Publications by authors named "Luca Giordano"

11 Publications

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Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active.

Gene Ther 2021 Mar 4. Epub 2021 Mar 4.

Faculty of Medicine and Health Technology, FI-33014 Tampere University Hospital, Tampere University of Applied Sciences (TUNI), Tampere, Finland.

Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.
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http://dx.doi.org/10.1038/s41434-021-00235-zDOI Listing
March 2021

An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma.

J Biol Chem 2020 11 8;295(46):15677-15691. Epub 2020 Sep 8.

Center for Metabolism and Mitochondrial Medicine, Division of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. Electronic address:

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.
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http://dx.doi.org/10.1074/jbc.RA120.015237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667980PMC
November 2020

Robotic-Assisted and Laparoscopic Sigmoid Resection.

JSLS 2020 Jul-Sep;24(3)

Department of Colon and Rectal Surgery, St. Joseph Hospital.

Background And Objectives: Published comparisons of minimally invasive approaches to colon surgery are limited. The objective of the current study is to compare the effectiveness of robotic-assisted and laparoscopic sigmoid resection.

Methods: A multicenter retrospective comparative analysis of perioperative outcomes from consecutive robotic-assisted and laparoscopic sigmoid resections performed between 2010 and 2015 by six general and colorectal surgeons, who are experienced in both robotic-assisted and laparoscopic surgical techniques and who had >50 annual case volumes for each approach. Baseline characteristics and surgical risk factors between the two groups were balanced using a propensity score methodology with inverse probability of treatment weighting. Mean standardized differences were reported, and in all instances, a -value < 0.05 was considered statistically significant.

Results: Three hundred thirty-six cases (robotic-assisted, n = 211; laparoscopic, n = 125) met eligibility criteria and were included in the study. Following weighting, patient demographics and baseline characteristics were comparable between the robotic-assisted (n = 344) and laparoscopic (n = 349) groups. The laparoscopic group was associated with shorter operating room and surgical times. The robotic-assisted group had lower estimated blood loss and shorter time to first flatus compared to the laparoscopic group. Rates of complications post discharge to 30 d tended to be lower for the RA group: 5.1% vs 8.6% [ = 0.0657]. The RA group also had lower rates of readmissions and reoperations: 4% vs 8% [ = 0.029] and 0.5% vs 5.1% [ = 0.0003], respectively.

Conclusions: Robotic-assisted sigmoid colon resection is clinically effective and provides a minimally invasive alternative to the laparoscopic approach with improved intraoperative and postoperative outcomes for colorectal patients.
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http://dx.doi.org/10.4293/JSLS.2020.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434398PMC
January 2021

Exploitation of Prunus mahaleb fruit by fermentation with selected strains of Lactobacillus plantarum and Saccharomyces cerevisiae.

Food Microbiol 2019 Dec 8;84:103262. Epub 2019 Jul 8.

CNR, Institute of Sciences of Food Production (ISPA), via Prov.le Lecce-Monteroni, 73100, Lecce, Italy. Electronic address:

The organoleptic attributes of Prunus mahaleb, a fruit representing a new source of bioactive compounds, are so pronounced that it can be consider non-edible. This study was designed to evaluate the acceptance of P. mahaleb fruits after fermentation with different Saccharomyces cerevisiae and Lactobacillus plantarum protechnological strains. Four different bacterial and one yeast strains, as single or mixed starter formulation, were used to inoculate an aqueous suspension of P. mahaleb fruits. The fermented fruits and fermentation broths were subjected to physico-chemical characterization and the organoleptic properties of both samples were also assessed by a hedonic panel. The obtained results indicated that all the employed strains were able to grow and to ferment the matrix. However, the mixed starter FG69 + Li180-7 (L. plantarum/S. cerevisiae) had the best impact on sensory characteristics of P. mahaleb fruit and fermented medium. The adopted protocol allowed us to attain edible fruits and a new fermented non-dairy drink with valuable probiotic health-promoting properties. In our knowledge, this is the first study concerning the exploitation of P. mahaleb fruits. This investigation confirmed the potential of yeasts and lactic acid bacteria co-inoculation in the design of starter tailored for this kind of food applications.
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http://dx.doi.org/10.1016/j.fm.2019.103262DOI Listing
December 2019

Intracorporeal versus extracorporeal anastomosis for minimally invasive right colectomy: A multi-center propensity score-matched comparison of outcomes.

