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Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.

The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast Saccharomyces cerevisiae an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules. The review also provides a summary of the main contributions to the understanding of mitochondrial diseases emerged from the study of this simple eukaryotic organism.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a ‘drug repurposing’ approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype. Six of them, able to reduce the mitochondrial DNA instability in yeast, have been then tested in Opa1 deleted mouse embryonic fibroblasts expressing the human OPA1 isoform 1 bearing the R445H and D603H mutations. Some of these molecules were able to ameliorate the energetic functions and/or the mitochondrial network morphology, depending on the type of OPA1 mutation. The final validation has been performed in patients’ fibroblasts, allowing to select the most effective molecules. Our current results are instrumental to rapidly translating the findings of this drug repurposing approach into clinical trial for DOA and other neurodegenerations caused by OPA1 mutations.

Thiosemicarbazones (TSC) and their metal complexes display diverse biological activities and are active against multiple pathological conditions ranging from microbial infections to abnormal cell proliferation. Ribonucleotide reductase (RNR) is considered one of the main targets of TSCs, yet, the existence of additional targets, differently responsible for the multifaceted activities of TSCs and their metal complexes has been proposed. To set the basis for a more comprehensive delineation of their mode of action, we chemogenomically profiled the cellular effects of bis(citronellalthiosemicarbazonato)nickel(II) [Ni(S-tcitr)2] using the unicellular eukaryote Saccharomyces cerevisiae as a model organism. Two complementary genomic phenotyping screens led to the identification of 269 sensitive and 56 tolerant deletion mutant strains and of 14 genes that when overexpressed make yeast cells resistant to an otherwise lethal concentration of Ni(S-tcitr)2. Chromatin remodeling, cytoskeleton organization, mitochondrial function and iron metabolism were identified as lead cellular processes responsible for Ni(S-tcitr)2 toxicity. The latter process, and particularly glutaredoxin-mediated iron loading of RNR, was found to be affected by Ni(S-tcitr)2. Given the multiple pathways regulated by glutaredoxins, targeting of these proteins by Ni(S-tcitr)2 can negatively affect various core cellular processes that may critically contribute to Ni(S-tcitr)2 cytotoxicity.

The mitochondrial ADP/ATP carrier is a nuclear encoded protein, which catalyzes the exchange of ATP generated in mitochondria with ADP produced in the cytosol. In humans, mutations in the major ADP/ATP carrier gene, ANT1, are involved in several degenerative mitochondrial pathologies, leading to instability of mitochondrial DNA. Recessive mutations have been associated with mitochondrial myopathy and cardiomyopathy whereas dominant mutations have been associated with autosomal dominant Progressive External Ophtalmoplegia (adPEO). Recently, two de novo dominant mutations, R80H and R235G, leading to extremely severe symptoms, have been identified. In order to evaluate if the dominance is due to haploinsufficiency or to a gain of function, the two mutations have been introduced in the equivalent positions of the AAC2 gene, the yeast orthologue of human ANT1, and their dominant effect has been studied in heteroallelic strains, containing both one copy of wild type AAC2 and one copy of mutant aac2 allele. Through phenotypic characterization of these yeast models we showed that the OXPHOS phenotypes in the heteroallelic strains were more affected than in the hemiallelic strain indicating that the dominant trait of the two mutations is due to gain of function.

Defects in nuclear-encoded proteins of the mitochondrial translation machinery cause early-onset and tissue-specific deficiency of one or more OXPHOS complexes. Here, we report a 7-year-old Italian boy with childhood-onset rapidly progressive encepha- lomyopathy and stroke-like episodes. Multiple OXPHOS defects and decreased mtDNA copy number (40%) were detected in muscle homogenate. Clinical features combined with low level of plasma citrulline were highly suggestive of mitochondrial en- cephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was ex- cluded. Targeted exome sequencing of genes encoding the mitochondrial proteome identified a damaging mutation, c.567 G > A, affecting a highly conserved amino acid residue (p.Gly189Arg) of the MRM2 protein. MRM2 has never before been linked to a hu- man disease and encodes an enzyme responsible for 2’-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA. We generated a knockout yeast model for the orthologous gene that showed a defect in respiration and the reduction of the 2’-O-methyl modification at the equivalent position (U2791) in the yeast mitochondrial 21S rRNA. Complementation with the mrm2 allele carrying the equivalent yeast mutation failed to rescue the respiratory phenotype, which was instead com- pletely rescued by expressing the wild-type allele. Our findings establish that defective MRM2 causes a MELAS-like phenotype, and suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation.

