Levels and Characteristics of mRNAs in Spores of Firmicute Species.

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation.

This study examined the microbicidal activity of 222-nm UV radiation (UV222), which is potentially a safer alternative to the 254-nm UV radiation (UV254) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV222 and at lower fluences than with UV254B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV222, as were spores lacking their DNA-protective proteins, the α/ß-type small, acid-soluble spore proteins. The spore cores' large amount of Ca2+-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV222, while spores' proteinaceous coat may have given some slight protection against UV222 Survivors among B. subtilis spores treated with UV222 acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV222 resistance. UV222-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV222 does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV222IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV254) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV222) may be less harmful to people than UV254 yet may still kill bacteria and at lower fluences than UV254 The present work has identified the damage by UV222 that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV222 also inactivates a herpesvirus.

Properties of Aged Spores of Bacillus subtilis.

Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.

Analysis of the mRNAs in Spores of Bacillus subtilis.

Large-scale shotgun sequencing (RNA-seq) analysis of mRNAs in dormant Bacillus subtilis spores prepared on plates or in liquid generally found the same ∼46 abundant mRNA species, with >250 mRNAs detected at much lower abundances. Knowledge of the amount of phosphate in a single B. subtilis spore allowed calculation of the amount of mRNA in an individual spore as ∼106 nucleotides (nt). Given the levels of abundant spore mRNAs compared to those of other mRNAs, it was calculated that the great majority of low-abundance mRNAs are present in only small fractions of spores in populations. Almost all of the most abundant spore mRNAs are encoded by genes expressed late in sporulation in the developing spore under the control of the forespore-specific RNA polymerase sigma factor, σG, and most of the encoded proteins are in spores. Levels of the most abundant spore mRNAs were also relatively stable for a week at 4°C after spore harvest. RNA-seq analysis of mRNAs in highly purified and less-well-purified spores made in liquid, as well as from spores that were chemically decoated to remove possible contaminating mRNA, indicated that low-abundance mRNAs in spores were not contaminants in purified spore preparations, and several sources of low-abundance mRNAs in spores are suggested. The function of at least the great majority of spore mRNAs seems most likely to be the generation of ribonucleotides for new RNA synthesis by their degradation early in spore revival.IMPORTANCE Previous work indicates that dormant Bacillus subtilis spores have many hundreds of mRNAs, some of which are suggested to play roles in spores' "return to life" or revival. The present work finds only ∼46 mRNAs at ≥1 molecule spore, with others in only fractions of spores in populations, often very small fractions. Less-abundant spore mRNAs are not contaminants in spore preparations, but how spores accumulate them is not clear. Almost all abundant spore mRNAs are synthesized in the developing spore late in its development, most encode proteins in spores, and abundant mRNAs in spores are relatively stable at 4°C. These findings will have a major impact on thinking about the roles that spore mRNAs may play in spore revival.

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development.

Ezh2 is a histone methyltransferase that suppresses osteoblast maturation and skeletal development. We evaluated the role of Ezh2 in chondrocyte lineage differentiation and endochondral ossification. Ezh2 was genetically inactivated in the mesenchymal, osteoblastic, and chondrocytic lineages in mice using the Prrx1-Cre, Osx1-Cre, and Col2a1-Cre drivers, respectively. WT and conditional knockout mice were phenotypically assessed by gross morphology, histology, and micro-CT imaging. Ezh2-deficient chondrocytes in micromass culture models were evaluated using RNA-Seq, histologic evaluation, and Western blotting. Aged mice with Ezh2 deficiency were also evaluated for premature development of osteoarthritis using radiographic analysis. Ezh2 deficiency in murine chondrocytes reduced bone density at 4 weeks of age but caused no other gross developmental effects. Knockdown of Ezh2 in chondrocyte micromass cultures resulted in a global reduction in trimethylation of histone 3 lysine 27 (H3K27me3) and altered differentiation in vitro RNA-Seq analysis revealed enrichment of an osteogenic gene expression profile in Ezh2-deficient chondrocytes. Joint development proceeded normally in the absence of Ezh2 in chondrocytes without inducing excessive hypertrophy or premature osteoarthritis in vivo In summary, loss of Ezh2 reduced H3K27me3 levels, increased the expression of osteogenic genes in chondrocytes, and resulted in a transient post-natal bone phenotype. Remarkably, Ezh2 activity is dispensable for normal chondrocyte maturation and endochondral ossification in vivo, even though it appears to have a critical role during early stages of mesenchymal lineage commitment.

Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice.

Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of Ezh2 early in the mesenchymal lineage (i.e. through excision via Prrx1 promoter-driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether Ezh2 controls osteoblastogenesis at later developmental stages beyond patterning. We show that Ezh2 loss in committed pre-osteoblasts by Cre expression via the osterix/Sp7 promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, in vivo Ezh2 loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, Ezh2 affects bone formation stage-dependently. We further show that Ezh2 loss in bone marrow-derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of Ezh2 cKO calvaria revealed that the cyclin-dependent kinase inhibitor Cdkn2a is the most prominent cell cycle target of Ezh2 Hence, genetic loss of Ezh2 in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that Ezh2 serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells.

Accumulation and Release of Rare Earth Ions by Spores of Bacillus Species and the Location of These Ions in Spores.

Two rare earth ions, Tb3+ and Dy3+, were incorporated into spores of Bacillus species in ≤5 min at neutral pH to 100 to 200 nmol per mg of dry spores, which is equivalent to 2 to 3% of the spore dry weight. The uptake of these ions had, at most, minimal effects on spore wet heat resistance or germination, and the ions were all released upon germination, probably by complex formation with the huge depot of dipicolinic acid (DPA) released when spores germinate. Adsorbed Tb3+/Dy3+ were also released by exogenous DPA within a few minutes and faster than in spore germination. The accumulation of Tb3+/Dy3+ was not reduced in Bacillus subtilis spores by several types of coat defects, significant modification of the spore cortex peptidoglycan structure, specific loss of components of the outer spore crust layer, or the absence of DPA in the spore core. All of these findings are consistent with Tb3+/Dy3+ being accumulated in spores' outer layers, and this was confirmed by transmission electron microscopy. However, the identity of the outer spore components binding the Tb3+/Dy3+ is not clear. These findings provide new information on the adsorption of rare earth ions by Bacillus spores and suggest this adsorption might have applications in capturing rare earth ions from the environment.IMPORTANCE Biosorption of rare earth ions by growing cells of Bacillus species has been well studied and has attracted attention for possible hydrometallurgy applications. However, the interaction of spores from Bacillus species with rare earth ions has not been well studied. We investigated here the adsorption and/or desorption of two rare earth ions, Tb3+ and Dy3+, by Bacillus spores, the location of the adsorbed ions, and the spore properties after ion accumulation. The significant adsorption of rare earth ions on the surfaces of Bacillus spores and the ions' rapid release by a chelator could allow the development of these spores as a biosorbent to recover rare earth ions from the environment.

Autologous Mesenchymal Stem Cells, Applied in a Bioabsorbable Matrix, for Treatment of Perianal Fistulas in Patients With Crohn's Disease.

In patients with Crohn's disease, perianal fistulas recur frequently, causing substantial morbidity. We performed a 12-patient, 6-month, phase 1 trial to determine whether autologous mesenchymal stem cells, applied in a bioabsorbable matrix, can heal the fistula. Fistula repair was not associated with any serious adverse events related to mesenchymal stem cells or plug placement. At 6 months, 10 of 12 patients (83%) had complete clinical healing and radiographic markers of response. We found placement of mesenchymal stem cell-coated matrix fistula plugs in 12 patients with chronic perianal fistulas to be safe and lead to clinical healing and radiographic response in 10 patients. ClinicalTrials.gov Identifier: NCT01915927.

Enhancer of Zeste Homolog 2 Inhibition Stimulates Bone Formation and Mitigates Bone Loss Caused by Ovariectomy in Skeletally Mature Mice.

