The composition of the maternal breastmilk microbiota influences the microbiota network structure during early infancy.

BACKGROUND/PURPOSE(S): Human breastmilk (BM) is important for microbiome maturation in infants across different body sites. Streptococcus and Staphylococcus are considered universally predominant genera in the BM microbiota. However, whether the differential abundance of Streptococcus and Staphylococcus in BM can differentially affect microbiome maturation in infants remains unclear. METHODS: We recruited exclusively breastfeeding mothers from among the donors of the human milk bank established at National Cheng-Kung University Hospital. The donor mothers provided 35 BM samples at three months (3 M; before introducing children to complementary feeding) and 23 BM samples at six months (6 M; after introducing children to complementary feeding) postpartum. At both time points, samples from different body sites, including nasal swabs, oral swabs and stool, were collected from the mothers and their infants. RESULTS: Maternal BMI was inversely associated with coagulase-negative Staphylococcus (CoNS) abundance in breastmilk. Staphylococcus caprae representation in BM CoNS showed a negative correlation with Streptococcus abundance. Network analysis revealed that infants fed Staphylococcus-dominated BM had better gut and nasal microbiota networks than infants fed Streptococcus-abundant BM during early infancy. CONCLUSION: Our work suggests that maternal metabolic status plays a crucial role in Staphylococcus/Streptococcus competition in BM, which in turn can impact the development of the infant microbiota. Our microbiota co-occurrence network analysis might serve as a helpful bioinformatic tool to monitor microbiota maturation during early infancy.

Gene therapy with a synthetic adeno-associated viral vector improves audiovestibular phenotypes in Pjvk-mutant mice.

Recessive PJVK mutations that cause a deficiency of pejvakin, a protein expressed in both sensory hair cells and first-order neurons of the inner ear, are an important cause of hereditary hearing impairment. Patients with PJVK mutations garner limited benefits from cochlear implantation; thus, alternative biological therapies may be required to address this clinical difficulty. The synthetic adeno-associated viral vector Anc80L65, with its wide tropism and high transduction efficiency in various inner ear cells, may provide a solution. We delivered the PJVK transgene to the inner ear of Pjvk mutant mice using the synthetic Anc80L65 vector. We observed robust exogenous pejvakin expression in the hair cells and neurons of the cochlea and vestibular organs. Subsequent morphologic and audiologic studies demonstrated significant restoration of spiral ganglion neuron density and hair cells in the cochlea, along with partial recovery of sensorineural hearing impairment. In addition, we observed a recovery of vestibular ganglion neurons and balance function to WT levels. Our study demonstrates the utility of Anc80L65-mediated gene delivery in Pjvk mutant mice and provides insights into the potential of gene therapy for PJVK-related inner ear deficits.

Generation and pathological characterization of a transgenic mouse model carrying a missense PJVK mutation.

Hearing loss is the most prevalent hereditary sensory disorder in children. Approximately 2 in 1000 infants are affected by genetic hearing loss. The PJVK gene, which encodes the pejvakin protein, has been linked to autosomal recessive non-syndromic hearing loss DFNB59. Previous clinical studies have revealed that PJVK mutations might be associated with a wide spectrum of auditory manifestations, ranging from hearing loss of pure cochlear origin to that involving the retrocochlear central auditory pathway. The phenotypic variety makes the pathogenesis of this disease difficult to determine. Similarly, mouse models carrying different Pjvk defects show phenotypic variability and inconsistency. In this study, we generated a knockin mouse model carrying the c.874G > A (p.G292R) variant to model and investigate the auditory and vestibular phenotypes of DFNB59.

Efficient in Utero Gene Transfer to the Mammalian Inner Ears by the Synthetic Adeno-Associated Viral Vector Anc80L65.

Sensorineural hearing loss is one of the most common sensory disorders worldwide. Recent advances in vector design have paved the way for investigations into the use of adeno-associated vectors (AAVs) for hearing disorder gene therapy. Numerous AAV serotypes have been discovered to be applicable to inner ears, constituting a key advance for gene therapy for sensorineural hearing loss, where transduction efficiency of AAV in inner ear cells is critical for success. One such viral vector, AAV2/Anc80L65, has been shown to yield high expression in the inner ears of mice treated as neonates or adults. Here, to evaluate the feasibility of prenatal gene therapy for deafness, we assessed the transduction efficiency of AAV2/Anc80L65-eGFP (enhanced green fluorescent protein) after microinjection into otocysts in utero. This embryonic delivery method achieved high transduction efficiency in both inner and outer hair cells of the cochlea. Additionally, the transduction efficiency was high in the hair cells of the vestibules and semicircular canals and in spiral ganglion neurons. Our results support the potential of Anc80L65 as a gene therapy vehicle for prenatal inner ear disorders.

