Saccharomyces cerevisiae has been efficiently used as a model organism to study mitochondria-related disorders thanks to its easy manipulation and well-known mitochondrial biogenesis and metabolism. It has been successfully exploited both to validate alleged pathogenic variants identified in patients and to discover potential beneficial molecules for their treatment. The so-called “drug drop test”, a phenotype-based high-throughput screening, especially if coupled with a drug repurposing approach, allows the identification of molecules with high translational potential in a cost-effective and time-saving manner. In addition to drug identification, S. cerevisiae can be used to point out the drug’s target or pathway. To date, drug drop tests have been successfully carried out for a variety of disease models, leading to very promising results. The most relevant aspect is that studies on more complex model organisms confirmed the effectiveness of the drugs, strengthening the results obtained in yeast and demonstrating the usefulness of this screening as a novel approach to revealing new therapeutic molecules for MDs.Mitochondrial diseases (MDs) refer to a group of clinically and genetically heterogeneous pathologies characterized by defective mitochondrial function and energy production. Unfortunately, there is no effective treatment for most MDs, and current therapeutic management is limited to relieving symptoms. The yeast  Saccharomyces cerevisiae is a single-cell eukaryotic microorganism commonly known as baker’s yeast. Despite its “simplicity”, it has strongly contributed to advancing our understanding of highly conserved basic cellular processes, such as cell cycle, vesicle trafficking, telomere function, mitochondrial function, autophagy, apoptosis, cytoskeletal dynamics, and aging ..De novo S. cerevisiae as a model system. The models described here are summarized in In this review, we will focus on drug drop tests for the identification of potential therapeutic compounds to treat mitochondrial diseases and on the characterization of the drugs through different analyses, exploiting yeast Yeast mutant cells defective in oxidative growth are spread at a proper concentration on square plates containing solid media supplemented with a non-fermentable carbon source, where they grow minimally, and, afterward, sterile filters are placed on the agar surface and spotted with the compounds to test. One filter is spotted with the solvent in which the molecules are solubilized as a negative control; in addition, wild type (wt) and/or a known positive compound can be spotted as positive controls of growth. The plates are then incubated for several days until a difference in growth between the positive control and the mutant strain is clearly observed. Depending on the presence or absence of a halo of growth and its position around the filters, the compounds are classified into different groups: (i) compounds with a rescuing effect that lead to the formation of a halo of enhanced growth near the filter; (ii) compounds toxic at high concentrations (near the filter) and active at lower concentrations (far from the filter), which generate an external crown of enhanced growth; (iii) compounds without any effect if no enhanced growth is observed; and (iv) toxic compounds that completely inhibit the basal growth of the mutant strain. The diffusion of the molecule around the filter in the agar medium creates a concentration gradient, making it unnecessary to find the specific optimal drug concentration by testing several dilutions of every compound. Primary screening allows us, in one simple experiment, to test a huge number of drugs across a large range of concentrations thanks to their diffusion. Only a few extremely toxic molecules should be diluted and further tested. To confirm the rescue effect observed during primary screening and to minimize the number of false-positive molecules, the active compounds (positive compounds) are subjected to a second step of analysis performed using the same conditions, except for the number of filters placed on each plate, which decreases from about 30 to 4 .Impairment of ATP synthase (complex V), the enzyme responsible for the mitochondrial synthesis of ATP and the energy-carrying molecule used to power many energy-requiring cellular reactions, determines a group of mitochondrial disorders. The ATP synthase-related disorders are due to defects in the structure or in the assembly of complex V and include Leigh syndrome , hypertrophic cardiomyopathy, and NARP  ,36,37. MATP6 is encoded by nucleotides 8527–9207 of the mitochondrial genome and, together with ATP8, make up the only mitochondrial ATP synthase subunits encoded by the mtDNA. Several mutations in ATP6 have been reported as causes of mitochondrial impairment. T8993G transversion was identified in a patient presenting mental retardation and ataxia without retinitis pigmentosa  and was Due to the high number of mutations identified in this gene and, more generally, related to ATP synthase deficiencies, it is important to identify new therapeutic approaches to treat the correlated syndromes. The conservation of mitochondrial functions and the capacity to manipulate both mitochondrial and nuclear genomes in yeast allowed us to largely study in this organism the consequences of ATP synthase dysfunctions, whose pathogenic mechanisms could not be ascribed to a mere lack or decrease in ATP production .S. cerevisiae mitochondrial genome is possible, yeast models for the most common ATP6 mutations found in NARP patients have been created and characterized [fmc1Δ strain, even if humans do not present an orthologue of this gene. FMC1 encodes a mitochondrial matrix protein required for the assembly and stabilization at high temperatures of the F1 subunit of the ATP synthase [fmc1Δ) is thermosensitive and is characterized by a strong reduction in assembled ATP synthase complexes. Nevertheless, the few ones present are fully functional. A similar condition was also found in patients carrying mutations in the nuclear genes ATP12 and TMEM70 and in patients heteroplasmic for ATP6 mutations, indicating that the null mutant fmc1Δ represents a suitable model to study human ATP synthase dysfunctions, regardless of the specific genetic cause. As a secondary effect, the loss of FMC1 negatively impacts respiratory chain complex assembly and activity and the mitochondrial membrane potential [Thanks to the biolistic technique, through which site-direct mutagenesis of the cterized ,59,60,61synthase . The nulotential .fmc1Δ strain on a glycerol-containing medium at 35 °C [fmc1Δ mutant [fmc1Δ strain and were used as positive controls during the high-throughput screening. In the primary screening, about ten molecules were identified as positive hits, among which was Chlorhexidine (CH), an antiseptic compound. CH was further tested on atp6-NARP models, showing a beneficial effect on the atp6-NARP T8993G and the atp6-NARP T8851C strains but not on the atp6-NARP T9176G strain, carrying the most severe mutation. This result could be due to the presence of a partially functioning ATP synthase in the former strains, so that the molecule could exert its beneficial effect [fmc1Δ strain, such as the presence of inclusion bodies and the absence of mitochondrial cristae, were partially rescued after CH treatment. To get insight into the effect of the CH treatment on cellular function, genome-wide analyses of the transcriptional responses were performed through high-resolution tiling microarrays. A total of 336 genes, mostly involved in mitochondrial respiration, showed significant differential expression in the fmc1Δ mutant, and in almost all cases, treatment with chlorhexidine determined the rescue of the transcriptional differences, although to a different extent for the different genes. Overall, the data suggested that CH improved the respiratory function of the fmc1Δ null mutant. In particular, it was proposed that CH could act both by increasing the number of ATP synthase complexes and the efficiency with which they are used, resulting in a modest but significant increase in ATP production that in turn leads to the rescue of the oxidative growth defect [Couplan and colleagues developed the drug drop test to find compounds active against ATP synthase-related disorders, exploiting the leaky oxidative growth defect of the at 35 °C . Around at 35 °C ,65. The Δ mutant ,67. Bothl effect . To deeph defect .atp6-T8993G-NARP cybrids, for which a severe growth defect was previously reported [To prove a possible translational use, the drugs identified through the yeast-based screening were also tested on human NARP-cybrids (cytoplasmic hybrids), used as human cell-based models. The results confirmed and strengthened the data obtained in yeast; in fact, DHLA, OA, and CH increased the growth rates of reported . The resreported ,70. Furtfmc1Δ strain [atp6-T8993G-NARP cybrids [TIM17 and TIM23 were identified. It was suggested that NaPT selectively and differentially modulates the import of proteins carrying pre-sequence signals through the Tim23 pathway. Supplementation of NaPT exerts a similar effect to those determined by TIM21 overexpression, a regulatory subunit of the Tim23 complex, leading to a change in Tim23 activity from matrix translocation towards inner membrane insertion [TIM21 overexpression improved respiratory chain complex assembly. Furthermore, it was demonstrated that the modulation of mitochondrial protein sorting alleviates not only primary ATP synthase deficiency but also several downstream-related phenotypes [In a second screening based on the same approach (drug repurposing and drug drop test) and on the same yeast mutant, Aiyar and colleagues identified the antiseptic Sodium pyrithione (NaPT) as able to rescue in a dose-dependent manner the oxidative growth defect of the Δ strain . This co cybrids ,65, sugg cybrids . By usin cybrids . Regardinsertion ,73. The enotypes ,61,72,74atp6-T8993G NARP patient-derived cybrids [Similar to what was observed in yeast, overexpression of TIM21 in human cells led to a significant increase in the survival of  cybrids , underlyANT1, also known as SLC25A4, encodes the adenine nucleotide translocator 1, which is one of the four isoforms of the mitochondrial ADP/ATP carrier. It is located in the inner mitochondrial membrane and is involved in the import of ADP into the mitochondrial matrix, where its conversion to ATP by the ATP synthase occurs. Subsequently, it is also involved in the transport of the newly produced ATP to the cytosol . AlthougThe first disease identified as being associated with ANT1 mutations was autosomal dominant progressive external ophthalmoplegia , characterized by multiple mitochondrial DNA deletions in skeletal muscle. The most common clinical features include the adult onset of weakness of the external eye muscles, ptosis, myopathy, and exercise intolerance . PathogeTo date, several degenerative mitochondrial pathologies caused by mutations in ANT1 are known; in particular, dominant missense point mutations  were identified in subjects affected by adult-onset adPEO ,86,87,88Saccharomyces cerevisiae, thanks to the high conservation of the genes encoding the ADP/ATP carrier in eukaryotes, including yeast. Yeast has three genes that are orthologous to ANT1: AAC1, AAC2, and AAC3. AAC2 encodes for the major isoform of the translocator [S. cerevisiae by using the aac2Δaac1Δ strains carrying the human equivalent mutations in AAC2 or in the chimeric gene [M114Paac2, to reproduce the p.Leu98Pro variant found in patients suffering from adPEO. The strain, characterized by severely affected but not completely abolished oxidative growth, was subjected to drug drop test screening to find molecules able to rescue the phenotype in yeast and therefore of potential therapeutic interest for the treatment of ANT1-related disorders [5 cells/plate to test the 1018 molecules from the Selleck FDA-approved chemical library. Positive compounds that induce a halo of enhanced growth around the corresponding filter were identified after 3–4 days of incubation at 28 °C. Eight active molecules  confirmed their positive effect in the secondary screening. Drugs used for chemotherapy, such as doxorubicin, epirubicin, and daunorubicin, are not suitable for long-term treatment of mitochondrial pathologies and therefore are not used for further characterization; despite this, they may lead to the identification of potential molecular targets or pathways targetable by non-toxic analogous molecules. The other five drugs were also able to act on two additional aac2 mutant strains characterized by a leaky oxidative growth defect, namely the strains presenting the mutations p.Ala128Pro and p.Ser303Met, corresponding to the human pathological mutations p.Ala114Pro and p.Val289Met, respectively. In contrast, the molecules were inactive in two mutant strains characterized by a strong oxidative growth defect and carrying p.Arg96His and p.Arg252Gly mutations, equivalent to the severe human dominant mutations p.Arg80His and p.Arg235Gly, respectively. The molecules were also inactive in the null mutant, indicating that the drugs were unable to bypass the Aac2 function.Due to the lack of expression of ANT1 in cultured human cell lines, such as fibroblasts and myoblasts, the impact of ANT1 mutations could not be studied in these cells . Studiesslocator , whereasslocator . In partric gene ,99,100. isorders . DiffereM114Paac2. They revealed a beneficial effect on respiratory activity but no beneficial action on mitochondrial membrane potential or the production of reactive oxygen species. Otilonium bromide showed a decoupling action, suggesting that the rescuing ability could be due to its capacity to reduce the electrochemical gradient across the inner mitochondrial membrane. Regarding the other drugs, the authors have speculated on a possible action on mitochondrial inner membrane lipid composition that could correct the proteostatic stress of the inner mitochondrial membrane (IMM) or the induction of an increase in Ca2+ levels that could, in turn, stimulate ATP-Mg/Pi carrier Sal1 transport activity [To deepen our understanding of the mechanism underlying the beneficial effect, the five effective drugs were also evaluated on different defective phenotypes of the haploid mutant strain activity .M114PAAC2/aac2, the model for the adPEO-associated human mutations p.Leu98Pro, and heteroallelic R96HAAC2/aac2, the model for the p.Arg80His de novo dominant missense mutation associated with a more severe phenotype, were used to assay the effect of the molecules. The drugs restored respiratory activity and reduced in both models the petite percentage, an index of mtDNA instability [In addition, Western blot analysis excluded the possibility that the treatment with molecules leads to an increase in Aac2 protein synthesis or to a stabilization of the protein. As the majority of ANT1 pathological mutations are dominant, heteroallelic strains could be used as a better model system to mimic the human heterozygous condition. Heteroallelic tability , which itability , thus extability .C. elegans ant1-1 by means of RNA interference demonstrated that the two tested drugs, otilonium bromide and pergolide mesylate, were able to decrease embryonic lethality, a worm-specific defect [Moreover, studies on c defect . This obS. cerevisiae BCS1, a gene involved in complex III assembly of the mitochondrial respiratory chain; it encodes a protein involved in the incorporation of the nuclearly-encoded Rieske subunit  in complex III, also known as the cytochrome bc1 complex. Mutations in this gene are the most common cause of complex III-related diseases, such as Bjornstad syndrome, GRACILE syndrome, and mitochondrial complex III deficiency type 1 (reviewed in [BCS1L is the homolog of iewed in ). Bjornsiewed in ,103. GRAiewed in . Mitochoiewed in ,106.S. cerevisiae, the impairment of Bcs1 determines the accumulation of an inactive form of complex III, pre-complex III (Pre-III), lacking Rip1, blocking the supercomplex III2-IV formation. Due to the dual genetic origin, the assembly of all the subunits into the mature and functional cytochrome bc1 complex is a complicated process that was largely studied in yeast  and, deiewed in . Thanks l mutant , confirmbcs1 mutants, Panozzo and collaborators performed a drug drop test [F342Cbcs1 strain (carrying the substitution p.Phe342Cys) [F342Cbcs1 strain, characterized by a mutation located in the AAA domain of the protein, was spread on plates containing glycerol, where the respiratory defect was visible, and incubated for several days at 25 °C. Two compounds, the antibiotics pentamidine and clarithromycin, were able to partially rescue the defect. To test the potential effectiveness of these molecules for the treatment of patients, they were tested on K192Pbcs1, mimicking the human p.Arg155Pro mutation, located in a Bcs1-specific domain and found in a patient presenting liver failure [F401Ibcs1, mimicking the human p.Phe368Ile mutation, located in the AAA-domain and identified in a patient presenting early-onset encephalopathy [bcs1Δ, showing no effect, indicating that the drugs do not act through a bypass mechanism. Additional analyses highlighted the large mitochondrial rRNA of the mitoribosome as the primary target of the effective antibiotics, disturbing mitochondrial protein synthesis and determining an unbalanced production of the OXPHOS subunits. In particular, differential effects on the mitochondrial synthesis of the various OXPHOS subunits were observed, leading to a stronger impairment in complex IV assembly. Genetic analyses confirmed the large mt-rRNA of the mitoribosome as the antibiotic target since the absence of the auxiliary factors Rrf1 or Mif3, which are involved in mitochondrial ribosome recycling or in the translation initiation step, resulted in unbalanced mitochondrial protein synthesis and was also able to ameliorate the OXPHOS defect of bcs1 mutants [In order to identify chemical compounds able to compensate for the respiratory deficiency in rop test . In parte342Cys) ,109. The failure , and on alopathy . The mol mutants .bcs1 mutants, possibly due to a slowed-down assembly, show an accumulation of inactive pre-complex III2 (Pre-III2) that can interact with complex IV and give rise to unfunctional supercomplex Pre-III2-IV. Treatment with specific concentrations of the antibiotics identified in the screening can dysregulate mitochondrial translation; this is supposed to be beneficial because the decrease in complex IV levels could partially prevent the trapping of Pre-III2, allowing the assembly process towards the fully assembled complex III and then the supercomplex III2-IV. It must be underlined that this compensatory mechanism could be beneficial only in the case of fine-tuning complex IV reduction; the decrease should be enough to allow at least some Pre-III to proceed toward assembly, but, in order to obtain enough active supercomplex III2-IV, a sufficient amount of cIV should be retained and associated with fully assembled III2. This could be obtained by deleting COX8, which codes for a non-essential complex IV subunit, leading to a 30% decrease in cytochrome aa3 [Yeast rome aa3 .2-IV. The binding of pentamidine, whose binding site is conserved from yeast to humans [C. elegans, allowing the formation of some functional complex III and supercomplex I-III2-IV [In humans, similar to in yeast, pre-complex III accumulates in patients and interacts with complex IV , prevento humans , could d-III2-IV .cristae, mtDNA stability, assembly of the respiratory supercomplexes, apoptosis, and mitophagy [OPA1 encodes a mitochondrial dynamin-like GTPase, a multifunctional protein involved in mitochondrial dynamics, including fusion, structural organization of itophagy ,117,118.itophagy ,120, a mitophagy ,122,123,itophagy ,129,130.itophagy . Usuallyitophagy ,132,133.itophagy ,133. Behitophagy ,135. Desitophagy ,137 or gitophagy , no apprS. cerevisiae can be exploited due to the presence of MGM1, the ortholog of OPA1, which is involved in mitochondrial morphology [cristae structure [I322Mmgm1), equivalent to the human p.Ile382Met variant [mgm1 pathogenic alleles show a strong increase in petite mutants that arise spontaneously after large deletions or loss of mtDNA, giving rise to respiratory deficiency [petite phenotype is an irreversible condition; thus, a thermosensitive mutation should be used in the attempt to identify possible beneficial molecules. The I322Mmgm1 strain showed a reduction in oxidative growth using an ethanol or glycerol carbon source at 37 °C. Two different libraries were tested through the drug drop test. Notably, the first library was the Selleck FDA-approved chemical library, for which a drug repurposing approach leads to a potential direct use on patients; the second was the NCI BioServices Diversity Set IV, which collects 1596 molecules with different structural properties, increasing the probability of positive molecule identification, albeit a direct use in clinics is not possible. In total, 22 molecules from the first library and 21 from the second were identified. One molecule, benzbromarone, belonging to both libraries, has been identified as positive in both cases, demonstrating the reproducibility of this test [MGM1/OPA1 chimeric gene  [S646chim3L strain. Between the latter 11 molecules, only benzbromarone, also identified in the other library, and ovalicin were considered for the subsequent characterization because the other molecules, for various reasons such as cytotoxicity, toxicity, instability in aqueous solutions, and unavailability, were not suitable for the treatment of mitochondrial diseases [MGM1 alter mtDNA stability, resulting in a petite frequency increase; therefore, the molecules identified through the screening were tested to assess whether the rescue of the oxidative growth defect may be due to an increase in mtDNA stability. The results allowed us to further restrict the number of molecules to be tested in higher models. In particular, six molecules  reduced the mitochondrial DNA instability of the I322Mmgm1 strain. To validate the effectiveness of these molecules on mammal models, OPA1-disease cell models, such