Background: Mutations in human gene encoding the mitochondrial DNA polymerase γ (HsPolγ) are associated with a broad range of mitochondrial diseases. Here we studied the impact on DNA replication by disease variants clustered around residue HsPolγ-K1191, a residue that in several family-A DNA polymerases interacts with the 3′ end of the primer. Methods Specifically, we examined the effect of HsPolγ carrying pathogenic variants in residues D1184, I1185, C1188, K1191, D1196, and a stop codon at residue T1199, using as a model the yeast mitochondrial DNA polymerase protein, Mip1p. Results The introduction of pathogenic variants C1188R (yV945R), and of a stop codon at residue T1199 (yT956X) abolished both polymerization and exonucleolysis in vitro. HsPolγ substitutions in residues D1184 (yD941), I1185 (yI942), K1191 (yK948) and D1196 (yD953) shifted the balance between polymerization and exonucleolysis in favor of exonucleolysis. HsPolγ pathogenic variants at residue K1191 (yK948) and D1184 (yD941) were capable of nucleotide incorporation albeit with reduced processivity. Structural analysis of mitochondrial DNAPs showed that residue HsPolγ-N864 is placed in an optimal distance to interact with the 3′ end of the primer and the phosphate backbone previous to the 3′ end. Amino acid changes in residue HsPolγ-N864 to Ala, Ser or Asp result in enzymes that did not decrease their polymerization activity on short templates but exhibited a substantial decrease for processive DNA synthesis. Conclusion Our data suggest that in mitochondrial DNA polymerases multiple amino acids are involved in the primer-stand stabilization.

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.

International audience; The cysteine-rich PLAC8 domain of unknown function occurs in proteins found in most Eukaryotes. PLAC8-proteins play important yet diverse roles in different organisms, such as control of cell proliferation in animals and plants or heavy metal resistance in plants and fungi. Mammalian Onzin can be either pro-proliferative or pro-apoptotic, depending on the cell type, whereas fungal FCR1 confers cadmium tolerance. Despite their different role in different organisms, we hypothesized common ancestral functions linked to the PLAC8 domain. To address this hypothesis, and to investigate the molecular function of the PLAC8 domain, murine Onzin and fungal FCR1 were expressed in the PLAC8-free yeast Saccharomyces cerevisiae. The two PLAC8-proteins localized in the nucleus and induced almost identical phenotypes and transcriptional changes when exposed to cadmium stress. Like FCR1, Onzin also reduced DNA damage and increased cadmium tolerance by a DUN1-dependent pathway. Both proteins activated transcription of ancient mitochondrial pathways such as leucine and Fe-S cluster biosynthesis, known to regulate cell proliferation and DNA repair in yeast. These results strongly suggest a common ancestral function of PLAC8 proteins and open new perspectives to understand the role of the PLAC8 domain in the cellular biology of Eukaryotes. The PLAC8 domain was described for the first time in the protein Onzin, the product of the human Placenta-Specific Gene 8 1. The same domain was later identified in many Eukaryotes, but its biological role remains elusive because PLAC8 proteins seem to play diverse roles in different organisms and cell types. The mammalian Onzin has been reported as a repressed target of the c-Myc oncoprotein 2 , with pro-proliferative anti-apoptotic effects in many cell types and a role in leukemia 3 , hepatic, pancreatic 4,5 and colon cancer progression 6,7 , but also in adipocyte growth 8. The same protein has pro-apoptotic activity in other cell types 9 , indicating that the final effect of Onzin is highly dependent on cell type. In plants, PLAC8 genes are also involved in cell proliferation because mutants are altered in organs size. The tomato Fruit Weight 2.2 (FW2.2) negatively influences fruit size and is downregulated in domesticated species 10. Similar function has been reported for FW2.2-like genes in other plant species, both dicots 11-14 and monocots 15,16. Another cellular role of PLAC8 proteins in plants and fungi is to increase resistance to heavy metals. First described in Arabidopsis thaliana, the Plant Cadmium Resistance (PCR) protein family includes proteins that confer resistance to cadmium or zinc. AtPCR1 was suggested to be-or to be part of-a cadmium transporter because of its localization on the plasma membrane in both A. thaliana and S. cerevisiae 17 , while AtPCR2 was associated to zinc transport 18. Although PLAC8 domain containing genes have been annotated in many fungal

