Analysis of gene expression profiles of Enterococcus faecalis induced by type I collagen

Enterococcus faecalis is an etiological agent of endodontic infections. The present study was performed to investigate the gene profiles of E. faecalis induced by type I collagen stimulation. E. faecalis ATCC 19433 was cultivated with [collagen (+)] or without type I collagen [collagen (−)], and transcriptome analysis was performed using high-throughput sequencing technology. A total of 3.6 gb of information was obtained by sequence analysis and 77 differentially expressed genes (DEGs) between the two culture conditions were identified. Among the 77 DEGs, 35 genes were upregulated in collagen (+) E. faecalis, whereas 42 genes were downregulated. Gene Ontology (GO) enrichment analysis was performed and 11 GO terms, including metalloendopeptidase activity (GO:0004222) and two related GO terms (GO:0031012, GO:0044421), were significantly enriched in the set of upregulated genes. We focused on an upregulated DEG belonging to the matrixin metalloprotease gene family, and matrix metalloprotease (MMP) activities of the bacterial cell were examined. The generic MMP, MMP-8, and MMP-9 activities of collagen (+) E. faecalis were significantly higher than those of collagen (−) E. faecalis. These results suggested that contact with type I collagen may alter the gene expression profile of E. faecalis, and upregulation of metalloprotease genes may result in enhanced MMP activities in E. faecalis.

A Library of Aspergillus niger Chassis Strains for Morphology Engineering Connects Strain Fitness and Filamentous Growth With Submerged Macromorphology

Submerged fermentation using filamentous fungal cell factories is used to produce a diverse portfolio of useful molecules, including food, medicines, enzymes, and platform chemicals. Depending on strain background and abiotic culture conditions, different macromorphologies are formed during fermentation, ranging from dispersed hyphal fragments to approximately spherical pellets several millimetres in diameter. These macromorphologies are known to have a critical impact on product titres and rheological performance of the bioreactor. Pilot productivity screens in different macromorphological contexts is technically challenging, time consuming, and thus a significant limitation to achieving maximum product titres. To address this bottleneck, we developed a library of conditional expression mutants in the organic, protein, and secondary metabolite cell factory Aspergillus niger. Thirteen morphology-associated genes transcribed during fermentation were placed via CRISPR-Cas9 under control of a synthetic Tet-on gene switch. Quantitative analysis of submerged growth reveals that these strains have distinct and titratable macromorphologies for use as chassis during strain engineering programs. We also used this library as a tool to quantify how pellet formation is connected with strain fitness and filamentous growth. Using multiple linear regression modelling, we predict that pellet formation is dependent largely on strain fitness, whereas pellet Euclidian parameters depend on fitness and hyphal branching. Finally, we have shown that conditional expression of the putative kinase encoding gene pkh2 can decouple fitness, dry weight, pellet macromorphology, and culture heterogeneity. We hypothesize that further analysis of this gene product and the cell wall integrity pathway in which it is embedded will enable more precise engineering of A. niger macromorphology in future.

Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue

Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.

In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Acquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis utilizes diverse genome encoded iron acquisition mechanisms to satisfy its need for this nutrient. Indeed, S. lugdunensis can usurp hydroxamate siderophores, and staphyloferrin A and B from S. aureus, using the fhuC ATPase-encoding gene. Acquisition of catechol siderophores and catecholamine stress hormones necessitates the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitates the feoAB locus. Heme iron is acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice we demonstrate that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci, we further demonstrate that only a strain mutated for all of isd, fhuC, sst-1, and feo, versus combination mutants carrying wild type copies of any one of those loci, was attenuated in its ability to proliferate to high numbers in kidneys. Taken together our data reveal that S. lugdunensis requires a repertoire of both heme and non-heme iron acquisition mechanisms to proliferate during systemic infection of mammals

