The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human faeces by application of next generation sequencing (NGS). However, ...the detected composition of the faecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for collection and storage of faecal samples (native and OMNIgene.GUT system) and bacterial DNA extraction (MP Biomedicals, QIAGEN and MO BIO kits), using two NGS platforms for 16S rRNA gene sequencing (Ilumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring Sutterella genus. MP provided superior DNA yield and quality, MO BIO depleted Gram positive organisms while using QIAGEN with OMNIgene.GUT resulted in greatest variability compared to other two kits. MiSeq and IT platforms in their supplier recommended setups provided comparable reproducibility of donor faecal microbiota. The differences included higher diversity observed with MiSeq and increased capacity of MiSeq to detect Akkermansia muciniphila, Odoribacteraceae, Erysipelotrichaceae and Ruminococcaceae (primarily Faecalibacterium prausnitzii). The results of our study could assist the investigators using NGS technologies to make informed decisions on appropriate tools for their experimental pipelines.
The knowledge on how gut microbes contribute to the inflammatory bowel disease (IBD) at the onset of disease is still scarce. We compared gut microbiota in newly diagnosed, treatment-naïve adult IBD ...(Crohn's disease (CD) and ulcerative colitis (UC)) to irritable bowel syndrome (IBS) patients and healthy group. Mucosal and fecal microbiota of 49 patients (13 UC, 10 CD, and 26 IBS) before treatment initiation, and fecal microbiota of 12 healthy subjects was characterized by 16S rRNA gene sequencing. Mucosa was sampled at six positions, from terminal ileum to rectum. We demonstrate that mucosal microbiota is spatially homogeneous, cannot be differentiated based on the local inflammation status and yet provides bacterial footprints superior to fecal in discriminating disease phenotypes. IBD groups showed decreased bacterial diversity in mucosa at all taxonomic levels compared to IBS. In CD and UC, Dialister was significantly increased, and expansion of Haemophilus and Propionibacterium characterized UC. Compared to healthy individuals, fecal microbiota of IBD and IBS patients had increased abundance of Proteobacteria, Enterobacteriaceae, in particular. Shift toward reduction of Adlercreutzia and butyrate-producing taxa was found in feces of IBD patients. Microbiota alterations detected in newly diagnosed treatment-naïve adult patients indicate that the microbiota changes are set and detectable at the disease onset and likely have a discerning role in IBD pathophysiology. Our results justify further investigation of the taxa discriminating between disease groups, such as H. parainfluenzae, R. gnavus, Turicibacteriaceae, Dialister, and Adlercreutzia as potential biomarkers of the disease.
The main goal of this study was the formation of bone tissue using dexamethasone (DEX)-loaded COCH₃-RADARADARADARADA-CONH₂ (RADA 16-I) scaffold that has the ability to release optimal DEX ...concentration under perfusion force. Bone-marrow samples were collected from three patients during a hip arthroplasty. Human mesenchymal stem cells (hMSCs) were isolated and propagated in vitro in order to be seeded on scaffolds made of DEX-loaded RADA 16-I hydrogel in a perfusion bioreactor. DEX concentrations were as follows: 4 × 10
, 4 × 10
and 4 × 10
M. After 21 days in a perfusion bioreactor, tissue was analyzed by scanning electron microscopy (SEM) and histology. Markers of osteogenic differentiation were quantified by real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. Minerals were quantified and detected by the von Kossa method. In addition, DEX release from the scaffold in a perfusion bioreactor was assessed. The osteoblast differentiation was confirmed by the expression analysis of osteoblast-related genes (alkaline phosphatase (ALP), collagen I (COL1A1) and osteocalcin (OC). The hematoxylin/eosin staining confirmed the presence of cells and connective tissue, while SEM revealed morphological characteristics of cells, extracellular matrix and minerals-three main components of mature bone tissue. Immunocytochemical detection of collagen I is in concordance with given results, supporting the conclusion that scaffold with DEX concentration of 4 × 10
M has the optimal engineered tissue morphology. The best-engineered bone tissue is produced on scaffold loaded with 4 × 10
M DEX with a perfusion rate of 0.1 mL/min for 21 days. Differentiation of hMSCs on DEX-loaded RADA 16-I scaffold under perfusion force has a high potential for application in regenerative orthopedics.
The healthy intestine represents a remarkable interface where sterile host tissues come in contact with gut microbiota, in a balanced state of homeostasis. The imbalance of gut homeostasis is ...associated with the onset of many severe pathological conditions, such as inflammatory bowel disease (IBD), a chronic gastrointestinal disorder increasing in incidence and severely influencing affected individuals. Despite the recent development of next generation sequencing and bioinformatics, the current scientific knowledge of specific triggers and diagnostic markers to improve interventional approaches in IBD is still scarce. In this review we present and discuss currently available and emerging therapeutic options in modulating composition and metabolic activity of gut microbiota in patients affected by IBD. Therapeutic approaches at the microbiota level, such as dietary interventions alone or with probiotics, prebiotics and synbiotics, administration of antibiotics, performing fecal microbiota transplantation (FMT) and the use of nematodes, all represent a promising opportunities towards establishing and maintaining of well-being as well as improving underlying IBD symptoms.