Protein domains are structurally and functionally distinct units responsible for particular protein functions or interactions. Although protein domains contribute to the overall protein function(s) and can be used for protein classification, about 20% of protein domains are currently annotated as “domains of an unknown function” (DUFs). DUF 614, a cysteine-rich domain better known as PLAC8 (Placenta-Specific Gene 8), occurs in proteins found in the majority of Eukaryotes. PLAC8-containing proteins play important yet diverse roles in different organisms, such as control of cell proliferation in animals and plants or heavy metal resistance in plants and fungi. For example, Onzin from Mus musculus is a key regulator of cell proliferation, whereas FCR1 from the ascomycete Oidiodendron maius confers cadmium resistance. Onzin and FCR1 are small, single-domain PLAC8 proteins and we hypothesized that, despite their apparently different role, a common molecular function of these proteins may be linked to the PLAC8 domain. To address this hypothesis, we compared these two PLAC8-containing proteins by heterologous expression in the PLAC8-free yeast Saccharomyces cerevisiae. When expressed in yeast, both Onzin and FCR1 improved cadmium resistance, reduced cadmium-induced DNA mutagenesis, localized in the nucleus and induced similar transcriptional changes. Our results support the hypothesis of a common ancestral function of the PLAC8 domain that may link some mitochondrial biosynthetic pathways (i.e. leucine biosynthesis and Fe-S cluster biogenesis) with the control of DNA damage, thus opening new perspectives to understand the role of this protein domain in the cellular biology of Eukaryotes.Author SummaryProtein domains are the functional units of proteins and typically have distinct structure and function. However, many widely distributed protein domains are currently annotated as “domains of unknown function” (DUFs). We have focused on DUF 614, a protein domain found in many Eukaryotes and better known as PLAC8 (Placenta-Specific Gene 8). The functional role of DUF 614 is unclear because PLAC8 proteins seem to play important yet different roles in taxonomically distant organisms such as animals, plants and fungi. We used S. cerevisiae to test whether these apparently different functions, namely in cell proliferation and metal tolerance, respectively reported for the murine Onzin and the fungal FCR1, are mediated by the same molecular mechanisms. Our data demonstrate that the two PLAC8 proteins induced the same growth phenotype and transcriptional changes in S. cerevisiae. In particular, they both induced the biosynthesis of the amino acid leucine and of the iron-sulfur cluster, one of the most ancient protein cofactors. These similarities support the hypothesis of an ancestral function of the DUF 164 domain, whereas the transcriptomic data open new perspectives to understand the role of PLAC8-proteins in Eukaryotes.

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1−/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions.

With a steadily increasing world population, a more efficient system of food production is of paramount importance. One of the major causes of food spoilage is the presence of fungal pathogens and the production and accumulation of mycotoxins. In the present work we report a study on the activity of a series of functionalized thiosemicarbazones (namely cuminaldehyde, trans -cinnamaldehyde, quinoline-2-carboxyaldehyde, 5-fluoroisatin thiosemicarbazone and 5-fluoroisatin N 4 -methylthiosemicarbazone), as antifungal and anti-mycotoxin agents, against the two major genera of cereal mycotoxigenic fungi, i.e. Fusarium and Aspergillus . These thiosemicarbazones display different patterns of efficacy on fungal growth and on mycotoxin accumulation depending on the fungal species. Some of the molecules display a greater effect on mycotoxin synthesis than on fungal growth.