as Opa1−/− mouse embryo fibroblasts (MEFs), expressing the human OPA1 isoform carrying p.Arg445His or p.Asp603His, associated with DOA-plus and DOA, respectively, and patients’ fibroblasts carrying the same mutations, were used. Specifically, five of the six molecules identified through yeast-based screening were also beneficial in MEFs , highlighting tolfenamic acid as the most promising molecule to be used for drug repurposing in a clinical trial for DOA or other neurodegenerative pathologies associated with mutations in the OPA1 gene [The yeast rphology ,140 and rphology ,142,143.tructure ,145,146. variant , relatedficiency . The pethis test . In ordehis test . The posof OPA1) . A variediseases . The nexPA1 gene . CellulaSchizosaccharomyces pombe, carrying the p.Pro300Ser variant in the msp1 gene (P300Smsp1), the ortholog of OPA1. About 1600 compounds were tested, and five of them  prevented the lethality associated with the loss of mtDNA maintenance. This highlights the usefulness of the test, which can also be tailored to organisms other than S. cerevisiae, in identifying new drugs for mitochondrial dysfunction rescue [Interestingly, in order to identify molecules able to suppress mitochondrial fusion and mtDNA maintenance defects, a drug drop test was also performed on the fission yeast n rescue . Two comn rescue . AlthougPKAN  belongs to the group of rare neurodegenerative diseases with brain iron accumulation (NBIA), characterized by a progressive iron accumulation in the basal ganglia and other areas of the brain. The consequences are extrapyramidal movements, such as parkinsonism and dystonia, along with pigmentary retinopathy and a classic MRI signature, the so-called ‘eye of the tiger’ . A huge Both PKAN and HARP syndrome are caused by mutations in the PANK2 gene, which encodes the pantothenate kinase, an essential regulatory enzyme involved in the first and limiting step in coenzyme A (CoA) biosynthesis and catalyzes the cytosolic phosphorylation of vitamin B5 (pantothenate), N-pantothenoylcysteine, and pantetheine . Four isS. cerevisiae has been successfully used to understand the molecular mechanisms underpinning several neurodegenerative diseases as well as iron-related disorders [CAB1. CAB1 encodes for the unique pantothenate kinase in yeast and co-localizes in the cytoplasm and in mitochondria, with a predominance in the cytoplasm [cab1 deletion. Therefore, a homologous approach was used, exploiting the yeast CAB1 gene that was mutagenized to mimic the human variants identified in patients and introduced into the cab1Δ strain through plasmid shuffling [G351Scab1) acts as a hypomorphic allele and presents a thermosensitive phenotype [N290Icab1 strain, mimicking the human pathogenic mutation p.Asn500Ile. Four molecules were able to restore the oxidative growth of this mutant, and the two compounds showing the better effect, 5,7 dichloro-8 hydroxyquinoline (CQCl) and nalidixic acid , were subjected to further studies to determine their rescue ability on other phenotypes due to CoA biosynthesis impairment. In particular, both molecules were able to rescue lipid peroxidation and the iron and ROS content, probably due to their ability to restore the physiological iron content. The authors speculate that the rescue ability could depend on the chelating properties of both CQCl and nalH, leading to excess iron chelation, a consequent reduction in oxidative stress, and maybe membrane damage. Moreover, nalidixic acid also showed a beneficial effect on other mutants , expanding its potential administration to a wider group of patients [Despite the lack of a neural system, isorders . Yeast cytoplasm . Ceccatehuffling . The yeahuffling ,172. Thehenotype . Thanks henotype . Eight cpatients . No studThe DNA polymerase γ, encoded by POLG, is the catalytic subunit of the only mitochondrial replicase present in animal mitochondria ,175. POLMIP1, encoding the mitochondrial polymerase, is the yeast S. cerevisiae ortholog of POLG [G651Smip1, harboring a mutation in the polymerase domain equivalent to the human p.Gly848Ser one, presents a thermosensitive phenotype, resulting in an oxidative growth defect on ethanol at 37 °C, and thus it was exploited to perform a drug drop test screening. A total of 1500 molecules from the TEBU-BIO and Prestwick chemical libraries were tested. Of the total, 30 drugs rescued the oxidative growth defect, and among them, Clofilium tosylate (CLO), an anti-arrhythmic drug presenting low toxicity and showing a strong and dose-dependent rescue of oxidative growth, was chosen to investigate its effect. CLO showed its ability to significantly reduce the petite frequency, a hallmark of mtDNA instability, of eleven different mutant strains. The mutations in these strains affect residues localized throughout the different Mip1 domains, thus suggesting a mutation-independent beneficial effect of the treatment. It should be noted that the more effective the molecule, the less the mtDNA stability in the mutant was impaired. Additionally, western blot results showed that CLO supplementation stabilizes Mip1 protein, increasing the amount of a mutant protein characterized by partial retention of catalytic activity. This increase could compensate, at least partially, for the defective polymerase activity, possibly by increasing mtDNA replication [ of POLG . Yeast h of POLG ), and, m of POLG . In factlication . DespiteC. elegans, patients’ fibroblasts, and zebrafish. In particular, CLO was able to rescue a C. elegans-specific defect, namely gonadal function compromise, which leads to sterility. Studies on C. elegans of two additional molecules, ibutilide and dofetilide, characterized by a chemical structure similar to CLO, allowed the animals to reach adulthood but showed less efficacy relative to CLO. The fibroblasts were derived from a patient with compound heterozygous POLG mutations located in the polymerase domain, leading to severe mtDNA depletion. In a quiescent experimental condition, CLO treatment led to a significant increase in mtDNA copy number and an increased POLG protein level, as observed in yeast. In zebrafish, supplementation with CLO improved the detrimental phenotypes observed in organisms harboring a homozygous hypomorphic non-lethal point mutation in polg. This results in an increase in mtDNA levels and complex I activity, as well as the rescue of the myofibril organization and the mitochondrial mass. Cardiac phenotypes such as atrial enlargement and tachycardia, induced by morpholino-mediated Polg deficiency, could be restored to normal values after treatment with CLO [The promising results obtained in yeast led to studies on different model systems, such as with CLO . Thus, twith CLO .cristae formation [MPV17 encodes a mitochondrial inner membrane protein involved in mitochondrial deoxynucleotide homeostasis and mtDNA maintenance . The MPVormation ,196,197.ormation ,198. Theormation . The manormation ,201,202.ormation . Navajo ormation . The simormation . Type 2Eormation ). Treatmormation , so it cSYM1 (Stress-inducible Yeast MPV17) was identified in 2004 as the yeast ortholog of MPV17; it is induced by heat stress and is necessary for growth on ethanol at 37 °C [petite frequency [at 37 °C ,207. Dueat 37 °C ,195,208.requency ,208.S. cerevisiae has been used to identify new potential molecules for the treatment of MPV17-related disorders; in particular, the R51Wsym1 strain was used to perform a drug drop test with the Selleck Chemicals FDA-approved library. The strain showed a reduction in oxidative growth on ethanol at 37 °C, which was rescued by eleven molecules. Moreover, six additional compounds belonging to the Prestwick and TEBU-BIO chemical libraries were tested, and one of them, imazalil, showed beneficial effects. Interestingly, half of the twelve molecules have the ergosterol pathway as a common target; therefore, only three of them were subjected to further investigations. By testing the potential beneficial effect on the sym1Δ strain in the drug drop test-like screening, all the molecules were positive, suggesting a bypass mechanism of action [petite frequency [−/− mice, and MPV17-deficient human fibroblasts [As no effective treatment exists , yeast Sf action . The nulrequency ,208. Furroblasts ,210,211.petite frequency in the R51Wsym1 and sym1Δ strains, confirming their rescuing activity of mtDNA instability, and an increase in the mitochondrial dTTP pool in sym1∆ [To get insight into the molecular effect of the beneficial molecules, their ability to rescue mtDNA instability and decrease dNTPs was measured. All the molecules determined a reduction in the in sym1∆ .The fact that half of the identified drugs modulate ergosterol biosynthesis suggests a beneficial effect by increasing the permeability of mitochondrial membranes. In mammalian cells, the ergosterol counterpart is cholesterol; hence, its moderate reduction could have beneficial effects in MPV17-related disorders. Since the cholesterol pathway is crucial for several cellular functions, it should be carefully modulated, possibly with no influence on collateral pathways such as the mevalonate pathway. For this reason, statins, the most commonly used cholesterol-lowering drugs, are not suitable; specific inhibitors of CYP51A1 (lanosterol 14-α-demethylase) could instead be a better target. Since antimycotic inhibitors of ergosterol synthesis have low activity on human CYP51A, other molecules should be considered.In addition to MPV17, mitochondrial DNA depletion syndromes (MDDS) are caused by mutations in several nuclear genes, including ABAT, AGK, DGUOK, MGME1, POLG, RNASEH, RRM2B, SUCLA2, SUCLG1, TFAM, TK2, TYMP, and TWNK, which are involved either in mtDNA replication or in the maintenance of a balanced mitochondrial nucleotide pool . MDRNR2/RRM2B and MIP1/POLG. In these models, an increase in the dNTP pool was shown to be beneficial for the maintenance of the mtDNA [To date, only 100 affected individuals have been identified with MPV17-related mtDNA maintenance defects. In light of this restricted number of patients, to evaluate whether the drugs may be beneficial for other mutations associated with MDDS and thus expand their potential therapeutic use, they were tested on two further yeast models: he mtDNA ,214.RNR2, the yeast ortholog of RRM2B, encoding the small p53-inducible ribonucleotide-diphosphate reductase, is involved in DNA repair and mtDNA synthesis in post-mitotic cells [S. cerevisiae by the homologous complementation approach. The Leu362Val substitution in yeast RNR2, equivalent to the human Leu317Val, showed the highest mtDNA maintenance defect and was thus used to test the SYM1-beneficial molecules. All the molecules, except Sertraline, determined a significant reduction in petite frequency, similar to that observed by overexpressing the RNR1 or RNR4 genes, which encode the large and small subunits of the RNR complex. Thus, increased levels of dNTPs  could prevent mtDNA instability and represent a potential target for the treatment of RRM2B-related pathologies [ic cells ; mutatioic cells . Severalhologies .MIP1, an ortholog of the human POLG, encoding