular signaling and has been associated with several disorders, namely Alzheimer’s disease and Down’s syndrome. Despite the growing association of SNXs with neurodegeneration, not much is known about their function in the nervous system. The aim of this work was to use the nematode Caenorhabditis elegans that encodes in its genome eight SNXs orthologs, to dissect the role of distinct SNXs, particularly in the nervous system. By screening the C. elegans SNXs deletion mutants for morphological, developmental and behavioral alterations, we show here that snx-3 gene mutation leads to an array of developmental defects, such as delayed hatching, decreased brood size and life span and reduced body length. Additionally, ?snx-3 worms present increased susceptibility to osmotic, thermo and oxidative stress and distinct behavioral deficits, namely, a chemotaxis defect which is independent of the described snx-3 role in Wnt secretion. ?snx-3 animals also display abnormal GABAergic neuronal architecture and wiring and altered AIY interneuron structure. Pan-neuronal expression of C. elegans snx-3 cDNA in the ?snx-3 mutant is able to rescue its locomotion defects, as well as its chemotaxis toward isoamyl alcohol. Altogether, the present work provides the first in vivo evidence of the SNX-3 role in the nervous system.     

<Emphasis Type="Italic">Cis</Emphasis>–<Emphasis Type="Italic">trans</Emphasis> interactions of cell surface receptors: biological roles and structural basis

Cell surface receptors bind ligands expressed on other cells (in trans) in order to communicate with neighboring cells. However, an increasing number of cell surface receptors are found to also interact with ligands expressed on the same cell (in cis). These observations raise questions regarding the biological role of such cis interactions. Specifically, it is important to know whether cis and trans binding have distinct functional effects and, if so, how a single cell discriminates between interactions in cis versus trans. Further, what are the structural features that allow certain cell surface receptors to engage ligand both on the same as well as on an apposed cell membrane? Here, we summarize known examples of receptors that display cis–trans binding and discuss the emerging diversity of biological roles played by these unconventional two-way interactions, along with their structural basis.     

Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rarα, and rarβ) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfβ2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rarβ, meis2, hoxb5, tgfβ2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgfβ2, and id2 spatial distribution; to increase rarβ, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal–distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.     

<Emphasis Type="Italic">O</Emphasis>-Mannosylation and human disease

Glycosylation of proteins is arguably the most prevalent co- and post-translational modification. It is responsible for increased heterogeneity and functional diversity of proteins. Here we discuss the importance of one type of glycosylation, specifically O-mannosylation and its relationship to a number of human diseases. The most widely studied O-mannose modified protein is alpha-dystroglycan (α-DG). Recent studies have focused intensely on α-DG due to the severity of diseases associated with its improper glycosylation. O-mannosylation of α-DG is involved in cancer metastasis, arenavirus entry, and multiple forms of congenital muscular dystrophy [1, 2]. In this review, we discuss the structural and functional characteristics of O-mannose-initiated glycan structures on α-DG, enzymes involved in the O-mannosylation pathway, and the diseases that are a direct result of disruptions within this pathway.     

Structural variations in wheat HKT1;5 underpin differences in Na<Superscript>+</Superscript> transport capacity

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na⁺) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na⁺-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K _m) for the Na⁺ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D⁴⁷¹/a gap and D⁴⁷⁴/G⁴⁷³ that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na⁺ exclusion in wheat that leads to an improved salinity tolerance in the field.     

Editing porcine <Emphasis Type="Italic">IGF2</Emphasis> regulatory element improved meat production in Chinese Bama pigs

Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR–Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3–3072 site using CRISPR–Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.     

Isolating <Emphasis Type="Italic">Escherichia coli</Emphasis> strains for recombinant protein production

Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> chromosomal passenger complex is essential for the organization of a functional mitotic spindle: a prerequisite for productive endodyogeny

The phylum Apicomplexa encompasses deadly pathogens such as malaria and Cryptosporidium. Apicomplexa cell division is mechanistically divergent from that of their mammalian host, potentially representing an attractive source of drug targets. Depending on the species, apicomplexan parasites can modulate the output of cell division, producing two to thousands of daughter cells at once. The inherent flexibility of their cell division mechanisms allows these parasites to adapt to different niches, facilitating their dissemination. Toxoplasma gondii tachyzoites divide using a unique form of cell division called endodyogeny. This process involves a single round of DNA replication, closed nuclear mitosis, and assembly of two daughter cells within a mother. In higher Eukaryotes, the four-subunit chromosomal passenger complex (CPC) (Aurora kinase B (ARKB)/INCENP/Borealin/Survivin) promotes chromosome bi-orientation by detaching incorrect kinetochore–microtubule attachments, playing an essential role in controlling cell division fidelity. Herein, we report the characterization of the Toxoplasma CPC (Aurora kinase 1 (Ark1)/INCENP1/INCENP2). We show that the CPC exhibits dynamic localization in a cell cycle-dependent manner. TgArk1 interacts with both TgINCENPs, with TgINCENP2 being essential for its translocation to the nucleus. While TgINCENP1 appears to be dispensable, interfering with TgArk1 or TgINCENP2 results in pronounced division and growth defects. Significant anti-cancer drug development efforts have focused on targeting human ARKB. Parasite treatment with low doses of hesperadin, a known inhibitor of human ARKB at higher concentrations, phenocopies the TgArk1 and TgINCENP2 mutants. Overall, our study provides new insights into the mechanisms underpinning cell cycle control in Apicomplexa, and highlights TgArk1 as potential drug target.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions

O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O-GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.     

The role of the <Emphasis Type="Italic">ATP2C1</Emphasis> gene in Hailey–Hailey disease

Hailey–Hailey disease (HHD) is a rare autosomal dominant acantholytic dermatosis, characterized by a chronic course of repeated and exacerbated skin lesions in friction regions. The pathogenic gene of HHD was reported to be the ATPase calcium-transporting type 2C member 1 gene (ATP2C1) located on chromosome 3q21–q24. Its function is to maintain normal intracellular concentrations of Ca²⁺/Mn²⁺ by transporting Ca²⁺/Mn²⁺ into the Golgi apparatus. ATP2C1 gene mutations are reportedly responsible for abnormal cytosolic Ca²⁺/Mn²⁺ levels and the clinical manifestations of HHD. Environmental factors and genetic modifiers may also affect the clinical variability of HHD. This article aims to critically discuss the clinical and pathological features of HHD, differential diagnoses, and genetic and functional studies of the ATP2C1 gene in HHD. Further understanding the role of the ATP2C1 gene in the pathogenesis of HHD by genetic, molecular, and animal studies may contribute to a better clinical diagnosis and provide new strategies for the treatment and prevention of HHD.