Several pathological mutations have been identified in human POLG gene, encoding for the catalytic subunit of Pol γ, the solely mitochondrial replicase in animals and fungi. However, little is known regarding non-pathological polymorphisms found in this gene. Here we studied, in the yeast model Saccharomyces cerevisiae, eight human polymorphisms. We found that most of them are not neutral but enhanced both mtDNA extended mutability and the accumulation of mtDNA point mutations, either alone or in combination with a pathological mutation. In addition, we found that the presence of some SNPs increased the stavudine and/or zalcitabine-induced mtDNA mutability and instability., Highlights • We studied the effects of 8 human polymorphisms in Pol γ in the model system yeast. • Most polymorphisms increase mtDNA extended and point mutability. • Treatment with NRTIs determines mtDNA instability in wt and mutant strains. • Some polymorphisms make Mip1 more sensitive to NRTIs-induced mtDNA toxicity.

The mitochondrial ADP/ATP carrier is a nuclear encoded protein, which catalyzes the exchange of ATP generated in mitochondria with ADP produced in the cytosol. In humans, mutations in the major ADP/ATP carrier gene, ANT1, are involved in several degenerative mitochondrial pathologies, leading to instability of mitochondrial DNA. Recessive mutations have been associated with mitochondrial myopathy and cardiomyopathy whereas dominant mutations have been associated with autosomal dominant Progressive External Ophtalmoplegia (adPEO). Recently, two de novo dominant mutations, R80H and R235G, leading to extremely severe symptoms, have been identified. In order to evaluate if the dominance is due to haploinsufficiency or to a gain of function, the two mutations have been introduced in the equivalent positions of the AAC2 gene, the yeast orthologue of human ANT1, and their dominant effect has been studied in heteroallelic strains, containing both one copy of wild type AAC2 and one copy of mutant aac2 allele. Through phenotypic characterization of these yeast models we showed that the OXPHOS phenotypes in the heteroallelic strains were more affected than in the hemiallelic strain indicating that the dominant trait of the two mutations is due to gain of function.

The human POLG gene encodes the catalytic subunit of mitochondrial DNA polymerase gamma (pol gamma). Mutations in pol gamma are associated with a spectrum of disease phenotypes including autosomal dominant and recessive forms of progressive external ophthalmoplegia, spino-cerebellar ataxia and epilepsy, and Alpers-Huttenlocher hepatocerebral poliodystrophy. Multiple deletions, or depletion of mtDNA in affected tissues, are the molecular hallmarks of pol gamma mutations. To shed light on the pathogenic mechanisms leading to these phenotypes, we have introduced in MIP1, the yeast homologue of POLG, two mutations equivalent to the human Y955C and G268A mutations, which are associated with dominant and recessive PEO, respectively. Both mutations induced the generation of petite colonies, carrying either rearranged (rho(-)) or no (rho(0)) mtDNA. Mutations in genes that control the mitochondrial supply of deoxynucleotides (dNTP) affect the mtDNA integrity in both humans and yeast. To test whether the manipulation of the dNTP pool can modify the effects of pol gamma mutations in yeast, we have overexpressed a dNTP checkpoint enzyme, ribonucleotide reductase, RNR1, or deleted its inhibitor, SML1. In both mutant strains, the petite mutability was dramatically reduced. The same result was obtained by exposing the mutant strains to dihydrolipoic acid, an anti-oxidant agent. Therefore, an increase of the mitochondrial dNTP pool and/or a decrease of reactive oxygen species can prevent the mtDNA damage induced by pol gamma mutations in yeast and, possibly, in humans.

