Regional-specific meniscal extracellular matrix hydrogels and their effects on cell-matrix interactions of fibrochondrocytes

Decellularized meniscal extracellular matrix (ECM) material holds great potential for meniscus repair and regeneration. Particularly, injectable ECM hydrogel is highly desirable for the minimally invasive treatment of irregularly shaped defects. Although regional-specific variations of the meniscus are well documented, no ECM hydrogel has been reported to simulate zonally specific microenvironments of the native meniscus. To fill the gap, different (outer, middle, and inner) zones of porcine menisci were separately decellularized. Then the regionally decellularized meniscal ECMs were solubilized by pepsin digestion, neutralized, and then form injectable hydrogels. The hydrogels were characterized in gelation behaviors and mechanical properties and seeded with bovine fibrochondrocytes to evaluate the regionally biochemical effects on the cell-matrix interactions. Our results showed that the decellularized inner meniscal ECM (IM) contained the greatest glycosaminoglycan (GAG) content and the least collagen content compared with the decellularized outer meniscal ECM (OM) and middle meniscal ECM (MM). The IM hydrogel showed lower compressive strength than the OM hydrogel. When encapsulated with fibrochondrocytes, the IM hydrogel accumulated more GAG, contracted to a greater extent and reached higher compressive strength than that of the OM hydrogel at 28 days. Our findings demonstrate that the regionally specific meniscal ECMs present biochemical variation and show various effects on the cell behaviors, thus providing information on how meniscal ECM hydrogels may be utilized to reconstruct the microenvironments of the native meniscus.

First Report of Sarcocystis Masoni in a Captive Alpaca (Vicugna Pacos) From China

Background: Sarcocystosis is a parasitic disease caused by intracellular protozoan parasite of the genus Sarcocystis. Tissue samples of alpacas (n = 4) from Henan province (China) were screened for Sarcocystis spp. infection by histological examination, pepsin digestion, and molecular assays.Results:Sarcocystis spp. was detected in heart, liver, spleen, lung, and kidney of an alpaca by molecular assays. Many sarcocysts with inflammation responses were observed in this alpaca myocardium, and they showed a high similarity to Sarcocystis masoni by sequence analysis.Conclusion: This study is the first to demonstrate Sarcocystis spp. infection in alpaca from China. The higher parasite load in the alpaca myocardium indicated that it had contact with an environment contaminated with sporocysts, and that the alpaca was susceptible to Sarcocystis spp.

A survey on the Frequency of Sarcocystis in Bandar Abbas, Iran in 2019 - 2020

Background: The zoonotic Sarcocystis parasite has an obligatory two-host life cycle that mainly involves herbivorous animals as intermediate hosts and carnivorous animals as definitive hosts. Objectives: Lack of reliable study and published data bout frequency of Sarcocystis in livestock of Hormozgan Province and consumed meat led us to investigate in abattoirs and slaughterhouses of Bandar Abbas, Iran, in 2019 - 2020. Methods: In this descriptive cross-sectional study, 400 meat samples of three types of animals (cow, sheep, goat) belonging to Hormozgan, Fars, and Kerman provinces were studied from September 2019 to January 2020 using naked eye examination for detection of macroscopic Sarcocystis cysts and pepsin digestion method accompanied by squeezing methods performed to examine striated muscles for microscopic cyst types. Isolated tissues of the esophagus, heart, and diaphragm of 400 slaughtered animals were examined for Sarcocystis. Results: The carcasses of all the animals were investigated to detect Sarcocystis macroscopic cysts, all of which were negative. However, microscopic examination of isolated tissues by pepsin digestion showed a total frequency of 92.25% in these animals. Analysis revealed that cows bred in Hormozgan and goats imported from Kerman are significantly infected and play an important role in the distribution of disease (P < 0.05). Conclusions: These results and obtained data indicated that a large volume of imported meats to Hormozgan is contaminated with this parasite, and more control should be applied over slaughtered livestock. Also, the people of the community who consume these meats should be given proper and complete training.

Can keratin scaffolds be used for creating three-dimensional cell cultures?

Three-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing.

“Dual protease type XIII/pepsin digestion offers superior resolution and overlap for the analysis of histone tails by HX-MS”

The N-terminal regions of histone proteins (tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications (PTMs) present on these regions contribute to transcriptional regulation. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed to analyze histones due to the poor N-terminal coverage obtained using pepsin. Here, we test the applicability of a dual protease type XIII/pepsin digestion column to this class of proteins. We optimize online digestion conditions using the H4 monomer, and extend the method to the analysis of histones in monomeric states and nucleosome core particles (NCPs). We show that the dual protease column generates many short and overlapping N-terminal peptides. We evaluate our method by performing hydrogen exchange experiments of NCPs for different time points and present full coverage of the tails at excellent resolution. We further employ electron transfer dissociation (ETD) and showcase an unprecedented degree of overlap across multiple peptides that is several fold higher than previously reported methods. The method we report here may be readily applied to the HX-MS investigation of histone dynamics and to the footprints of histone binding proteins on nucleosomes.