PLoS One 2018 24;13(10):e0206277. Epub 2018 Oct 24.

Department of Surgery, Division of Colon and Rectal Surgery, University of California Irvine, Irvine, California, United States of America.

Background: The primary objective of this study was to retrospectively compare short-term outcomes of intracorporeal versus extracorporeal anastomosis for minimally invasive laparoscopic and robotic-assisted right colectomies for benign and malignant disease. Recent studies suggest potential short-term outcomes advantages for the intracorporeal anastomosis technique.

Methods: This is a multicenter retrospective propensity score-matched comparison of intracorporeal and extracorporeal anastomosis techniques for laparoscopic and robotic-assisted right colectomy between January 11, 2010, and July 21, 2016.

Results: After propensity score-matching, there were a total of 1029 minimal invasive surgery cases for analysis-379 right colectomies (335 robotic-assisted and 44 laparoscopic) done with an intracorporeal anastomosis and 650 right colectomies (253 robotic-assisted and 397 laparoscopic) done with an extracorporeal anastomosis. There were no significant differences in any preoperative patient characteristics between groups. The minimally invasive intracorporeal anastomosis group had significantly longer operative times (p<0.0001), lower conversion to open rate (p = 0.01), shorter hospital length of stay (p = 0.02) and lower complication rate from after discharge to 30-days (p = 0.04) than the extracorporeal anastomosis group.

Conclusions: This comparison shows several clinical outcomes advantages for the intracorporeal anastomosis technique in minimally invasive right colectomy. These data may guide future refinements in minimally invasive training techniques and help surgeons choose among different minimally invasive options.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206277PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200279PMC
April 2019

Alternative Oxidase Attenuates Cigarette Smoke-induced Lung Dysfunction and Tissue Damage.

Am J Respir Cell Mol Biol 2019 05;60(5):515-522

1 Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.

Cigarette smoke (CS) exposure is the predominant risk factor for the development of chronic obstructive pulmonary disease (COPD) and the third leading cause of death worldwide. We aimed to elucidate whether mitochondrial respiratory inhibition and oxidative stress are triggers in its etiology. In different models of CS exposure, we investigated the effect on lung remodeling and cell signaling of restoring mitochondrial respiratory electron flow using alternative oxidase (AOX), which bypasses the cytochrome segment of the respiratory chain. AOX attenuated CS-induced lung tissue destruction and loss of function in mice exposed chronically to CS for 9 months. It preserved the cell viability of isolated mouse embryonic fibroblasts treated with CS condensate, limited the induction of apoptosis, and decreased the production of reactive oxygen species (ROS). In contrast, the early-phase inflammatory response induced by acute CS exposure of mouse lung, i.e., infiltration by macrophages and neutrophils and adverse signaling, was unaffected. The use of AOX allowed us to obtain novel pathomechanistic insights into CS-induced cell damage, mitochondrial ROS production, and lung remodeling. Our findings implicate mitochondrial respiratory inhibition as a key pathogenic mechanism of CS toxicity in the lung. We propose AOX as a novel tool to study CS-related lung remodeling and potentially to counteract CS-induced ROS production and cell damage.
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http://dx.doi.org/10.1165/rcmb.2018-0261OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503618PMC
May 2019

Expression of the Alternative Oxidase Influences Jun N-Terminal Kinase Signaling and Cell Migration.

Mol Cell Biol 2018 12 28;38(24). Epub 2018 Nov 28.

Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland

Downregulation of Jun N-terminal kinase (JNK) signaling inhibits cell migration in diverse model systems. In pupal development, attenuated JNK signaling in the thoracic dorsal epithelium leads to defective midline closure, resulting in cleft thorax. Here we report that concomitant expression of the alternative oxidase (AOX) was able to compensate for JNK pathway downregulation, substantially correcting the cleft thorax phenotype. AOX expression also promoted wound-healing behavior and single-cell migration in immortalized mouse embryonic fibroblasts (iMEFs), counteracting the effect of JNK pathway inhibition. However, AOX was not able to rescue developmental phenotypes resulting from knockdown of the AP-1 transcription factor, the canonical target of JNK, nor its targets and had no effect on AP-1-dependent transcription. The migration of AOX-expressing iMEFs in the wound-healing assay was differentially stimulated by antimycin A, which redirects respiratory electron flow through AOX, altering the balance between mitochondrial ATP and heat production. Since other treatments affecting mitochondrial ATP did not stimulate wound healing, we propose increased mitochondrial heat production as the most likely primary mechanism of action of AOX in promoting cell migration in these various contexts.
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http://dx.doi.org/10.1128/MCB.00110-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275184PMC
December 2018