Aspergillus flavus is an opportunistic mold that represents a serious threat for human and animal health due to its ability to synthesize and release, on food and feed commodities, different toxic secondary metabolites. Among them, aflatoxin B1 is one of the most dangerous since it is provided with a strong cancerogenic and mutagenic activity. Controlling fungal contamination on the different crops that may host A. flavus is considered a priority by sanitary authorities of an increasing number of countries due also to the fact that, owing to global temperature increase, the geographic areas that are expected to be prone to experience sudden A. flavus outbreaks are widening. Among the different pre- and post-harvest strategies that may be put forward in order to prevent fungal and/or mycotoxin contamination, fungicides are still considered a prominent weapon. We have here analyzed different structural modifications of a natural-derived compound (cuminaldehyde thiosemicarbazone) for their fungistatic and anti-aflatoxigenic activity. In particular, we have focused our attention on one of the compound that presented a prominent anti-aflatoxin specificity, and performed a set of physiological and molecular analyses, taking also advantage of yeast (Saccharomyces cerevisiae) cell as an experimental model.

Addressing the problem of proctologic sequelae after Scopinaroʼs classical BPD, we elongated the common limb from 50 to 200 cm at the expense of the alimentary limb and simultaneously, with the aim of avoiding weight regain, reduced the gastric pouch from 500 to 40 ml. After increased experience with the new procedure, we observed a favourable tendency towards further weight loss. Thus, we subsequently extended the indication to the procedure to patients with unsatisfactory weight loss after Scopinaroʼs classical BPD (SBPD). We retrospectively reviewed our clinical experience with the new procedure. From March 2008 to December 2014, 38 patients were submitted to the revisional procedure. The indication to surgical revision was proctologic in 26 patients and unsatisfactory weight loss in 12. After the revisional procedure, a significant reduction in bowel movements per day was observed, together with a significant reduction in body weight (from preoperative 87.1 ± 21 to 69.2 ± 13.5 kg at post-operative year 1 and 68.1 ± 11.9 kg at year 5; p

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.

Biliopancreatic diversion (BPD) is a bariatric technique burdened, in some instances, by clinical evidence of malabsorption and malnutrition, and by intractable diarrhea. The objective of this study was to assess metabolic and nutritional effects on patients undergoing BPD and BPD plus revisional surgery because of side effects. Thirty-five consecutive BPD patients underwent revisional surgery (elongation of the common limb from 50 to 200 cm and reduction of the gastric pouch from 500 to 40 ml) after a median 48-month period [48.3 ± 9.17 months (mean ± SD)] and were observed for a total period of 116.2 ± 6.21 months; 88 patients only undergoing BPD (controls) were observed for 120 months. Revisional surgery significantly improved side effects of BPD, with resolution of clinical symptoms in most instances. After revisional surgery, patients had a further decrease of body weight. The effect on disappearance of diabetes mellitus (DM) was remarkable, with no difference between revisional surgery and BPD. Triglycerides and transaminases decreased in a similar way, while cholesterol levels differed significantly. Estimated glomerular filtration rate improved. Nutritional parameters were similarly affected. This study suggests that it is possible to maintain the clinical and metabolic effects of BPD after a revisional procedure that leads to lesser malabsorption and to a greater restriction of the stomach. In particular, the positive effects on DM still persist after revisional surgery. This approach should be kept in mind in the presence of significant side effects due, inter alia, to excessive malabsorption.