Perturbations in skeletal development and bone degeneration may result in reduced bone mass and quality, leading to greater fracture risk. Bone loss is mitigated by bone protective therapies, but there is a clinical need for new bone-anabolic agents. Previous work has demonstrated that Ezh2 (enhancer of zeste homolog 2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitors. Here, we investigated whether inhibition of Ezh2 can be leveraged for bone stimulatory applications. Pharmacologic inhibition and siRNA knockdown of Ezh2 enhanced osteogenic commitment of MC3T3 preosteoblasts. Next generation RNA sequencing of mRNAs and real time quantitative PCR profiling established that Ezh2 inactivation promotes expression of bone-related gene regulators and extracellular matrix proteins. Mechanistically, enhanced gene expression was linked to decreased H3K27 trimethylation (H3K27me3) near transcriptional start sites in genome-wide sequencing of chromatin immunoprecipitations assays. Administration of an Ezh2 inhibitor modestly increases bone density parameters of adult mice. Furthermore, Ezh2 inhibition also alleviated bone loss in an estrogen-deficient mammalian model for osteoporosis. Ezh2 inhibition enhanced expression of Wnt10b and Pth1r and increased the BMP-dependent phosphorylation of Smad1/5. Thus, these data suggest that inhibition of Ezh2 promotes paracrine signaling in osteoblasts and has bone-anabolic and osteoprotective potential in adults.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2.

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Methylenetetrahydrofolate Reductase CpG Islands: Epigenotyping.

BACKGROUND: Determination of the differential DNA methylation patterns of methylenetetrahydrofolate reductase (MTHFR) that are associated with differential MTHFR activity is important to understand the pathogenesis of ischemic stroke. However, to date, no data are available on the differential DNA methylation profiles of Kelantanese Malays. Therefore, we developed a rapid and efficient serial pyrosequencing assay to determine differential DNA methylation profiles of MTHFR, which help to further our understanding of the pathogenesis of ischemic stroke. The developed assay also served as the validation platform for our previous computational epigenetic research on MTHFR. METHODS: Polymerase chain reaction primers were designed and validated to specifically amplify the cytosine that is followed by guanine residues (CpGs) A and B regions. Prior epigenotyping on 110 Kelantanese Malays, the serial pyrosequencing assays for the CpGs A and B regions were validated using five validation controls. The mean values of the DNA methylation profiles of CpGs A and B were calculated. RESULTS: The mean DNA methylation levels for CpGs A and B were 0.984 ± 0.582 and 2.456 ± 1.406, respectively. The CpGs 8 and 20 showed the highest (5.581 ± 4.497) and the lowest (0.414 ± 2.814) levels of DNA methylation at a single-base resolution. CONCLUSION: We have successfully developed and validated a pyrosequencing assay that is fast and can yield high-quality pyrograms for DNA methylation analysis and is therefore applicable to high throughput study. Using this newly developed pyrosequencing assay, the MTHFR DNA methylation profiles of 110 Kelantanese Malays were successfully determined. It also validated our computational epigenetic research on MTHFR.

Histone deacetylase inhibition destabilizes the multi-potent state of uncommitted adipose-derived mesenchymal stromal cells.

Human adipose-derived mesenchymal stromal cells (AMSCs) grown in platelet lysate are promising agents for therapeutic tissue regeneration. Here, we investigated whether manipulation of epigenetic events by the clinically relevant histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) alters differentiation of AMSCs. The multipotency of AMSCs was validated by their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. High-throughput RNA sequencing and RT-qPCR established that human histone deacetylases (HDAC1 to HDAC11, and SIRT1 to SIRT7) are differentially expressed in AMSCs. SAHA induces hyper-acetylation of histone H3 and H4, stimulates protein expression of the HDAC-responsive gene SLC9A3R1/NHERF1 and modulates the AKT/FOXO1 pathway. Biologically, SAHA interferes with osteogenic, chondrogenic and adipogenic lineage commitment of multipotent AMSCs. Mechanistically, SAHA-induced loss of differentiation potential of uncommitted AMSCs correlates with multiple changes in the expression of principal transcription factors that control mesenchymal or pluripotent states. We propose that SAHA destabilizes the multi-potent epigenetic state of uncommitted human AMSCs by hyper-acetylation and perturbation of key transcription factor pathways. Furthermore, AMSCs grown in platelet lysate may provide a useful biological model for screening of new HDAC inhibitors that control the biological fate of human mesenchymal stromal cells.

RUNX3 facilitates growth of Ewing sarcoma cells.

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma.

In silico analyses reveal common cellular pathways affected by loss of heterozygosity (LOH) events in the lymphomagenesis of Non-Hodgkin's lymphoma (NHL).

