Uncompensated polychromatic analysis of mitochondrial membrane potential using JC-1 and multilaser excitation

The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-etraethylbenzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (δψm). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, δψm along with other biological parameters, such as apoptosis and the production of reactive oxygen species

Endoscopic third ventriculostomy in obstructive infantile hydrocephalus: Remarks about the so-called 'unsuccessful cases'

Background: The failure rate following endoscopic third ventriculostomy (ETV) in infants younger than 2 years of age has been reported to be higher compared with that of older children, and it is unclear whether ETV might be superior to shunt placement in this age group. Methods: Between 2003 and 2009, 23 patients younger than 6 months and without a previous history of shunting underwent ETV in our institution. A review of the literature was performed on the basis of publications presenting detailed data on age and etiology in every single patient. Results: In our own patients, total success rate was 39.1%. In the successful cases, median age was 140 days, whereas in the unsuccessful cases it was 47 days. The difference between the two groups was statistically significant (p = 0.01). The median ages of both successful and unsuccessful groups corresponded to data gained from an analysis of the literature (p = 0.04). At a median follow-up of 47 months, 2 out of 14 patients shunted after a failed ETV were revised for ventriculoperitoneal shunt malfunction. Conclusion: The impact of age on ETV failure in infants is clear and becomes crucial during the first 2 months of life, even when excluding etiological factors. Nevertheless, age cannot be considered the only parameter of the decision-making process, especially in these very young patients. Probably, the definition of 'unsuccessful ETV' should be reevaluated in light of decreased risk of shunt malfunction observed after a failed ETV. Copyright © 2011 S. Karger AG, Basel

Emerging role of Lon protease as a master regulator of mitochondrial functions

Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi

Interfering with ROS metabolism in cancer cells: the potential role of quercetin.

Abstract: A main feature of cancer cells, when compared to normal ones, is a persistent pro-oxidative state that leads to an intrinsic oxidative stress. Cancer cells have higher levels of reactive oxygen species (ROS) than normal cells, and ROS are, in turn, responsible for the maintenance of the cancer phenotype. Persistent ROS stress may induce adaptive stress responses, enabling cancer cells to survive with high levels of ROS and maintain cellular viability. However, excessive ROS levels render cancer cells highly susceptible to quercetin, one of the main dietary flavonoids. Quercetin depletes intracellular glutathione and increases intracellular ROS to a level that can cause cell death

Neural crest derived niche of human dental pulp stem cells promotes peripheral nerve regeneration and remyelination in animal model of critical sized sciatic nerve injury

ABSTRACT Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The use of stem cells, easily accessible, capable of rapid expansion in culture as well as fully integrate into the host tissue and capable to differentiate in myelinating cells of the peripheral nervous system, represent an attractive therapeutic approach for the treatment of nerve injuries. Farther, stem cells sources sharing the same embryological origin of Schwann cells, might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal sub-population of STRO-1+/c-Kit+/CD34+ hDPSCs (1, 2), most of which being positive for neural crest (P75NTR) and neural progenitor cells (nestin) markers, to differentiate into Schwann cells-like cells in vitro and to promote axonal regeneration in vivo. As a matter of fact, following culture in appropriate induction medium, STRO-1+/c-Kit+/CD34+ hDPSCs were able to commit towards Schwann cells express- ing P75NTR, GFAP and S100b. After transplantation in animal model of sciatic nerve defect, hDPSCs promoted axonal regeneration from proximal to distal stumps, providing guidance to newly formed myelinated nerve fibers, which led to functional recovery as measured by sustained gait improvement. Particularly, transplanted hDP- SCs engrafted into critical sized sciatic nerve defect, as revealed by the positive stain- ing against human nuclei, showed the expression of typical Schwann cells markers, S100b and GFAP. In conclusion this study demonstrates that STRO-1+/c-Kit+/CD34+ hDPSCs, associated to neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues

Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations

Human dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency

Increased Plasma Levels of Mitochondrial DNA and Normal Inflammasome Gene Expression in Monocytes Characterize Patients With Septic Shock Due to Multidrug Resistant Bacteria

Introduction: The activity and regulation of inflammasome is receiving increasing attention in septic shock. Moreover, there is a growing body of evidence suggesting that mitochondrial DNA (mtDNA) can play a role as biomarker of disease severity and even mortality both in adults and children in critically ill setting. However, no data are available on the amount of circulating mtDNA and inflammasome gene expression in multi-drug resistant (MDR) bacteria septic shock. For this reason, the aim of this study was to determine whether plasma mtDNA levels and inflammasome gene expression in monocytes could be related to severity in patients admitted to intensive care unit (ICU) with septic shock due to MDR pathogens. Materials and Methods: Peripheral blood mononuclear cells (PBMC) and plasma were isolated from up to 20 ml of venous blood by density gradient centrifugation in patients admitted to ICU with the diagnosis of septic shock due to MDR-bacteria. Then, CD14+ monocytes were sorted, and RNA and DNA were extracted. NLRP3, PYCARD, AIM2 and NAIP expression level was analyzed by RT-PCR. Plasma circulating mtDNA levels were quantified by digital droplet PCR. Basal and outcome characteristics of the patients were collected. Age-matched healthy subjects were chosen as controls. Results: Nineteen patients with septic shock and 20 healthy subjects were enrolled in the study. A small trend toward an increased expression of inflammasome genes was observed in septic shock patients, who also displayed a marked tendency to an increased expression of IL-18 and IL-1\u3b2 genes. Circulating mtDNA levels were significantly higher in septic shock patients if compared to healthy subjects, and patients who died in ICU were characterized by higher level of mtDNA if compared to those who were dismissed after 7 days. No correlations were found between mtDNA and inflammasome level and other clinical variables. Conclusion: Despite many limitations, our data suggest that in patients with septic shock caused by MDR pathogens the expression of main inflammasome genes was comparable to that of healthy patients without infection. Furthermore, our data evidence a possible role of mtDNA as a prognostic marker of severity in septic shock from MDR

Impaired Mitochondrial Morphology and Functionality in Lonp1wt/- Mice

LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations ofLONP1have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in whichLonp1was ablated. The homozygousLonp(-/-)mouse was not vital, while the heterozygousLonp1(wt/-)showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) fromLonp1(wt/-)mice showed a reduced expression ofLonp1andTfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of theLonp1gene leads to impairment of mitochondrial ultrastructure and functions in vivo

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and \u201cCD4 Easy Count Kit-Dry\u201d, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the "gold standard" for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the "CD4% Count Kit-Dry"