the catalytic subunit of mtDNA polymerase γ, is involved in mtDNA replication, and its role in mitochondrial dysfunction was presented in the previous paragraph. The pathogenic mechanism in POLG deficiency is due to faulty polymerase activity, leading to mtDNA depletion/deletion. It was demonstrated in yeast that the mtDNA defects associated with G651Smip1, equivalent to the human POLG p.Gly848Ser pathogenic variants, one of the most common POLG mutations described previously, could be corrected by increasing the amount of dNTPs, the substrates of the enzyme [SYM1-beneficial molecules were able to reduce the petite frequency of the G651Smip1 strain [e enzyme . Six SYMS strain . This reS strain .In the case of MDDS due to impairment of the mtDNA synthesis machinery, such as for POLG-related diseases, or due to nucleotide metabolism, such as for RRM2B and MPV17-related diseases, a proposed therapeutic option is the supplementation of deoxyribonucleosides ,218,219.Barth syndrome , also known as 3-methylglutaconic aciduria type II, is a recessive X-linked disorder caused by mutations in the TAFAZZIN gene. This pathology, first described by Barth and collaborators , is charS. cerevisiae has been exploited through the years to study the synthesis and remodeling of cardiolipin and, therefore, how mitochondria are influenced by the misregulation of these processes [TAZ1 is the yeast ortholog of TAFAZZIN and encodes the lysophosphatidylcholine acyltransferase required for the normal phospholipid content of mitochondrial membranes, particularly in regulating cardiolipin content. The yeast strain presenting the deletion of TAZ1 (taz1Δ) showed monolysocardiolipin accumulation and low levels of cardiolipin [taz1Δ yeast strain, identified ODC1  as a potential therapeutic target for several mitochondrial diseases, including BTHS [ODC1 expression [taz1Δ. After incubation at 36 °C on an ethanol-containing medium, a halo of enhanced growth around the filter was visible, demonstrating the beneficial effect of this molecule and its potential use for the treatment of tafazzin-related disorders.The yeast rocesses ,234,235.diolipin ,237,238 diolipin ,240,241.ing BTHS . Oleic apression  and was taz1Δ mutant strain, cytosolic protein synthesis inhibitors showed their ability to rescue the oxidative growth phenotype; in particular, cycloheximide, anisomycin, and emetine, which were rationally selected and tested, suppressed the oxidative defect in a dose-dependent manner. Cycloheximide was beneficial at concentrations 20–30 times lower than the other protein synthesis inhibitors tested and determined the best growth improvement, making it the most interesting compound [Furthermore, using a drug drop test-like technique on the compound . Therefocompound .ODC1, and thus the transport of oxodicarboxylic acids across the IMM, and modulation of cytosolic protein synthesis were identified as potential targets for the treatment of other mitochondrial diseases S. cerevisiae can be considered a powerful ally both for modeling mitochondrial disorders and for searching for beneficial drugs by means of the drug drop test method. In fact, thanks to all the advantages discussed in the introduction, such as the flexibility of genetic tools combined with the high conservation of protein sequence and function, this organism can be considered an excellent “mitochondrial disease model”. In particular, both nDNA and mtDNA mutations can be easily reproduced in S. cerevisiae thanks to efficient homologous recombination and plasmid transformation, or biolistic transformation. Over the years, a variety of yeast disease models have been produced  are a group of rare diseases with a prevalence of approximately 1.6 in 5000 live births, characterized by progressive and incurable defects that can result in premature death. The genetic, clinical, biochemical, and molecular heterogeneity of MDs and the limited availability of preclinical models have strongly hampered the development of effective therapeutic interventions. In fact, there is no resolutive treatment for almost any MD, and current therapeutic management is predominantly focused on relieving symptoms and treating complications. These actions are important to improve the quality of life of patients and their families; however, these conditions are usually progressive, leading to major disabilities. Even if, in recent decades, significant progress has been made (reviewed in ), pharmaiewed in ,247,248)iewed in . Furtheriewed in , flavonoiewed in , and copiewed in  to supprompounds . Yeast aompounds .C. elegans, zebrafish, patients’ fibroblasts)  , demonstOf course, not all the drugs identified could be considered good candidates to be used for long-term administration, which is instead required for mitochondrial pathologies. For example, the use of chemotherapy medications, characterized by high toxicity and lots of side effects, cannot be pursued. Nevertheless, the identification of these types of molecules can be fruitful in discovering potential molecular targets or pathways that can be modulated by non-toxic analogous molecules in order to develop usable and safe medications. Similarly, some drugs could be expected to be inactive in mammals due to the non-conservation of the binding site; this is, for instance, the case with clarithromycin. Again, the identification of this molecule in yeast is not useless, as other rationally selected drugs acting on the same target could be used instead. Additionally, the direct use of azoles, antifungal molecules targeting the ergosterol/cholesterol pathway and inhibiting lanosterol 14-α-demethylase, in patients is not tenable. In fact, despite the degree of conservation between fungal and human enzymes, they have weak inhibitory activity on the human enzyme. It would be interesting to test a synthetic, potent, and stoichiometric human CYP51 inhibitor that was recently identified .In several cases, it was shown that the molecules were active in yeast, although it does not possess the known molecular target of these drugs, suggesting they could act on one or more unknown secondary targets. In fact, it is currently accepted that a drug acts on a multitude of targets in the cell, potentially regulating several different pathways and leading to off-target effects.The identification of the target/pathway by which the drug can rescue the yeast defect could be of great interest not only from a therapeutical point of view but also because this could help to elucidate the molecular pathogenic mechanism of the disease. Target identification can be addressed using the yeast collection of heterozygous deletants by systematic chemical-genomic profiling. Haploinsufficiency of a heterozygous deletion mutant, pointed out by a marked sensitivity to a compound, indicates that the deleted gene is a molecular target of the chemical ,257,258.fmc1Δ and taz1Δ mutants; and nalidixic acid was identified as an active compound for PANK2-related and OPA1-related diseases. This highlights the possibility of cross-reactions and common pathways as possible intervention points. This hypothesis could be directly addressed by testing chemicals that were active in a model on other models, as performed by di Punzio and collaborators. They showed that half of the drugs identified as active on yeast MPV17-disease models are also effective on two other disease models characterized by the same molecular defect, i.e., mtDNA depletion [Considering the rarity of patients affected by a specific mutation, an interesting approach is to look for molecules that are effective for a group of diseases, widening their possible application. Looking at the results obtained on different yeast-disease models, it is possible to notice that some molecules come up independently in different screenings. For example, sertraline was identified both in ANT1-related and MPV17-related screenings; oleic acid was demonstrated to be beneficial in epletion .Interestingly, a variety of chemicals identified are supposed to act directly or indirectly on the membrane composition or the membrane permeability property, highlighting IMM as a major target for MDs treatment.In addition to the screening of a complete library, a drug drop test can also be used to directly test a small number of rationally selected chemicals, as performed for TAFAZZIN-related diseases ,34.Saccharomyces cerevisiae has proven to be an invaluable model system to rapidly and economically discover beneficial molecules with high translational potential for the pharmacological treatment of MDs. To date, the phenotype-based high-throughput screening, called the drug drop test, has been successfully carried out for a variety of disease models, leading to very promising results, strengthened by the fact that studies in more complex eukaryotic model organisms, such as C. elegans or zebrafish, and studies in cell models, such as patients’ fibroblasts, confirmed the effectiveness of the drugs. The data presented in this review underline the usefulness of yeast screening as a novel approach to revealing new potential therapeutic molecules for MDs. In In the last few years, the yeast


ND2, ND4, ND6 and COIII, have experienced positive selection, suggesting their important roles in enhancing the efficiency of ATP production to cope with the increased energy requirements for life in terrestrial environments. These results strongly suggest that the adaptive evolution of mitochondrial genes has played a key role in terrestrial adaptions in Amblyopinae, as well as in Oxudercinae, and provide new insights into the molecular mechanisms underlying the water-to-land transition in vertebrates.The water-to-land transition is one of the most important events in evolutionary history of vertebrates. However, the genetic basis underlying many of the adaptations during this transition remains unclear. Mud-dwelling gobies in the subfamily Amblyopinae are one of the teleosts lineages that show terrestriality and provide a useful system for clarifying the genetic changes underlying adaptations to terrestrial life. Here, we sequenced the mitogenome of six species in the subfamily Amblyopinae. Our results revealed a paraphyletic origin of Amblyopinae with respect to Oxudercinae, which are the most terrestrial fishes and lead an amphibious life in mudflats. This partly explains the terrestriality of Amblyopinae. We also detected unique tandemly repeated sequences in the mitochondrial control region in Amblyopinae, as well as in Oxudercinae, which mitigate oxidative DNA damage stemming from terrestrial environmental stress. Several genes, such as  The water-to-land transition is one of the most important events in evolutionary history, as it led to an explosive radiation of tetrapods, which is the most successful group of vertebrates on land . PreviouOdontamblyopus and Taenioides) are so well adapted to terrestrial environments that they possess unique abilities to breathe air via their richly vascularized inner epithelia in the buccal-opercular cavity to cope with the hypoxia conditions in their tidal mudflat habitat [Eel gobies  are the largest group of marine