Establishment of an induced pluripotent stem cell (iPSC) line from a 7-year-old male patient with profound hearing loss carrying c.235delC in GJB2 gene.

Gap junction protein beta 2 gene (GJB2) mutations are the most frequent cause of hereditary hearing impairment. The recessive c.235delC mutation in the GJB2 gene is the most common mutation causing severe to profound sensorineural hearing loss in the Asian population. The induced pluripotent stem cell (iPSC) line was generated using the integration-free Sendai virus method from peripheral blood mononuclear cells (PBMCs) of a hearing-impaired patient with homozygous GJB2 c.235delC mutation. This cell line may serve as a cellular model for studying the pathogenic mechanisms of deafness caused by GJB2 mutations.

Different mechanisms involved in the berberine-induced antiproliferation effects in triple-negative breast cancer cell lines.

BACKGROUND/AIM: Berberine (BBR) is known to be effective at inhibiting cell proliferation and promoting apoptosis in various cancer cells. However, the effects of BBR on triple-negative breast cancer (TNBC) cells remain unclear. The aim of this study was to investigate the cell inhibition effects of BBR on different subtypes of TNBC cells. METHODS: Using human TNBC cell lines of different subtypes, namely, MDA-MB-231, MDA-MB-468, MDA-MB-453, and BT-549 as in vitro models, antiproliferative effects of BBR were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue exclusion assay, and clonogenic assay. Furthermore, cell apoptosis and autophagy were analyzed by flow cytometry, immunofluorescent staining, and LC3 I/II-targeted Western blotting. Various cell growth-related signaling pathways (AKT/ERK/p38) and the expression of proteins present in various cell cycle kinase complexes were analyzed by Western blotting. RESULTS: BBR concentration-dependently suppressed cell proliferation in MDA-MB-468 (0, 3, 6, and 12 µM) and MDA-MB-231 (0, 6.25, 12.5, and 25 µM). The inhibitory effect was not brought about by inducing cell apoptosis, necrosis, or autophagy. Cell cycle analysis disclosed an increased S+G2/M fraction among the BBR-treated MDA-MB-231 and MDA-MB-453 cells; while with the BBR-treated MDA-MB-468 and BT-549 lines, an increased G0/G1 fraction was found. In MDA-MB-231 and MDA-MB-453 cells, by Western blotting, BBR decreased the expression of Cyclin A and CDK1, On the other hand, in BBR-treated MDA-MB-468 and BT-549 cells, there was a decrease in Cyclin D and CDK4 expression. CONCLUSION: Our results demonstrate that the antiproliferation effects of BBR occur via different mechanisms in different subtypes of TNBC cells, which suggests that BBR has potential as a personalized treatment for TNBC patients.

The Deubiquitinase OTU5 Regulates Root Responses to Phosphate Starvation.

Phosphorus, taken up by plants as inorganic phosphate (Pi), is an essential but often growth-limiting mineral nutrient for plants. As part of an orchestrated response to improve its acquisition, insufficient Pi supply triggers alterations in root architecture and epidermal cell morphogenesis. Arabidopsis (Arabidopsis thaliana) mutants defective in the expression of the OVARIAN TUMOR DOMAIN-CONTAINING DEUBIQUITINATING ENZYME5 (OTU5) exhibited a constitutive Pi deficiency root phenotype, comprising the formation of long and dense root hairs and attenuated primary root growth. Quantitative protein profiling of otu5 and wild-type roots using the isobaric tag for relative and absolute quantification methodology revealed genotype- and Pi-dependent alterations in protein profiles. In otu5 plants, Pi starvation caused a short-root-hair phenotype and decreased abundance of a suite of Pi-responsive root hair-related proteins. Mutant plants also showed the accumulation of proteins involved in chromatin remodeling and altered distribution of reactive oxygen species along the root, which may be causative for the alterations in root hair morphogenesis. The root hair phenotype of otu5 was synergistic to that of actin-related protein6 (arp6), harboring a mutation in the SWR1 chromatin-remodeling complex. Genetic analysis of otu5/arp6 double mutants suggests independent but functionally related roles of the two proteins in chromatin organization. The root hair phenotype of otu5 is not caused by a general up-regulation of the Pi starvation response, indicating that OTU5 acts downstream of or interacts with Pi signaling. It is concluded that OTU5 is involved in the interpretation of environmental information, probably by altering chromatin organization and maintaining redox homeostasis.

