Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study.

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.

Yeast expression of mammalian Onzin and fungal FCR1 suggests ancestral functions of PLAC8 proteins in mitochondrial metabolism and DNA repair.

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.

Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome.

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 encephalomyopathy 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 encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was excluded. 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 human 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 completely 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.

Structural modification of cuminaldehyde thiosemicarbazone increases inhibition specificity toward aflatoxin biosynthesis and sclerotia development in Aspergillus flavus.

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.

Revision of Biliopancreatic Diversion for Side Effects or Insufficient Weight Loss: Codification of a New Procedure.

PURPOSE: Addressing the problem of proctologic sequelae after Scopinaro's 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 Scopinaro's classical BPD (SBPD). METHODS: We retrospectively reviewed our clinical experience with the new procedure. RESULTS: 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 < 0.001) and a parallel reduction in BMI (from preoperative 33.03 ± 7.6 to 26.8 ± 4.1 at post-operative year 1 and 26.9 ± 2.8 at year 5; p < 0.001). Mean excess BMI percent loss was 49.5 ± 94.6% at post-operative month 3, 76.51 ± 74.9% at year 1 and 76.2 ± 31.3% at year 5. Nutritional and metabolic parameters remained stable. Similar results were observed, analysing separately both groups of patients. CONCLUSIONS: Our preliminary data suggest that the proposed procedure could represent a safe and effective revisional tool to treat invalidating proctologic sequelae after SBPD, or when weight loss may be deemed unsatisfactory.

Clinical and Metabolic Effects of Biliopancreatic Diversion Persist After Reduction of the Gastric Pouch and Elongation of the Common Alimentary Tract. Preliminary Report in a Series of Patients with a 10-Year Follow-Up.

BACKGROUND: Biliopancreatic diversion (BPD) is a bariatric technique burdened, in some instances, by clinical evidence of malabsorption and malnutrition, and by intractable diarrhea. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number.

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.

Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles.

Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1.

Roux-en-Y reconstruction at greater curvature in biliopancreatic diversion: effects on early postoperative functional recovery.

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.

Laparoscopic versus open biliopancreatic diversion: a prospective comparative study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The mitochondrial disulfide relay system protein GFER is mutated in autosomal-recessive myopathy with cataract and combined respiratory-chain deficiency.

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.

KNQ1, a Kluyveromyces lactis gene encoding a transmembrane protein, may be involved in iron homeostasis.

The original purpose of the experiments described in this article was to identify, in the biotechnologically important yeast Kluyveromyces lactis, gene(s) that are potentially involved in oxidative protein folding within the endoplasmic reticulum (ER), which often represents a bottleneck for heterologous protein production. Because treatment with the membrane-permeable reducing agent dithiothreitol inhibits disulfide bond formation and mimics the reducing effect that the normal transit of folding proteins has in the ER environment, the strategy was to search for genes that conferred higher levels of resistance to dithiothreitol when present in multiple copies. We identified a gene (KNQ1) encoding a drug efflux permease for several toxic compounds that in multiple copies conferred increased dithiothreitol resistance. However, the KNQ1 product is not involved in the excretion of dithiothreitol or in recombinant protein secretion. We generated a knq1 null mutant, and showed that both overexpression and deletion of the KNQ1 gene resulted in increased resistance to dithiothreitol. KNQ1 amplification and deletion resulted in enhanced transcription of iron transport genes, suggesting, for the membrane-associated protein Knq1p, a new, unexpected role in iron homeostasis on which dithiothreitol tolerance may depend.

Complete loss-of-function of the heart/muscle-specific adenine nucleotide translocator is associated with mitochondrial myopathy and cardiomyopathy.

Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no ophthalmoplegia. A muscle biopsy showed the presence of numerous ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.

MIG1-dependent and MIG1-independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p.

Imp2p (Yil154c) is a transcriptional activator involved in glucose derepression of the maltose, galactose and raffinose utilization pathways and in resistance to thermal, oxidative or osmotic stress. We analysed the role of Imp2 in the regulation of GAL genes. Imp2 was shown to have a positive effect on glucose derepression of Leloir pathway genes and their activator gene GAL4. The effect of Imp2 on galactose metabolism was shown to be partially dependent on Mig1p. The Mig1-independent role depends on Nrg1p. However, disruption of both MIG1 and NRG1 only partially relieves the glucose repression of GAL genes in the Deltaimp2 mutant, indicating that Imp2 must also have other function(s). Moreover, the interaction between IMP2 and GAL6/BLH1, a recently isolated gene involved in the regulation of GAL genes that shares with Imp2 the ability to protect cells from the glycopeptide bleomycin, was also analysed. The results suggest a major role of Imp2 in a GAL6-independent pathway.