fishes showing unique adaptations to life in terrestrial habitats ,7,8,9. T habitat ,8. There habitat ,10,11, fMitochondrial energy metabolism plays an important role in mediating adaptation to the terrestrial environment by aquatic animals through aerobic respiration . The adeHere, we sequenced the complete mitogenome sequences of six Amblyopinae species and compared them with those of their closest terrestrial and non-terrestrial relatives to clarify the molecular basis underlying adaptation to terrestrial habitats in bony fishes during the early water-to-land transition. Given that signatures of adaptive evolution have been observed in the mitochondrial genes of other terrestrial marine animals ,15, we hAla, Asn, Cys, Gln, Glu, Pro, Ser, and Tyr) genes are encoded on the light strand. The arrangement of genes is similar to that observed in the mitogenomes of other goby species. We characterized the main features of the mitogenomes in Amblyopinae using these six new mitogenomes, as well as six other mitogenomes deposited in Genbank. A noteworthy feature of the Amblyopinae mitogenomes is the 50–53 bp putative origin of L-strand replication (OL) located between tRNA-Asn and tRNA-Cys in a cluster of five tRNA genes , which is known as the WANCY region [Odontamblyopus, in which the sequences start more than 140 bp behind in the mitogenome (started with -AAAGATTT)  , possiblAmblyotrypauchen, Paratrypauchen, Ctenotrypauchen, Trypauchen, and Taenioides were more closely related to non-Amblyopinae species in the subfamily Oxudercinae than to species in the genus Odontamblyopus comprising their sister clade. The observation that, in both trees, species in genera Amblyotrypauchen, Paratrypauchen, Ctenotrypauchen, Trypauchen, and Taenioides are clustered with non-Amblyopinae species rather than Amblyopinae species in the genus Odontamblyopus provides strong support for the paraphyletic origins of Amblyopinae. This suggests that these two subfamilies should be merged and reveals an expansion of phenotypic variation within the “terrestrial goby” clade, as has been suggested by Steppan [Periophthalmus appear to have evolved slightly earlier (34.5 My vs. 29.1 My) than the rest of the species, which indicates that they comprise a basal lineage in terrestrial gobies. We constructed the phylogeny of Amblyopinae using concatenated sequences of 13 coding sequences (CDSs) using our six newly sequenced mitogenomes and 57 published mitogenomes sequences from Gobioidei. The mitogenomic phylogenetic analyses yielded trees with consistent topologies and with high levels of support based on both ML and BI inference methods . Both th Steppan . Using fCOI, COIII, ND1, ND2, ND4, ND5, and ND6) of the clade had significantly (p < 0.05) higher ω values than other Gobioidei species, which indicates that these genes have been positively selected in terrestrial gobies (COIII (G162S), ND2 , ND4 , and ND6 (Y78F) were inferred as positively selected sites in the terrestrial goby branch with posterior probabilities greater than 95%  of TRS with some Oxudercinae species, rather than with the genus Odontamblyopus, which again supports a paraphyletic origin of Amblyopinae  estimated for the lineage appears to support the latter, more detailed analyses are needed to clarify the evolutionary scenarios in these paraphyletic clades. To detect the signs of adaptive evolution acting on OXPHOS genes, we first constructed the phylogenetic trees of Amblyopinae based on the ML and BI inference methods. The result yielded trees with consistent topologies, and all branches were highly supported. In contrast to the monophyly inferred from traditional taxonomy of subfamily Amblyopinae, both the ML and BI trees strongly supported the paraphyletic origins of Amblyopinae with respect to Oxudercinae, which is consistent with the results obtained from several recent studies using both nuclear and mitochondrial DNA ,28,29. Alyopinae . These fCOI, COIII, ND1, ND2, ND4, ND5, and ND6) in these paraphyletic clades had significantly higher ω values (p < 0.05) than other Gobioidei species, suggesting a signal for positive selection  with posterior probabilities greater than 95%, further suggesting that the positive selection has acted on these genes in this paraphyletic clade  in the mitochondrial electron transport chain ,31, and ronments ,32,33. Wronments ,35 and tronments . Howeverronments  and Zhu ronments  revealeddal zone , which s shrimps  and high shrimps . Therefo shrimps . Several shrimps ,41. Thisl gobies . These cl gobies . SignifiIn conclusion, our results provide new insights into the paraphyletic origin of Amblyopinae with respect to Oxudercinae, and suggest that these two subfamilies should be merged. New elements of TRS and episodes of positive selection in the mitogenome have occurred in the branches of the paraphyletic clades (Amblyopinae and Oxudercinae), indicating that they might play a role in adaptation to terrestrial habitats. The increased demand for energy and the need to cope with stresses associated with mudflat habitats might have induced physiological changes in these paraphyletic clades and triggered functional adaptations at the mitochondrial level. More in-depth analyses can take into account how these observed TRS and adaptively selected codons affect protein function and enhance oxygen utilization during the OXPHOS process. New genomic information including more mitochondrial and nuclear sequences is necessary for this clade in the future, which will likely reveal even more genes involved in the metabolic and structural processes essential for the colonization of the terrestrial realm.Amblyotrypauchen arctocephalus [Ctenotrypauchen chinensis [Paratrypauchen microcephalus [Taenioides gracilis [T. anguillaris [T. sp. Thailand [2, 0.2 mM dNTPs, 0.2 mM of each primer, and 4.0 U Taq DNA polymerase  using a PTC-200  PCR machine. The standard PCR conditions were as follows: 5 min initial denaturation at 94 °C, 40 cycles of 1 min at 94 °C for denaturation, 1 min at 45.7–54.1 °C for annealing, 1 min at 72 °C for extension, and a final extension at 72 °C for 5 min. All sets of PCR included a negative control reaction in which all reagents were included except for the template DNA. PCR products were sequenced using Sanger sequencing at Invitrogen Ltd., Shanghai, China. We sequenced the mitochondrial genomes of six species in four genera of the subfamily Amblyopinae: cephalus  . The complete mitogenome was annotated using Sequin software (version 15.10), which is a mitogenome toolkit . The bouhttps://www.ncbi.nlm.nih.gov/genbank/, accessed on 22 June 2022) for phylogenetic analysis , and Clupeiformes (Alosa sapidissima) were used as the outgroups.Fifty-seven Gobioidei mitogenomes were downloaded from GenBank  as foreground, and the other non-terrestrial gobies within the same family of Gobionellidae as the background. Given that positive selection may act in very short episodes during the evolution of a protein and affect only a few sites along a few lineages. We also used a branch-site model to identify sites under positive selection along lineages of interest. We compared model A  against the null model . Positive selected sites with a posterior ratio greater than 95% were determined using Bayes Empirical Bayes analysis in CODEML [The nonsynonymous to synonymous ratio ω (dn/ds) indicates changes in selection pressure at the protein level. A dn/ds ratio of 1, <1, or >1 in PCGs typically indicates neutral mutations, negative (purifying) selection, and positive selection, respectively . The CODn CODEML .http://web.expasy.org/protparam/, accessed on 24 July 2022) to identify significant physicochemical changes induced by the putative selection. We also constructed the crystal structure of positively selected genes and mapped selected sites onto this structure. The automated mode program  in the SWISS-MODEL server was used to predict and visualize the 3D structures of the proteins using the optimal templates.To gain insight into the functional significance of the putatively selected sites, genes identified as positively selected in the branch-site test were analyzed in Expasy (


Recently, research has focused on the modulation of the gut microbiota because of its central role in several digestive physiological functions and its involvement in the onset of not only gastrointestinal but also systemic diseases. Supplementing rabbit diets with nutraceutical substances could be a strategy to prevent dysbiosis, strengthen the immune system, and reduce mortality during the critical weaning period. Bovine colostrum (BC) is a by-product of the dairy industry and is very rich in compounds with several biological activities. Its use as an intestinal microbiota modulator in rabbits has never been investigated. This study evaluates the effects of diet supplementation with two different percentages of BC (2.5 and 5%) on luminal and mucosa-associated microbiota and its metabolism-associated pathways in the jejunum, caecum, and colon of rabbits. Although our results showed no effect of BC on microbiota biodiversity, there were significant differences between experimental groups in the microbial composition, mainly at the level of sub-dominant components depending on the dose of supplementation. The metabolism-associated pathways have also been affected, and particularly interesting are the results on the amino acids and lactose metabolism. Overall, findings suggest that BC could be used as a supplement in rabbit feed, although its effects on productive and reproductive performances, intestinal disease resistance, and economic aspects need to be further evaluated.n = 7) receiving a commercial feed (CTRL group) and the same diet supplemented with 2.5% and 5% BC , from 35 (weaning) to 90 days of age (slaughtering). At slaughter, the digestive tract was removed from each animal, then both content and mucosa-associated microbiota of jejunum, caecum, and colon were collected and analysed by Next Generation 16SrRNA Gene Sequencing. Significant differences were found in the microbial composition of the three groups , especially in the caecum and colon of the 2.5% BC group. The relative abundance analysis showed that the families most affected by the BC administration were Clostridia UCG-014, Barnesiellaceae, and Eggerthellaceae. A trend was also found for Lachnospiraceae, Akkermansiaceae, and Bacteroidaceae. A functional prediction has revealed several altered pathways in BC groups, with particular reference to amino acids and lactose metabolism. Firmicutes:Bacteroidetes ratio decreased in caecum luminal samples of the 2.5% BC group. These findings suggest that BC supplementation could positively affect the intestinal microbiota. However, further research is needed to establish the optimal administration dose.BC is a nutraceutical that can modulate intestinal microbiota. This study investigates the effects of BC diet supplementation on luminal and mucosa-associated microbiota in the jejunum, caecum, and colon of rabbits. Twenty-one New Zealand White female rabbits were divided into three experimental groups ( Rabbits are herbivores with a