Therapeutic Mesenchymal Stem/Stromal Cells: Value, Challenges and Optimization

Cellular therapy aims to replace damaged resident cells by restoring cellular and molecular environments suitable for tissue repair and regeneration. Among several candidates, mesenchymal stem/stromal cells (MSCs) represent a critical component of stromal niches known to be involved in tissue homeostasis. In vitro, MSCs appear as fibroblast-like plastic adherent cells regardless of the tissue source. The therapeutic value of MSCs is being explored in several conditions, including immunological, inflammatory and degenerative diseases, as well as cancer. An improved understanding of their origin and function would facilitate their clinical use. The stemness of MSCs is still debated and requires further study. Several terms have been used to designate MSCs, although consensual nomenclature has yet to be determined. The presence of distinct markers may facilitate the identification and isolation of specific subpopulations of MSCs. Regarding their therapeutic properties, the mechanisms underlying their immune and trophic effects imply the secretion of various mediators rather than direct cellular contact. These mediators can be packaged in extracellular vesicles, thus paving the way to exploit therapeutic cell-free products derived from MSCs. Of importance, the function of MSCs and their secretome are significantly sensitive to their environment. Several features, such as culture conditions, delivery method, therapeutic dose and the immunobiology of MSCs, may influence their clinical outcomes. In this review, we will summarize recent findings related to MSC properties. We will also discuss the main preclinical and clinical challenges that may influence the therapeutic value of MSCs and discuss some optimization strategies.

Extracellular Vesicles Derived From Stem Cells in Intervertebral Disc Degeneration

Intervertebral disc degeneration (IVDD) is the leading cause of low back pain related to degradation of cartilaginous tissues, mainly resulting from oxidative stress, cell apoptosis, and extracellular matrix degradation. Extracellular vesicles (EVs) exist in all bodily fluids and can be produced by all types of cells. Stem cell-derived EVs (SC-EVs), which are the main paracrine components of stem cells, have gained significant attention in the field of regenerative medicine. Over the past years, accumulating evidence indicates the therapeutic and diagnostic potentials of EVs in IVDD. The main mechanisms involve the induction of regenerative phenotypes, apoptosis alleviation, and immune modulation. In addition, the efficiency of SC-EVs can be enhanced by choosing appropriate donor cells and cell phenotypes, optimizing cell culture conditions, or engineering EVs to deliver drugs and targeting molecules. Given the importance and novelty of SC-EVs, we give an overview of SC-EVs and discuss the roles of SC-EVs in IVDD.

Enhanced Production of Bacterial Cellulose from Miscanthus as Sustainable Feedstock through Statistical Optimization of Culture Conditions

Biorefineries are attracting attention as an alternative to the petroleum industry to reduce carbon emissions and achieve sustainable development. In particular, because forests play an important role in potentially reducing greenhouse gas emissions to net zero, alternatives to cellulose produced by plants are required. Bacterial cellulose (BC) can prevent deforestation and has a high potential for use as a biomaterial in various industries such as food, cosmetics, and pharmaceuticals. This study aimed to improve BC production from lignocellulose, a sustainable feedstock, and to optimize the culture conditions for Gluconacetobacter xylinus using Miscanthus hydrolysates as a medium. The productivity of BC was improved using statistical optimization of the major culture parameters which were as follows: temperature, 29 °C; initial pH, 5.1; and sodium alginate concentration, 0.09% (w/v). The predicted and actual values of BC production in the optimal conditions were 14.07 g/L and 14.88 g/L, respectively, confirming that our prediction model was statistically significant. Additionally, BC production using Miscanthus hydrolysates was 1.12-fold higher than in the control group (commercial glucose). Our result indicate that lignocellulose can be used in the BC production processes in the near future.

Berberine alleviates LPS-induced apoptosis, oxidation, and skewed lineages during mouse preimplantation development

Female infertility is a heterogeneous disorder with a variety of complex causes, including inflammation and oxidative stress, which are also closely associated with the pathogenesis of Polycystic Ovary Syndrome (PCOS). As a new treatment for PCOS, berberine (BER), a natural compound from Berberis, has been clinically applied recently. However, the mechanisms underlying the association between BER and embryogenesis are still largely unknown. In this study, effects of BER on preimplantation development was evaluated by using both normal and inflammatory culture conditions induced by lipopolysaccharide (LPS) in the mouse. Our data first suggest that BER itself (25 nM) does not affect embryo quality or future developmental potency, moreover, it can effectively alleviate LPS-induced embryonic damage by mitigating apoptosis via ROS−/caspase-3-dependent pathways and by suppressing pro-inflammatory cytokines via inhibition of NF-κB signaling pathway during preimplantation embryo development. In addition, skewed cell lineage specification in inner cell mass (ICM) and primitive endoderm (PE) caused by LPS can also be successfully rescued with BER. In summary, these findings for the first time demonstrate the non-toxicity of low doses of BER and its anti-apoptotic and anti-oxidative properties on embryonic cells during mammalian preimplantation development.

A Novel Gene, Le-Dd 10, Is Involved in Fruiting Body Formation of Lentinula Edodes

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened by using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd 1~21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.