Delayed gastric emptying after distal gastrectomy and reconstruction of alimentary tract with a gastroenteric anastomosis can significantly influence early and late postoperative course and the length of hospital stay. The purpose of this study was to compare the effect on postoperative functional recovery of two different Roux-en-Y reconstructions: at the gastric greater curvature and at the transected gastric staple line in the Scopinaro’s biliopancreatic diversion. We conducted comparative study; 80 patients were enrolled and divided in two groups: group A (RY-GC) and group B (RY-SL) with 40 patients in each group. We compared the early postoperative functional recovery for both groups measuring four parameters: gastric stasis indicated with the volume of the gastric fluid collected per 24 h, day of removal of the nasogastric tube, day of starting the oral intake, and day of hospital discharge. There was statistically significant (p < 0.001) reduction in gastric fluid volume in favor of the RY-GC group starting from the first postoperative day resulting in earlier removal of nasogastric tube with earlier starting of oral feeding than RY-SL group, with no symptoms of stasis required nutrition suspension; while three patients in RY-SL group experienced persistence of nausea and vomiting and needed nutrition suspension for several days. There was statistically significant (p < 0.001) reduction in the hospital stay for RY-GC group. Roux-en-Y reconstruction at the greater curvature ensures a rapid functional recovery with early hospital discharge. The use of stapler devices made this method easier and safer and no complications have arisen with mechanical anastomoses.

Bariatric surgery is playing an increasingly important role in our society. The surgical approach should be chosen in consistent with the patients' problem. The purpose of this study was to compare surgical outcomes in patients who underwent laparoscopic Scopinaro's biliopancreatic diversion (BPD) versus open BPD in our institute experience. Eighty patients were enrolled and divided in two groups: laparoscopic group (VL) and open group (OP), with 40 patients in each group during two calendar years 2006–2007. We performed the same technique for both groups using the same staplers for anastomosis, with the same measurements for alimentary and common limbs in both groups. We compared the following variables in the two groups: operative time, intra and early postoperative complications, postoperative pain, consumption of analgesics, recovery of intestinal function, days to removal of NG tube and start of oral intake, hospital stay, and late complication—incisional hernia incidence. We found statistically significant reduction in favor of laparoscopic group regarding reduction in postoperative pain, consumption of analgesics, incidence of incisional hernia, operative time, and hospital stay—with early removal of NG tube and early oral intake. We did not find any statistically significant difference regarding intra, immediate and early postoperative complications and recovery of intestinal functions. Laparoscopic BPD is a safe technique; has good results without affecting the duration of the intervention; and ensures less postoperative pain with rapid functional recovery, less hospital stay, and drastic reduction of incisional hernia incidence.

A set of shuttle yeast vectors containing the dominant selectable markers KanMX4 or HphMX4 cassettes, conferring resistance to geneticin and hygromycin B, respectively, was constructed. Dominant selectable markers are useful for genetic manipulation of natural, wine and industrial strains which do not contain any auxotrophic markers as well as of strains which cannot grow on synthetic mineral medium. Vectors were characterized by (i) copy number, (ii) mitotic stability both in selective and non-selective conditions, (iii) the efficiency and frequency of transformations, (iv) optimal adaptation times in non-selective media, (v) optimum conditions for transformation of various laboratory, commercial and wine strains, and (vi) expression level of an inserted gene. Furthermore we produced GFP-containing vectors that can be used for protein subcellular localization in prototrophic strains.

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.

Synteny analysis is combined with sequence similarity and motif identification to trace the evolution of the putative monocarboxylate (lactate/pyruvate) transporters Jen1p and the dicarboxylate (succinate/fumarate/malate) transporters Jen2p in Hemiascomycetes yeasts and Euascomycetes fungi. It is concluded that a precursor form of Jen1p, named here preJen1p, arose by the duplication of an ancestral Jen2p, during the speciation of Yarrowia lipolytica, which was transferred into a new syntenic context. The Jen1p transporters differentiated from preJen1p in Kluyveromyces lactis, before the Whole Genome Duplication (WGD), and are conserved as a single copy in the Saccharomyces species. In contrast, the ancestral Jen2p was definitively lost just prior to the WGD and is absent in Saccharomyces.