Inflammatory bowel diseases are a group of chronic inflammatory conditions that affect gastrointestinal tract due to inapt and continuous immune activation in response to a myriad of predisposing ...factors (most notably genetics, environmental impact and gut microbiota composition). It has been shown that vitamin D status can also play a role in the disease pathogenesis, as its deficiency is commonly observed in two major forms of inflammatory bowel diseases - Crohn's disease and ulcerative colitis. Mounting evidence supports the concept of intricate relationship between gut dysbiosis and vitamin D metabolism, while suboptimal levels of this vitamin have been linked to increased clinical disease relapse rates, inadequate response to drugs, as well as decreased quality of life in patients with Crohn's disease and ulcerative colitis. Consequently, the pertinent question is whether increased vitamin D supplementation and (on a population level) food fortification may bring significant benefit to the affected individuals. In this short review we discuss the synthesis, functions, status and food sources of vitamin D, appraise biotechnological facets of vitamin D status analysis and food fortification, and concentrate on novel developments in the field that describe its influence on intestinal microbiota and inflammatory bowel disease.
Stem cells in bone regeneration PANEK, MARINA; MARIJANOVIĆ, INGA; IVKOVIĆ, ALAN
Periodicum biologorum,
03/2015, Letnik:
117, Številka:
1
Journal Article
Odprti dostop
Bone defects, including normal fracture healing as well as healing problems represent a global health problem. The need for better treatment of bone defects is one of the central issues of tissue ...engineering and regenerative medicine. Regenerative orthopedics has several approaches – activation of endogenous stem cells, stem cell therapy and tissue engineering. Development of new treatments is mainly focused on the tissue engineering strategies that include stem cells, bioactive signals and appropriate scaffold support.
The aim of this review is to describe a variety of stem cells that have an
ability to become bone cells and therefore are of central importance for bone tissue engineering. Several cell types have been proposed as starting material - embryonic stem cells, induced pluripotent stem cells and adult stem cells. Due to ethical and safety issues, embryonic and induced pluripotent stem cells may be more suitable for studying human development and tissue formation under diverse experimental conditions, and represent an excellent base for understanding human diseases and development of innovative therapeutic solutions. Among adult stem cells, mesenchymal stem cells are the most suitable for bone tissue engineering. They can be isolated from variety of mesenchymal tissues and can differentiate into osteoblasts when given appropriate mechanical support and osteoinductive signal.
The near future of bone healing and regeneration is closely related to
advances in tissue engineering. The optimization of protocols of bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries.
Upalne bolesti crijeva predstavljaju skup kroničnih upalnih stanja s negativnim utjecajem na probavni sustav, a javljaju se uslijed neadekvatne i kontinuirane aktivacije imunosnog sustava kao odgovor ...na čitav niz predisponirajućih čimbenika (i to prvenstveno genetike, utjecaja okoline te sastava crijevne mikrobiote). Pokazalo se da status vitamina D također može imati određenu ulogu u razvoju bolesti, budući da se njegov manjak često viđa u Crohnovoj bolesti i ulceroznom kolitisu. U porastu je broj dokaza koji podupiru koncept uske povezanosti disbioze crijeva i metabolizma vitamina D; nadalje, suboptimalne razine ovog vitamina povezuju se s povećanom učestalošću kliničkih relapsa bolesti, neadekvatnim odgovorom na lijekove, kao i sa smanjenom kvalitetom života oboljelih od Chronove bolesti i ulceroznog kolitisa. Shodno tome, nameće se pitanje mogu li intervencije poput pojačanog nadomještanja vitamina D te (na razini populacije) obogaćivanje hrane njime dovesti do blagotvornih učinaka za oboljele osobe. U ovom kratkom preglednom radu donosimo presjek sinteze, funkcija, statusa i nutritivnih izvora vitamina D, analiziramo biotehnološke aspekte određivanja razine vitamina D i obogaćivanje hrane, ali se koncentriramo i na neka sasvim nova otkrića koja se bave utjecajem vitamina D na crijevnu mikrobiotu te na upalnu bolest crijeva.
Omega-3 (ω-3) and omega-6 (ω-6) are polyunsaturated fatty acids (PUFAs) that play critical role in human health and have to be provided by food. In the brain, PUFAs are also precursors of ...endocannabinoids. The aim of this chapter is to review the existing literature on how dietary PUFAs impact on the endocannabinoid system in the brain and what are the consequences for brain function and dysfunction. In this chapter, we will first describe how PUFAs enter the brain, what are their metabolism processes and roles in brain function. We will describe the pathways from PUFAs to endocannabinoid production. Then, we will review the literature on how dietary ω-6/ω-3 ratio impacts the endocannabinoid system, in terms of endocannabinoid levels, proteins and endocannabinoid-dependent synaptic plasticity. In the next part, we will describe what we know about the interactions between PUFAs and endocannabinoids in neurological and neuropsychiatric disorders. Finally, we will conclude on the possible implications of the interactions between dietary PUFAs and endocannabinoids in the normal and pathological brain. In particular, we will discuss how dietary PUFAs, as homeostatic regulators of endocannabinoids, can constitute interesting therapeutic strategies for the prevention and/or treatment of neurological disorders with endocannabinoids impairment.
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