digestive system anatomically and physiologically evolved to obtain nutrients from low-calories and fibre-rich food thanks to the intense fermentative activity in the large intestine, particularly in the caecum ,2. A eubNatural substances integrated into the diet can favour the development of beneficial gut microbial flora and, therefore, adequately stimulate the immune system not only in human beings but also in animals. In rabbits, this could contribute to the reduction of the use of antibiotics and the improvement in animal welfare as well as the profitability of the farm . MoreoveAmong the natural substances which can be supplemented to the feed, bovine colostrum (BC) has recently drawn the attention of the scientific community. Colostrum is the secretion produced by the mammary glands immediately after delivery , which iSacculus rotundus level [With regards to rabbits, the effects of BC supplementation have been assessed on meat quality  and diarus level .In this study, we hypothesized that BC-enriched diets in rabbits can influence gut bacterial richness, diversity, and functional potential and that these modifications are dose-dependent. The purpose of this experimental trial was to discern the luminal and mucosal microbiota composition, as well as their metabolism-associated pathways, in different digestive tract sections  of rabbits fed with diets supplemented with two percentages of BC. The experimental trial was conducted in the facilities of the Department of Agricultural, Food and Environmental Sciences of the University of Perugia, Italia. In accordance with the European and Italian laws (EU Directive 2010/63 and Decreto Legislativo 26/2014) regarding the protection of animals used for scientific purposes, the rabbits were maintained under the supervision of a responsible veterinarian, and the experimental protocol was approved by the Ethical Committee of the Department of Veterinary Medicine of the University of Milano with the code OPBA_42_2021. All efforts were made to minimize animal discomfort and to reduce the number of experimental animals.n = 7 animals, CTRL) was fed with a commercial feed including the following ingredients: dehydrated alfalfa meal (43.0%), wheat bran (30.0%), barley (9.5%), sunflower meal (4.6%), rice bran (4.0%), soybean meal (4.0%), calcium carbonate (2.2%), cane molasses (2.0%), soybean oil (0.4%), and salt (0.3%). The other two groups were fed with the same diet, to which, before pelleting, was added lyophilized BC at the rate of 2.5%  and 5% . The lyophilized BC was derived from high-quality colostrum from multiparous dairy cows. The basic composition and IgG concentration were evaluated before lyophilisation: total solids 22.1%, fat 4.5%, total protein 14.2%, lactose 2.9%, and IgG 3.3 g/100 mL. According to dietary treatment, 21 New Zealand White female rabbits were randomly assigned to three groups from 35 days (weaning) until 90 days of age (slaughtering). The control group  in order to collect the mucosa-attached bacteria for mucosa-associated microbiota determination. The achieved samples were stored in sterile 1.5 mL tubes at −80 °C until analysis.Rabbits were slaughtered following the EU Regulations currently in force in an authorized slaughterhouse. The animals were first stunned by mechanical stunning and then slaughtered by jugulation. The digestive tract was removed from each animal, and the content of the different intestinal tract sections  was collected. Specifically, regarding the colon, the anatomical region sampled was the proximal part between the The bacterial DNA was extracted from each sample of intestinal contents by using the commercial QIAamp PowerFecal Pro DNA Kit , as already described . The NanBacterial DNA was amplified and sequenced as described in Cremonesi et al., 2022 . BrieflyRaw reads from each sample were subjected to a preliminary filtering pipeline that comprised the merging of the two paired reads coming from the same fragment in one single sequence by PandaSeq  and the Functional predictions from the 16S rRNA-derived microbial profiles were estimated by applying PICRUSt2  on the t-test , with 999 random permutations, was employed in order to assess whether the samples belonging to one experimental class were more or less diverse than those of a different class.Biodiversity analysis  was performed using several metrics . A non-parametric permutation-based Microbial profile analysis  was based on unweighted and weighted UniFrac distances  and reprComposition analysis in terms of taxa relative abundances was performed by grouping the classified zOTUs to different taxonomic levels . A Kruskal–Wallis test was employed to assess whether there was a significant difference among levels of the experimental categories on the relative abundance of the taxa, on the predicted pathway abundances, and on Firmicutes:Bacteroidetes ratio evaluation; Dunn’s post hoc pairwise test was employed if necessary. All statistical analyses and plots were performed using Matlab .https://www.ncbi.nlm.nih.gov/bioproject/PRJNA915237, accessed on 5 January 2023).The data presented in this study are openly available in NCBI Short Read Archive (SRA) under experiment IDs SRR22879769-SRR22879893 . The analysis of the rarefactions curves for both the chao1 and the observed species metrics showed that the majority of the samples had a tendency toward reaching a plateau, thus suggesting that the depth of coverage was sufficient to describe the biological diversity within the samples . Sample 114  had a very low number of raw reads  and was therefore discarded. The average read depth per sample was 22,374 ± 9372. All the downstream analyses, then, were performed on 125 samples (excluding sample 114), and all samples were normalized to the least sequenced sample . Dubosiella, Akkermansia, Bacteroides, Methanobrevibacter, Ruminococcus, Marvinbryantia, and Alistipes, which, however, made up only 15.5% of the relative abundance on average. The major genera found in the samples were Jejunum lumen samples were characterized by a relatively high content of Firmicutes (~80%), Actinobacteriota (~7%), Euryarchaeota (~6%), and Patescibacteria (~2.5%), whereas Bacteroidota were very low (<0.5%); at the family level, these samples were rich in Eubacteriaceae (~58%), Erysipelotrichaceae (~12%), Methanobacteriaceae (~6%), and Saccharimonadaceae (~2.5%).Furthermore, the corresponding jejunum mucosa samples had about 35% of the overall abundance (on average) composed by “Unclassified” and “Bacteria” unclassified at lower levels; Firmicutes composed about 55% of the relative abundance, whereas Proteobacteria and Actinobacteriota accounted for about 4.5% and 2.5%, respectively. This was reflected also at the family level, where apart from Eubacteriaceae (accounting for ~40% of the relative abundance) and Erysipelotrichaceae (~4%), all the other families were scarcely present.The microbiota of the caecum lumen samples was mainly composed of Firmicutes (~65%), Bacteroidota (~19%), Actinobacteriota (~2.6%), and Verrucomicrobiota (~6.5%) at the phylum level, and by Eubacteriaceae (~25%) and members of Lachnospiraceae, Oscillospiraceae, Ruminococcaceae, Akkermansiaceae, Bacteroidaceae, Christensenellaceae, as well as Muribaculaceae at the family level, each accounting for 3.9%-9.4% of the relative abundance.Caecum mucosa samples were characterized by Firmicutes (~68%), Bacteroidota (~7%), and Verrucomicrobiota (~3%), with a higher abundance of unclassified bacteria (accounting for 7.7–18.0%), as previously observed in mucosa samples; at the family level, these samples were mainly constituted by Eubacteriaceae (~30%), Lachnospiraceae, Erysipelotrichaceae, Oscillospiraceae, and Ruminococcaceae, each in the range of 3.4–9.8% of the overall abundance.Colon lumen samples had about ~66% Firmicutes, ~19% Bacteroidota, ~6% Verrucomicrobiota, and ~2% Euryarchaeota, whereas, at the family level, they were composed of Lachnospiraceae (9%), Ruminococcaceae (6.5%), Oscillospiraceae (6.5%), Christensenellaceae (3.5%), Akkermansiaceae (6%), Muribaculaceae (5%), and Bacteroidaceae (6%).Finally, colon mucosa samples, besides ~50% Firmicutes, ~5% Proteobacteria, and ~3% Bacteroidota, had an additional ~35% composed of “Unclassified” bacteria; the abundance of Eubacteriaceae was the lowest (~20%), with a consistent presence of Lachnospiraceae (~8%), Oscillospiraceae (~6.5%), Ruminococcaceae (~3%), and Christensenellaceae (~2.3%).n = 21), jejunum mucosa samples had the lowest biodiversity , followed by the jejunum lumen samples; on the other hand, caecum and colon (both lumen and mucosa) had a higher diversity  for all comparisons, except for caecum and colon lumen samples, which were similar (n = 42 per group), with the biodiversity of jejunum samples lower than the caecum and colon samples for the PD whole tree  and weighted (p ≤ 0.02) UniFrac distances (versus the mucosa samples (n = 63 per group), with lumen samples having a higher biodiversity than mucosa samples   .n = 7), i.e., considering differences only in the diet supplementation, provided that the intestinal tract  and site (lumen and mucosa) were the same. As far as the alpha-diversity analysis, there was a non-significant difference among samples according to the BC supplementation. However, according to the chao1 metric, in samples with increasing percentage of BC, the biodiversity increased in the jejunum lumen and in caecum mucosa, whereas it decreased in jejunum mucosa and caecum lumen samples; no trend was observed for colon samples (both mucosa and lumen) , a comparison of the microbiota composition in rabbits was investigated. Results were computed stratifying samples in diet groups homogeneous for the intestinal tract and site sampled (d lumen) . The tenp ≤ 0.002 for all pairwise comparisons) in caecum and colon lumen samples. At the same time, in colon mucosa samples, significant differences were reported for CTRL vs. 5% BC and 2.5% BC vs. 5% BC, whereas no difference was observed between CTRL and 2.5% BC. No significant differences were reported for caecum mucosa and jejunum (both lumen and mucosa) samples (n = 42) did not show different biodiversity for any of the tested metrics, whereas the microbial profiles resulted somehow different for the unweighted UniFrac  , on unweighted UniFrac distance, of the three diet groups  resulted in significantly different ( samples . As a wh≤ 0.014) .Regarding the relative abundance analysis, due to the reduced number of samples per group, the significantly altered taxa were few, and some of them were at relatively low relative abundances. This is somehow expected, also considering the results from the beta-diversity analysis, for which the differences were observed for the unweighted UniFrac distance, suggesting modifications in the sub-dominant components of the microbiota. The following data only consider the taxa present at a relative abundance > 1% on average in at least one of the experimental groups, and the indication of an “increase” or a “reduction” is provided using the CTRL diet as a reference. The reported data are for the phylogenetic level of families