Saccharomyces cerevisiae is a good model with which to study the effects of morphologic differentiation on the ecological behaviour of fungi. In this work, 33 morphologic mutants of a natural strain of S. cerevisiae, obtained with UV mutagenesis, were selected for their streak shape and cell shape on rich medium. Two of them, showing both high sporulation proficiency and constitutive pseudohyphal growth, were analysed from a genetic and physiologic point of view. Each mutant carries a recessive monogenic mutation, and the two mutations reside in unlinked genes. Flocculation ability and responsiveness to different stimuli distinguished the two mutants. Growth at 37 °C affected the cell but not the colony morphology, suggesting that these two phenotypes are regulated differently. The effect of ethidium bromide, which affects mitochondrial DNA replication, suggested a possible “retrograde action” of mitochondria in pseudohyphal growth.

In Kluyveromyces lactis , the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by Kl ZWF1 . We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae , Kl ZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae , the Kl zwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield.

Mitochondria are organelles that have their own DNA (mitochondrial DNA, mtDNA) whose maintenance is necessary for the majority of ATP production in eukaryotic cells. Defects in mtDNA maintenance or integrity are responsible for numerous diseases. The DNA polymerase γ (POLG) ensures proper mtDNA replication and repair. Mutations in POLG are a major cause of mitochondrial disorders including hepatic insufficiency, Alpers syndrome, progressive external ophthalmoplegia, sensory neuropathy and ataxia. Mutations in POLG are also associated with parkinsonism. To date, no effective therapy is available. Based on the conservation of mitochondrial function from yeast to human, we used Saccharomyces cerevisiae and Caenorhabditis elegans as first pass filters to identify a chemical that suppresses mtDNA instability in cultured fibroblasts of a POLG-deficient patient. We showed that this unsuspected compound, clofilium tosylate (CLO), belonging to a class of anti-arrhythmic agents, prevents mtDNA loss of all yeast mitochondrial polymerase mutants tested, improves behavior and mtDNA content of polg-1-deficient worms and increases mtDNA content of quiescent POLG-deficient fibroblasts. Furthermore, the mode of action of the drug seems conserved as CLO increases POLG steady-state level in yeast and human cells. Two other anti-arrhythmic agents (FDA-approved) sharing common pharmacological properties and chemical structure also show potential benefit for POLG deficiency in C. elegans. Our findings provide evidence of the first mtDNA-stabilizing compound that may be an effective pharmacological alternative for the treatment of POLG-related diseases.

Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1.

Biliopancreatic diversion (BPD), as proposed by Scopinaro, is a malabsorptive procedure for the treatment of morbid obesity, which has shown excellent results in terms of percentage excess weight loss (EWL) and improvement of co-morbidities.

Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities.

In Kluyveromyces lactis , galactose transport has been thought to be mediated by the lactose permease encoded by LAC12 . In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1 , previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12 , and the double mutant hgt1lac12 , suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.

Plug insertion for primary femoral hernia repair may cause p.o. discomfort. The Kugel technique may avoid this problem. Patients' satisfaction to the Kugel and the plug techniques is compared in the present study. Demographics, surgical, outcome and analgesic consumption data of 26 patients treated for with the plug technique (P group) are compared with 24 operated with the Kugel patch (K group). Patients' p.o. discomfort to the two procedures was measured with quantitative (VAS score) and a qualitative (the short form of McGill pain questionnaire, SF-MPQ) methods, and compared. P group presented higher early p.o. pain (P

A large monoinstitutional series adopting the Kugel retroparietal technique for inguinal hernia surgery is analysed. Our aim is to assess the "mini-invasiveness" of this technique. Six hundred and twenty patients (pts) affected by monolateral inguinal hernia were treated with a preperitoneal alloplasty with a posterior approach (Kugel hernia repair, KHR) between January 2002 and September 2004. The surgical incision extension was 3.5 cm on average (range 2-4.5). The mean operation time was 33 min (range 20-45). Spinal anaesthesia and ambulatory procedure were applied in 595 cases (96%). Postoperative complications affected 20 pts (3%). The postoperative pain was well controlled. No chronic neuropathic pain was registered at follow-up. Patients resumed work after an average of 9 days (range 7-12) from operation. Recurrence rate was 0.8%. Conclusions. The Kugel hernia repair satisfies the standards to be awarded as a "mini-invasive" technique.

