MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.

DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2 and MBD4 and show that MBD2 acts as a TE repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused activation at a small subset of TEs in the vegetative cell of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. TE activation in mbd2 became more significant in the mbd5 mbd6 and adcp1 mutant backgrounds, suggesting that MBD2 acts redundantly with other silencing pathways to repress TEs. Overall, our study identifies MBD2 as a methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

The Extremes of Constipation: A Case of Stercoral Perforation From Fecal Impaction in a Teenager.

Stercoral perforation is a rare sequela of poorly controlled constipation that is more commonly seen in older, bedridden patients than in pediatric patients. We present the case of a 13-year-old patient requiring a divided sigmoid colostomy following rectal perforation, one of the few examples in the pediatric literature of stercoral perforation from chronic constipation. The current report highlights the importance of appropriate treatment of functional constipation at onset and the life-threatening complications that can occur without appropriate follow-up.

Vacuum-Assisted Closure Treats Refractory Esophageal Leak in a Pediatric Patient.

Esophageal perforations can have iatrogenic and non-iatrogenic causes. Early identification is a predictor of good outcomes. When identified, perforations can be managed conservatively with wide drainage or repaired surgically. Endoscopic esophageal vacuum-assisted closure may be used as a definitive treatment, particularly in scenarios where conservative management and primary surgical repair fail to achieve complete healing. We present such a scenario advocating for the consideration of endoscopic esophageal vacuum-assisted closure in patients with refractory esophageal leaks.

Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing in the vegetative cell of developing pollen.

Silencing of transposable elements (TEs) drives the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here, we show, via single-nucleus RNA-seq of developing male gametophytes, that these repressors are critical for TE silencing in the pollen vegetative cell, a companion cell important for fertilization that undergoes chromatin decompaction. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We show that TEs repressed by MBD5/6 gain chromatin accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system particularly important when chromatin compaction is compromised.

Culture duration modulates collagen hydrolysate-induced tissue remodeling in chondrocyte-seeded agarose hydrogels.

Media supplementation with collagen hydrolysate was hypothesized to increase the collagen content in engineered cartilage. By d28, hydrolysate at 0.5 mg/mL increased type II collagen content and 1 mg/mL increased mechanical properties, total collagen content, and type II collagen content over controls. By d42, however, controls possessed the highest GAG content and compressive Young's modulus. Real-time PCR found that 1 mg/mL increased type II collagen gene expression in d0 constructs, but increased MMP expression with no effect on type II collagen on d28. A 10 mg/mL concentration produced the lowest tissue properties, the lowest type II collagen gene expression on d0, and the highest MMP gene expression on d28. These results indicate that the duration of culture modulates the response of chondrocytes to collagen hydrolysate in 3D culture, transforming the response from positive to negative. Therefore, collagen hydrolysate as a media supplement is not a viable long-term method to improve the collagen content of engineered cartilage tissue.

Dynamic deformational loading results in selective application of mechanical stimulation in a layered, tissue-engineered cartilage construct.

The application of dynamic physiologic loading to a bilayered chondrocyte-seeded agarose construct with a 2% (wt/vol) top layer and 3% (wt/vol) bottom layer was hypothesized to (1) improve overall construct properties and (2) result in a tissue that mimics the mechanical inhomogeneity of native cartilage. Dynamic loading over the 28 day culture period was found to significantly increase bulk mechanical and biochemical properties versus free-swelling culture. The initial depth-distribution of the compressive Young's modulus (EY) reflected the intrinsic properties of the gel in each layer and a similar trend to the native tissue, with a softer 2% gel layer and a much stiffer 3% gel layer. After 28 days in culture, free-swelling conditions maintained this general trend while loaded constructs possessed a reverse profile, with significant increases in EY observed only in the 2% gel. Histological analysis revealed preferential matrix formation in the 2% agarose layer, with matrix localized more pericellularly in the 3% agarose layer. Finite element modeling revealed that, prior to significant matrix elaboration, the 2% layer experiences increased mechanical stimuli (fluid flow and compressive strain) during loading that may enhance chondrocyte stimulation and nutrient transport in that layer, consistent with experimental observations. From these results, we conclude that due to the limitations in 3% agarose, the use of this type of bilayered construct to construct depth-dependent inhomogeneity similar to the native tissue is not likely to be successful under long-term culture conditions. Our study underscores the importance of other physical properties of the scaffold that may have a greater influence on interconnected tissue formation than intrinsic scaffold stiffness.