since it is the lowest for which the majority of the taxa are properly defined . Jejunum lumen samples showed a significant increase of Clostridia in 2.5% BC and 5% BC groups, together with a tendency towards a reduction of Eubacteriaceae and Erysipelotrichaceae in the 2.5% BC group, as well as an increase of Oscillospiraceae and Ruminococcaceae in 2.5% BC group. Jejunum mucosa samples showed a tendency towards an increase of unclassified bacteria and Clostridiaceae in the 2.5% BC group, together with a tendency towards a reduction of Eubacteriaceae (in 2.5% BC) and Lachnospiraceae (in both 2.5% BC and 5% BC groups). Caecum lumen samples showed a significant increase of Erysipelotrichaceae (in 5% BC), Eggerthellaceae (in 2.5% BC and 5% BC), and family UCG-014 of Clostridia (in 2.5% BC and 5% BC), as well as a tendency towards an increase of Bacteroidaceae (in 5% BC); on the other hand, we found a significant decrease of Barnesiellaceae (in 2.5% BC) as well as of UCG-010 and vadinBB60 families of Clostridia (both in 5% BC). Caecum mucosa samples had a significant increase of Clostridia (in 2.5% BC) and a tendency towards the increase of Lachnospiraceae (in 2.5% BC), Akkermansiaceae (in 2.5% BC and 5% BC), Bacteroidaceae (in 2.5% BC), Barnesiellaceae (in 2.5% BC), and towards the reduction of Eubacteriaceae (in 5% BC) and unclassified bacteria (in 2.5% BC). Colon lumen samples were characterized by a significant increase of Eggerthellaceae (in 2.5% BC and 5% BC) and by a tendency towards an increase of Akkermansiaceae (in 5% BC), Bacteroidaceae (in 2.5% BC), and Rikenellaceae (in 5% BC); on the other hand, Ruminococcaeceae (in 2.5% BC) and Monoglobaceae (in 2.5% BC and 5% BC) were significantly reduced. Colon mucosa samples highlighted a tendency towards an increase of Eubacteriaceae (in 5% BC) and Campylobacteriaceae (in 2.5% BC and 5% BC) and a tendency towards a reduction of unclassified bacteria (in 5% BC) D. The coThe PICRUSt2-based functional prediction from the 16S rRNA microbial profiles of the samples revealed several bacterial metabolism-associated pathways significantly altered in the rabbits fed a BC-supplemented diet . The effIn the jejunum lumen, processes belonging to pathways of proteinogenic amino acid  biosynthesis and NAD synthesis were depleted in BC groups, whereas phylloquinone biosynthesis was enriched in 2.5% BC samples; no differences were observed for 5% BC samples. On the other hand, in jejunum mucosa samples, several level-4 pathways were significantly more abundant in 2.5% BC and 5% BC, such as the biosynthesis of peptidoglycans, NAD, geranylgeranyl pyrophosphate (GGPP) (cofactor-biosynthesis group), lactose, and diterpenoids as well as isoprenoids , both belonging to the terpenoid-biosynthesis group and the degradation of lysine (involving L-lysine fermentation to acetate and butanoate).In the caecum lumen, several altered pathways were reported for 2.5% BC samples and included the increase of histidine and lysine degradation and of sugar nucleotides and vitamin B6 biosynthesis ; at the same time, pathways related to nucleotide biosynthesis , and proteinogenic amino acid synthesis  were depleted. On the other hand, results for 5% BC samples were contrasting, with phenylalanine and tyrosine synthesis increased. In caecum mucosa samples, contrastingly, only one level-4 pathway was altered, i.e., histidine degradation, which resulted in more abundant samples from BC-supplemented rabbits than those fed the CTRL diet.Colon lumen samples were characterized by a significant increase in quinone biosynthesis, including DHNA, demethylmenaquinone , menaquinone (pathways of menaquinol 6–13), phylloquinone, vitamin B6 biosynthesis, and glutamate degradation in both 2.5% BC and 5% BC, whereas the biosynthesis of glycogen, acetylmuramoyl-pentapeptide, GGPP, purine nucleotides salvage, and isoprenoids was depleted. Finally, colon mucosa samples showed a significant increase in histidine and lysine degradation, and many level-4 pathways were depleted in BC-supplemented samples. The latter included biosynthesis of glycogen, cofactor-biosynthesis members , unsaturated fatty acids biosynthesis -dodec-5-enoate biosynthesis), phospholipid biosynthesis , purine nucleotides salvage , and isoprenoids, together with a reduction of glycogen degradation. Notably, two pathways, biotin and stearate biosynthesis, showed a discordant behaviour in BC-supplemented samples, being increased in 2.5% BC and decreased in 5% BC compared to the CTRL diet.p = 0.0112, Dunn pairwise test). On the other hand, regardless of being from the colon or mucosa, jejunum samples had a notably higher ratio   ratio was found to be very different in the mucosa and luminal samples (in both caecum and colon), with the former characterized by a higher F:B . The F:B ratio was found to be significantly reduced in caecum lumen samples of the 2.5% BC group ( groups) .This is the first study investigating the effects of BC diet supplementation on the intestinal microbial community and its metabolism-associated pathways in rabbits. Moreover, for the first time, the differences among luminal and mucosa-associated microbiota in the jejunum, caecum, and colon of rabbits have also been investigated.Akkermansia and Ruminococcus agree with other investigations [First of all, the taxonomic composition of gut microbiota independently from the experimental group was investigated. In jejunum lumen samples, about 80% of the microbial population belonged to the Firmicutes phylum. The most represented family was Eubacteriaceae, followed by Erysipelotrichaceae. This family is involved in lipid metabolism , confirmigations ,41. The Regardless of the experimental group, the alpha-diversity showed higher values in the caecum and colon compared to the jejunum. This was a foreseeable result, considering that the rabbit is a hindgut fermenter, and the biggest microbial population lives in the caecum and colon ,9,40. AsThe analysis of the beta-diversity indicated that a different microbial composition characterizes the different tracts of the rabbit intestine, except for colon and caecum luminal samples, which showed similar results. This is probably due to the similar physiological fermentative function of these two tracts, which, therefore, have a homogenous microbial population . InteresIn this study, the effect of the BC diet supplementation on the intestinal rabbit microbiota biodiversity and microbial composition has been evaluated for the first time. The alpha-diversity index indicated several differences due to the BC administration, although only as a trend toward statistical significance. Findings suggested a dose-dependent effect of BC on the microbiota’s biodiversity of jejunum and caecum samples, but the direction of these changes  was different between lumen and mucosa. In particular, the microbiota’s biodiversity in the caecum mucosa tended to increase as the dose of BC supplementation increased. Similar results were found after BC administration on mice’s gut microbiota, mainly due to an increase in short-chain fatty acids-producing microorganisms . In the The relative abundance analysis showed that the greater modulation by BC mainly interested the sub-dominant components of the microbiota. Few but interesting differences were, however, found for the phyla. Specifically, Firmicutes decreased in the colon luminal samples of both BC groups, probably as a result of the reduced fibre content of BC diets. Campylobacter jejuni-infected turkeys [At the family level, the most important taxa showing significant differences among groups were Clostridia UCG-014, Barnesiellaceae, and Ruminococcaceae. Clostridia UCG-014 increased in the caecum luminal samples of BC groups. This family is positively considered as one of the main bacteria involved in the production of tryptophan metabolites, which in turn are pivotal for the regulation of gastrointestinal homeostasis in humans ,60. They turkeys . Barnesi turkeys  and in w turkeys . Convers turkeys ,9,40. ThOther minor families were modulated by the BC administration. Specifically, Eggerthellaceae, a polyphenol-degradating family specifically correlated to lipid metabolism, has increased its prevalence in both BC groups. This could be due to a higher lipid content of the BC-supplemented diets compared to the control group. Its increment has been previously linked to weight loss and intestinal histomorphology restoration in obese mice . ConsideIn this study, the gut microbiota metabolism-associated pathways were also investigated to better understand the influence that BC supplementation could have on the functional potential of the microbiota. Genes of microorganisms encode for several enzymes, which are involved in protein, lipid, carbohydrate, and nucleotide metabolism. For this reason, microbiota alterations, also due to diet modifications, are linked to gut microbiota metabolic activity variations. In our study, biosynthesis of several pathways, such as that of cofactors , diterpenoids, and sugar nucleotides, was generally increased after BC supplementation. These modifications could be related to the increment of some bacterial populations, such as Eggerthellaceae, which are involved in the synthesis of some of these cofactors . On the Finally, the Firmicutes:Bacteroidetes ratio (F:B), an important index for the evaluation of the eubiosis, has also been evaluated. Its alterations have been linked to the presence of inflammatory and metabolic diseases in humans, being a good indicator of microbiota homeostasis . SpecifiFurther research will be needed to better investigate the effects of the BC diet supplementation on the histological structure of the rabbit intestine, the immune response, metabolic diseases, and the animals’ productive and reproductive performances. Furthermore, it should be noted that functional predictions and the related pathways analyses performed in this study cannot substitute whole shotgun metagenomics in the evaluation of the actual functions and pathways altered by the BC-supplementation in the diet. As a matter of fact, differential abundance testing results are known to vary between shotgun metagenomics data and amplicon-based metagenome predictions based on the same samples, especially for community-wide pathway predictions ,73. MoreThe results of this investigation have evidenced that dietary supplementation with BC could modulate the gut microbiota and its metabolic-associated pathways in fattening rabbits. Although microbial diversity was not strongly modified, the 2.5% BC supplementation changed the phylogenetic microbial composition, especially in the caecum and colon. Clostridia UCG-014, Barnesiellaceae, and Eggerthellaceae were the families most affected in their prevalence by the dietary treatment and altogether suggest a positive microbiota modulation exerted by the BC administration, even if the consequences of these changes should be better investigated. Further research is needed to clarify the BC dose to be added to feed and the other effects that BC inclusion could have on the rabbit besides intestinal microbial composition changes.