The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1 , an orthologue of Saccharomyces cerevisiae ERO1 , was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1 , the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis , they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1 , KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae , K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.

Autosomal dominant and recessive forms of progressive external ophthalmoplegia (adPEO and arPEO) are mitochondrial disorders characterized by the presence of multiple deletions of mitochondrial DNA in affected tissues. Four adPEO-associated missense mutations have been identified in the ANT1 gene. In order to investigate their functional consequences on cellular physiology, we introduced three of them at equivalent positions in AAC2, the yeast orthologue of human ANT1. We demonstrate here that expression of the equivalent mutations in aac2-defective haploid strains of Saccharomyces cerevisiae results in (a) a marked growth defect on non-fermentable carbon sources, and (b) a concurrent reduction of the amount of mitochondrial cytochromes, cytochrome c oxidase activity and cellular respiration. The efficiency of ATP and ADP transport was variably affected by the different AAC2 mutations. However, irrespective of the absolute level of activity, the AAC2 pathogenic mutants showed a significant defect in ADP versus ATP transport compared with wild-type AAC2. In order to study whether a dominant phenotype, as in humans, could be observed, the aac2 mutant alleles were also inserted in combination with the endogenous wild-type AAC2 gene. The heteroallelic strains behaved as recessive for oxidative growth and petite-negative phenotype. In contrast, reduction in cytochrome content and increased mtDNA instability appeared to behave as dominant traits in heteroallelic strains. Our results indicate that S. cerevisiae is a suitable in vivo model to study the pathogenicity of the human ANT1 mutations and the pathophysiology leading to impairment of oxidative phosphorylation and damage of mtDNA integrity, as found in adPEO.

In the yeast Saccharomyces cerevisiae, the first step in lactate metabolism is its transport across the plasma membrane, a proton symport process mediated by the product of the gene JEN1. Under aerobic conditions, the expression of JEN1 is regulated by the carbon source: the gene is repressed by glucose and induced by non-fermentable substrates. JEN1 expression is also controlled by oxygen availability, but is unaffected by the absence of haem biosynthesis. JEN1 is negatively regulated by the repressors Mig1p and Mig2p, and requires Cat8p for full derepression. In this report we demonstrate that, in addition to these regulators, the Hap2/3/4/5 complex interacts specifically with a CAAT-box element in the JEN1 promoter, and acts to derepress JEN1 expression. We also provide evidence for transcriptional stimulation of JEN1 by the protein kinase Snf1p. Data are presented which provide a better understanding of the molecular mechanisms implicated in the co-regulation of genes involved in the metabolism of lactate.

The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the KlCAT8 gene (a homologue of S. cerevisiae CAT8 , encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S. cerevisiae . While CAT8 controls the key enzymes of gluconeogenesis in S. cerevisiae , KlCAT8 of K. lactis does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193–203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2 , were specifically regulated by KlCAT8 , but very differently from the S. cerevisiae counterparts. KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8. Also, KlJEN1 , encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8 .

In yeast the utilization of lactate requires two enzymes, the D and L-lactate ferricytochrome c oxidoreductase (D and L-LCR), which stereospecifically oxidize D- and L-lactate to pyruvate. These enzymes are nuclearly encoded and localized in mitochondria. In the yeast Kluyveromyces lactis, a mutant devoid of D- and L-LCR activities and unable to grow on racemic lactate was isolated. Transformation of the mutant with a K. lactis genomic library allowed the isolation of the KlCYB2 gene, restoring the growth on lactate and the L-LCR activity. The KlCYB2 gene and its flanking regions were sequenced (Accession No. AJ243324; EMBL/GenBank databases). The deduced amino acid sequence is highly homologous to the corresponding Saccharomyces cerevisiae and Hansenula anomala protein sequences previously characterized. The homology is missed in the N-terminal region, corresponding to the presequence cleaved during import into mitochondria. Analysis of KlCYB2 gene expression indicated that, in contrast to S. cerevisiae, the major regulatory feature is induction by lactate.