Deubiquitinating Enzyme OTU5 Contributes to DNA Methylation Patterns and Is Critical for Phosphate Nutrition Signals.

Phosphate (Pi) starvation induces a suite of adaptive responses aimed at recalibrating cellular Pi homeostasis. Plants harboring a mutation in OVARIAN TUMOR DOMAIN-CONTAINING DEUBIQUITINATING ENZYME5 (OTU5) showed altered DNA methylation of root hair-related genes and altered Pi-responsive root traits. Unlike the wild type, homozygous otu5 mutants did not respond to Pi starvation by increased lateral root formation and increased root hair length but formed very short root hairs when grown on low-Pi media. Under Pi-replete conditions, otu5 plants developed more root hairs than the wild type due to attenuated primary root growth, a phenotype that resembled that of Pi-deficient plants. Growth of plants on low-Pi media altered both H3K4 and H3K27 trimethylation levels at the transcriptional start site of a subset of genes encoding key players in Pi homeostasis, which was correlated with mRNA abundance changes of these genes. Pi starvation had a minor impact on DNA methylation. Differentially methylated regions were enriched in transposable elements, suggesting that DNA methylation associated with low Pi supply is required for maintaining genome integrity. It is concluded that DNA methylation and histone methylation constitute critical, interdependent regulatory components that orchestrate the activity of a subset of Pi-responsive genes.

Systems-wide analysis of manganese deficiency-induced changes in gene activity of Arabidopsis roots.

Manganese (Mn) is pivotal for plant growth and development, but little information is available regarding the strategies that evolved to improve Mn acquisition and cellular homeostasis of Mn. Using an integrated RNA-based transcriptomic and high-throughput shotgun proteomics approach, we generated a comprehensive inventory of transcripts and proteins that showed altered abundance in response to Mn deficiency in roots of the model plant Arabidopsis. A suite of 22,385 transcripts was consistently detected in three RNA-seq runs; LC-MS/MS-based iTRAQ proteomics allowed the unambiguous determination of 11,606 proteins. While high concordance between mRNA and protein expression (R = 0.87) was observed for transcript/protein pairs in which both gene products accumulated differentially upon Mn deficiency, only approximately 10% of the total alterations in the abundance of proteins could be attributed to transcription, indicating a large impact of protein-level regulation. Differentially expressed genes spanned a wide range of biological functions, including the maturation, translation, and transport of mRNAs, as well as primary and secondary metabolic processes. Metabolic analysis by UPLC-qTOF-MS revealed that the steady-state levels of several major glucosinolates were significantly altered upon Mn deficiency in both roots and leaves, possibly as a compensation for increased pathogen susceptibility under conditions of Mn deficiency.

PFT1-controlled ROS balance is critical for multiple stages of root hair development in Arabidopsis.

Reactive oxygen species (ROS) have been shown to play key roles in cellular decision making and signal integration in multicellular organisms. In roots, ROS levels are managed by the action of peroxidases and NAPDH oxidases, resulting in a distinct spatial distribution of hydrogen peroxide (H2O2) and superoxide (O2(-)) that is critical for the balance between cell proliferation and differentiation. In addition, ROS is required for the determination of the cell shape of root hairs. Mutations in the Mediator subunit MED25/PFT1 result in compromised root hair development, due to altered expression of a suite of H2O2-producing class III peroxidases. pft1-1 mutants form shorter root hairs than wild-type plants. Analysis of pft1-1 cross-sections revealed that also root hair initiation is compromised, probably by impeding local cell wall loosening. It is suggested that ROS homeostasis is critical throughout the development of root hairs, controlling various processes via PFT1-regulated transcription of genes encoding redox-active enzymes.