BACKGROUND: The analysis of cellular networks and pathways involved in oncogenesis has increased our knowledge about the pathogenic mechanisms that underlie tumour biology and has unmasked new molecular targets that may lead to the design of better anti-cancer therapies. Recently, using a high resolution loss of heterozygosity (LOH) analysis, we identified a number of potential tumour suppressor genes (TSGs) within common LOH regions across cases suffering from two of the most common forms of Non-Hodgkin's lymphoma (NHL), Follicular Lymphoma (FL) and Diffuse Large B-cell Lymphoma (DLBCL). From these studies LOH of the protein tyrosine phosphatase receptor type J (PTPRJ) gene was identified as a common event in the lymphomagenesis of these B-cell lymphomas. The present study aimed to determine the cellular pathways affected by the inactivation of these TSGs including PTPRJ in FL and DLBCL tumourigenesis. RESULTS: Pathway analytical approaches identified that candidate TSGs located within common LOH regions participate within cellular pathways, which may play a crucial role in FL and DLBCL lymphomagenesis (i.e., metabolic pathways). These analyses also identified genes within the interactome of PTPRJ (i.e. PTPN11 and B2M) that when inactivated in NHL may play an important role in tumourigenesis. We also detected genes that are differentially expressed in cases with and without LOH of PTPRJ, such as NFATC3 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3). Moreover, upregulation of the VEGF, MAPK and ERBB signalling pathways was also observed in NHL cases with LOH of PTPRJ, indicating that LOH-driving events causing inactivation of PTPRJ, apart from possibly inducing a constitutive activation of these pathways by reduction or abrogation of its dephosphorylation activity, may also induce upregulation of these pathways when inactivated. This finding implicates these pathways in the lymphomagenesis and progression of FL and DLBCL. CONCLUSIONS: The evidence obtained in this research supports findings suggesting that FL and DLBCL share common pathogenic mechanisms. Also, it indicates that PTPRJ can play a crucial role in the pathogenesis of these B-cell tumours and suggests that activation of PTPRJ might be an interesting novel chemotherapeutic target for the treatment of these B-cell tumours.

High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells.

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.

Computational epigenetic profiling of CpG islets in MTHFR.

Computational epigenetics is a new area of research focused on exploring how DNA methylation patterns affect transcription factor binding that affect gene expression patterns. The aim of this study was to produce a new protocol for the detection of DNA methylation patterns using computational analysis which can be further confirmed by bisulfite PCR with serial pyrosequencing. The upstream regulatory element and pre-initiation complex relative to CpG islets within the methylenetetrahydrofolate reductase gene were determined via computational analysis and online databases. The 1,104 bp long CpG island located near to or at the alternative promoter site of methylenetetrahydrofolate reductase gene was identified. The CpG plot indicated that CpG islets A and B, within the island, contained 62 and 75 % GC content CpG ratios of 0.70 and 0.80-0.95, respectively. Further exploration of the CpG islets A and B indicates that the transcription start sites were GGC which were absent from the TATA boxes. In addition, although six PROSITE motifs were identified in CpG B, no motifs were detected in CpG A. A number of cis-regulatory elements were found in different regions within the CpGs A and B. Transcription factors were predicted to bind to CpGs A and B with varying affinities depending on the DNA methylation status. In addition, transcription factor binding may influence the expression patterns of the methylenetetrahydrofolate reductase gene by recruiting chromatin condensation inducing factors. These results have significant implications for the understanding of the architecture of transcription factor binding at CpG islets as well as DNA methylation patterns that affect chromatin structure.

High-resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to Non-Hodgkin's lymphoma.

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin's lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

Extrusion 3D-printing and characterization of poly(caprolactone fumarate) for bone regeneration applications.