Characterization of Drosophila ATPsynC mutants as a new model of mitochondrial ATP synthase disorders.

PLoS One 2018 10;13(8):e0201811. Epub 2018 Aug 10.

Dipartimento di Biologia, Università degli Studi di Bari "Aldo Moro", Bari, Italy.

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201811PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086398PMC
February 2019

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber's hereditary optic neuropathy.

Brain 2014 Feb 24;137(Pt 2):335-53. Epub 2013 Dec 24.

1 Department of Radiology, Oncology and Pathology, Sapienza, University of Rome, Rome, Italy.

Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.
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http://dx.doi.org/10.1093/brain/awt343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3914475PMC
February 2014

Generation of diversity by somatic mutation in the Camelus dromedarius T-cell receptor gamma variable domains.

Eur J Immunol 2012 Dec 25;42(12):3416-28. Epub 2012 Oct 25.

Department of Biology, University of Bari, Bari, Italy.

In jawed vertebrates the V-(D)-J rearrangement is the main mechanism generating limitless variations of antigen-specific receptors, immunoglobulins (IGs), and T-cell receptors (TCRs) from few genes. Once the initial diversity is established in primary lymphoid organs, further diversification occurs in IGs by somatic hypermutation, a mechanism from which rearranged TCR genes were thought to be excluded. Here, we report the locus organization and expression of the T-cell receptor gamma (TCRG) genes in the Arabian camel (Camelus dromedarius). Expression data provide evidence that dromedary utilizes only two TCRG V-J genomic arrangements and, as expected, CDR3 contributes the major variability in the V domain. The data also suggest that diversity might be generated by mutation in the productively rearranged TCRGV genes. As for IG genes, the mutational target is biased toward G and C bases and (A/G/T)G(C/T)(A/T) motif (or DGYW). The replacement and synonymous substitutions (R/S) ratios in TCRGV regions are higher for CDR than for framework region, thus suggesting selection toward amino acid changes in CDR. Using the counterpart human TCR γδ receptor as a template, structural models computed adopting a comparative procedure show that nonconservative mutations contribute to diversity in CDR2 and at the γδ V domain interface.
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http://dx.doi.org/10.1002/eji.201142176DOI Listing
December 2012

Portal vein thrombosis following splenectomy: identification of risk factors.

Am Surg 2003 Nov;69(11):951-6

Center for Minimally Invasive Surgery, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA.

Portal vein thrombosis (PVT) following splenectomy is a potentially life-threatening complication, and the true incidence of PVT in splenectomized patients is unknown. The objective of this study was to determine the incidence of symptomatic PVT after splenectomy. The hospital database was searched to identify cases of PVT associated with splenectomy from January 1990 to May 2002. Six hundred eighty-eight patients underwent splenectomy during this period, 321 of them for hematologic diseases. Eleven of the 688 patients had PVT associated with splenectomy, and the charts of these patients were reviewed. Six patients developed PVT after splenectomy. Five had hematologic diseases. Symptoms were abdominal pain (6), ileus (5), fever (3), or diarrhea (2). Diagnosis was confirmed by computed tomography (CT) (4), duplex ultrasonography (1), and magnetic resonance imaging (1). The indications for splenectomy included hemolytic anemia (3), thalassemia (1), and myelofibrosis (1). One patient had an incidental splenectomy during gastrectomy. There were four laparoscopic and two open splenectomies. The median interval between splenectomy and diagnosis of PVT was 40 days (range, 13-741). One patient died of pulmonary embolism. Five of six patients with postsplenectomy PVT had splenomegaly and hemolysis. We conclude that the risk of PVT is higher in patients with hematologic conditions associated with splenomegaly and hemolysis.
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November 2003
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