Polycaprolactone fumarate (PCLF) is a cross-linkable PCL derivative extensively considered for tissue engineering applications. Although injection molding has been widely used to develop PCLF scaffolds, platforms developed using such technique lack precise control on architecture, design, and porosity required to ensure adequate cellular and tissue responses. In particular, the scaffolds should provide a suitable surface for cell attachment and proliferation, and facilitate cell-cell communication and nutrient flow. 3D printing technologies have led to new architype for biomaterial development with micro-architecture mimicking native tissue. Here, we developed a method for 3D printing of PCLF structures using the extrusion printing technique. The crosslinking property of PCLF enabled the unique post-processing of 3D printed scaffolds resulting in highly porous and flexible PCLF scaffolds with compressive properties imitating natural features of cancellous bone. Generated scaffolds supported excellent attachment and proliferation of mesenchymal stem cells (MSC). The high porosity of PCLF scaffolds facilitated vascularized membrane formation demonstrable with the stringency of the ex ovo chicken chorioallantoic membrane (CAM) implantation. Furthermore, upon implantation to rat calvarium defects, PCLF scaffolds enabled an exceptional new bone formation with a bone mineral density of newly formed bone mirroring native bone tissue. These studies suggest that the 3D-printed highly porous PCLF scaffolds may serve as a suitable biomaterial platform to significantly expand the utility of the PCLF biomaterial for bone tissue engineering applications.

Biodegradable poly(caprolactone fumarate) 3D printed scaffolds for segmental bone defects within the Masquelet technique.

Segmental bone defects, often clinically treated with nondegradable poly(methylmethacrylate) (PMMA) in multistage surgeries, present a significant clinical challenge. Our study investigated the efficacy of 3D printed biodegradable polycaprolactone fumarate (PCLF)/PCL spacers in a one-stage surgical intervention for these defects, focusing on early bone regeneration influenced by spacer porosities. We compared nonporous PCLF/PCL and PMMA spacers, conventionally molded into cylinders, with porous PCLF/PCL spacers, 3D printed to structurally mimic segmental defects in rat femurs for a 4-week implantation study. Histological analysis, including tissue staining and immunohistochemistry with bone-specific antibodies, was conducted for histomorphometry evaluation. The PCLF/PCL spacers demonstrated compressive properties within 6 ± 0.5 MPa (strength) and 140 ± 15 MPa (modulus). Both porous PCLF/PCL and Nonporous PMMA formed collagen-rich membranes (PCLF/PCL: 92% ± 1.3%, PMMA: 86% ± 1.5%) similar to those induced in the Masquelet technique, indicating PCLF/PCL's potential for one-stage healing. Immunohistochemistry confirmed biomarkers for tissue regeneration, underscoring PCLF/PCL's regenerative capabilities. This research highlights PCLF/PCL scaffolds' ability to induce membrane formation in critical-sized segmental bone defects, supporting their use in one-stage surgery. Both solid and porous PCLF/PCL spacers showed adequate compressive properties, with the porous variants exhibiting BMP-2 expression and woven bone formation, akin to clinical standard PMMA. Notably, the early ossification of the membrane into the pores of porous scaffolds suggests potential for bone interlocking and regeneration, potentially eliminating the need for a second surgery required for PMMA spacers. The biocompatibility and biodegradability of PCLF/PCL make them promising alternatives for treating critical bone defects, especially in vulnerable patient groups.

Visible light-induced 3D bioprinted injectable scaffold for minimally invasive tissue regeneration.

Pre-formed hydrogel scaffolds have emerged as favorable vehicles for tissue regeneration, promoting minimally invasive treatment of native tissue. However, due to the high degree of swelling and inherently poor mechanical properties, development of complex structural hydrogel scaffolds at different dimensional scales has been a continuous challenge. Herein, we take a novel approach at the intersections of engineering design and bio-ink chemistry to develop injectable pre-formed structural hydrogel scaffolds fabricated via visible light (VL) induced digital light processing (DLP). In this study, we first determined the minimum concentration of poly(ethylene glycol) diacrylate (PEGDA) to be added to the gelatin methacrylate (GelMA) bio-ink in order to achieve scalable and high printing-fidelity with desired cell adhesion, viability, spreading, and osteogenic differentiation characteristics. Despite the advantages of hybrid GelMA-PEGDA bio-ink in improving scalability and printing-fidelity, compressibility, shape-recovery, and injectability of the 3D bioprinted scaffolds were compromised. To restore these needed characteristics for minimally invasive tissue regeneration applications, we performed topological optimization to design highly compressible and injectable pre-formed (i.e., 3D bioprinted) microarchitectural scaffolds. The designed injectable pre-formed microarchitectural scaffolds showed a great capacity to retain the viability of the encapsulated cells (>72 % after 10 cycles of injection). Lastly, ex ovo chicken chorioallantoic membrane (CAM) studies revealed that the optimized injectable pre-formed hybrid hydrogel scaffold is biocompatible and supports angiogenic growth.