Development of endovascular interventional techniques gradually replaced traditional open surgery and has become the preferred treatment for renal aneurysms. This study aimed to analyze the clinical characteristics of renal artery aneurysm (RAA) and the safety and efficacy of intravascular interventional treatment.We retrospectively analyzed the clinical characteristics and imaging data of 23 aneurysms in 18 patients with RAA. The technical success rate, complication rate, mortality rate, reintervention rate, and use of embolization materials were evaluated.In 18 patients with RAA , a total of 23 aneurysms were found . Among them, 11 cases (61.1%) were discovered accidentally, and the remaining patients were diagnosed due to the following major complaints: four cases (22.2%) presented low back pain, two (11.1%) were due to high blood pressure, and one (5.5%) had low back pain with gross hematuria. A total of 14 aneurysms in 13 patients received endovascular interventional therapy. The technical success rate of 13 patients with renal aneurysms was 100%. Three of the 18 patients were lost to follow-up, and the remaining were followed up for 4–89 months. There was no recurrence of the aneurysm or displacement of the stent or coil.Endovascular treatment for RAA has a high success rate, low complication rate, and low reintervention rate. It has the advantage of less trauma and is flexible and more targeted for different types of renal aneurysms. Aneurysm-like lesions of renal blood vessels are abnormal expansions of the vascular lumen, which can be divided into true aneurysms and pseudoaneurysms according to the integrity of the vessel wall. True aneurysms retain all three layers of the vascular wall , while pseudoaneurysms cause the continuity of the artery wall to be interrupted by various factors and blood spills into the surrounding tissues to form a hematoma . This stRenal artery aneurysms (RAAs) are rare, accounting for 1% of all aneurysms and 15%–22% of visceral aneurysms , 4. MassWith the development of vascular and interventional radiology, interventional therapy has become less invasive, with fewer complications, and has become the first choice for the clinical treatment of RAA. This study included 18 patients with true renal aneurysms. This study aimed to retrospectively analyze clinical characteristics and imaging data, evaluate the safety and efficacy of endovascular interventional therapy, and share the clinical experience with the readers.We searched for data on aneurysms using the keyword “renal artery aneurysms” in the imaging radiology system from July 2014 to mid-June 2021. According to pathogenesis, clinical, and morphological criteria, a total of 18 patients with true renal aneurysms, including 23 aneurysms, were enrolled after excluding pseudo-renal aneurysms. I In order to ensure more complete and rich data, the selected patients with aneurysms were reviewed and not eliminated again: the included patients with renal aneurysms included patients with indications for treatment after evaluation by experts. If the complex renal aneurysms were not suitable for endovascular interventional treatment, they should be referred to surgical treatment. According to medical records, sex, age, comorbidities, clinical manifestations, number and location of aneurysms, treatment measures and materials, and prognosis of the patients were recorded. We recorded the incidence of complications, mortality, and reintervention rate after endovascular treatment.(1) Stent implantation: A guiding catheter is introduced. After the renal aneurysm was confirmed by angiography, a microguide wire was introduced into the distal end of the renal artery branch through the main trunk, and a self-expanding stent system  was introduced to completely cover the neck of the aneurysm. The stent was then released, and an angiographic review was performed. After observing the condition of the aneurysm, a balloon was used to fully expand the stent when necessary.(2) Coil embolization: The 5F Cobra catheter was inserted into the neck of the aneurysm, and coils  were introduced into the aneurysm for embolization until the aneurysm was densely packed and there was no obvious flowing blood flow in the aneurysm lumen. Angiography confirmed that the parent artery was unobstructed, the aneurysm lumen disappeared, and the distal branch of the renal artery was not lost, indicating a successful operation .(3) Parent artery embolization: After selecting the cannula of the parent artery and angiography to further clarify the relationship between the renal aneurysm and the parent artery, a microcatheter and microguide wire were introduced to superselect the neck of the aneurysm. Simple coil embolization or liquid embolic agents N-butyl-2-cyanoacrylate (NBCA)  or Onyx  Stent-assisted coiling embolization: A guiding catheter is introduced. After the renal aneurysm was confirmed by angiography, the microcatheter was introduced into the aneurysm lumen, the micro-guidewire was passed through the aneurysm body to the distal end of the renal artery branch, and the bare stent was introduced along the guidewire and positioned to the opening of the aneurysm. The coil was then inserted into the aneurysm body through a microcatheter to embolize the aneurysm.(5) Liquid embolic agent embolization: The use of liquid embolic agents by experienced operators is a safe and effective method. As with other embolic materials, embolization also requires embolization of the distal and proximal ends of the parent artery. To reduce collateral ischemic events, it was recommended to use a balloon to temporarily seal the aneurysm neck . Use antUnder local anesthesia, guided by a DSA , the Seldinger technique was used to puncture the right or left femoral artery and insert a 5F Cobra catheter  for abdominal aortic angiography and bilateral renal arteriography. To determine the size, location, size of aneurysm neck, number of renal aneurysms, and their relationship with parent artery, whether branched blood vessels, vascular malformations, vascular stenosis, and whether they are consistent with preoperative computed tomography angiography (CTA). Combined with DSA results, renal aneurysms were classified according to Rundback et al. : type I,In 18 patients with RAA , a total of 23 aneurysms were found  in which 11 (61.1%) were asymptomatic, which was accidentally discovered. The remaining patients had the following major complaints: four cases (22.2%) were diagnosed with low back pain, and one case (5.5%) showed low back pain with gross hematuria. The most common complication was hypertension, with nine cases (50%). Dyslipidemia was observed in four cases (22.2%). Three patients (16.7%) had a history of tobacco use. One patient (5.5%) had a splenic aneurysm. One patient (5.5%) had bilateral duplication of renal malformations and left adrenal pheochromocytoma. There were 15 aneurysms on the left side (63.6%) and eight on the right side (36.4%), of which one was bilateral with three aneurysms, three were unilateral with two aneurysms, and the rest were unilateral with single aneurysms . Among tFive patients had not undergone endovascular interventional treatment for the following reasons. Case 5 was a 62-year-old male with lower back pain. DSA suggested an aneurysm in the right renal parenchyma with a diameter of approximately 1 cm. Since the aneurysm was not large in diameter, embolization may have caused a partial loss of renal function. Embolization was not performed after consultation with the patient. After 56 months, the aneurysm had not ruptured or enlarged. Case 7 was a 32-year-old male, an aneurysm was found due to increased blood pressure. The DSA showed an aneurysm  at the distal end of the right renal artery main trunk, accompanied by localized stenosis of approximately 90% of the right upper pole accessory renal artery. An increase in blood pressure is considered due to stenosis of the accessory renal artery. The aneurysm was not large in diameter; therefore, no intervention was performed. Case 8 was a 48-year-old female with left renal artery main and inferior posterior branch aneurysms, which were accidentally discovered during CT. The diameters were 3.8 and 2.5 cm, respectively. DSA showed difficulty in endovascular treatment; therefore, a surgical intervention was recommended. The follow-up patient underwent a kidney transplant at the outer hospital. Case 11 was an asymptomatic 56-year-old female; DSA revealed an aneurysm at the end of the right renal artery , involving multiple graded arteries. Therefore, surgical intervention was recommended. Case 12 was a 42-year-old male with elevated blood pressure. CT showed three bilateral aneurysms, of which two were on the left . As multiple branches were involved, surgical intervention was recommended. Case 18 was a 46-year-old female with two unilateral aneurysms, which were accidentally found, of which the small size was approximately 0.5 cm and was not treated.The remaining 13 patients with 14 aneurysms  were treated endovascularly. Three aneurysms were treated with simple coil embolization, two with coils plus liquid embolic agent to embolize the aneurysm lumen, four underwent embolization of the parent artery embolization, one implanted with a bare stent, and two implanted ball-expanded stents, two used stent-assisted coil embolization. The technical success rate of the 13 patients with renal aneurysms was 100% and four (30.7%) had minor complications  after treatment. No serious complications or deaths occurred during treatment. After treatment, symptoms of low back pain were relieved in four patients, and blood pressure decreased in two cases. Four out of 18 patients were lost to follow-up, and the remaining patients were followed up for 4–89 months. There was no recurrence of the aneurysm or displacement of the stent or coil. Further details are provided in Case 1 was a 47-year-old female with a major complaint of “repeated left back pain” for 1 month. Before the operation, CT showed an aneurysm of the proximal left renal artery posterior segment, approximately 1.4 cm in diameter . DSA shoCase 12 was a 42-year-old male who presented elevated blood pressure. Preoperative CT indicated two tumor-like expansions in the main left renal artery, the larger one involved the proximal segment of each branch, and the widest part was approximately 2.5 cm. And CT also indicated limited expansion of the main right renal artery approximately 0.6 cm wide . DSA shoCase 10 was a 41-year-old male with a sore left waist for half a month. Preoperative CT showed that the central part of the left renal artery was enlarged in a tumor-like shape, with a size of approximately 1.0 × 1.4 cm, with a vaguely visible intima. The proximal and distal blood vessels of the left renal artery were irregularly narrowed; the narrowest part was approximately 80% . It is cCase 6 was a 64-year-old female with an abnormal vascular cluster in the left renal parenchyma that was accidentally detected during CT examination. The size is approximately 4.9 cm × 4.5 cm × 4.9 cm. Considering vascular malformations, arteriovenous malformation (AVM) is more likely . DSA shoCase 3 was a 40-year-old female with a mass in the right kidney on physical examination. Preoperative CT examination revealed that the right kidney was enlarged, showing masses and earthworm-shaped blood vessel masses . DSA shoCase 13 was a 63-year-old man. A left renal aneurysm was found during CT examination due to a left pheochromocytoma. The maximum inner diameter was approximately 12.7 mm, the opening was approximately 2.5 mm wide, and the vessel wall showed shell-like calcification . DSA shoRenal aneurysms (RAAs) are rare clinical entities, and there are few reports on their natural course. Henriksson et al.  reportedRAA can be caused by many factors, such as congenital disease, dilatation after stenosis, arteritis, and infection, among which the most common is atherosclerosis and fibromuscular dysplasia (FMD) , 13. HenAneurysms can be diagnosed and evaluated using various imaging methods, each with its advantages. DSA is an invasive test, but it can not only determine the location, size, and number of aneurysms, but also provide opportunities for interventional therapy. It is also the gold standard for diagnosing renal aneurysms (PMID: 24363127). CTA is the first choice for diagnosing RAA. It displays the main renal artery and branches of grades 1–3 in a three-dimensional manner and obtains all information on the renal artery and renal vein . HoweverPregnancy is believed to be associated with renal aneurysm rupture and mortality. The important reasons for the increase and rupture of aneurysms during pregnancy are mainly due to increased blood volume, increased intra-abdominal pressure, changes in hormone metabolism, and hyperdynamic status. Most studies have shown that most RAA ruptures occur in the third trimester –20. PrevSolitary kidneys with RAAs are clinically rare. As patients with isolated kidneys have poor renal compensatory ability, once pathological changes occur, renal function should be protected as much as possible during treatment to avoid renal parenchymal damage. In all surgical procedures, up to 5% of cases have undergone unplanned nephrectomy; therefore, endovascular techniques should be considered the preferred technique for isolated cases of renal aneurysm .The main purpose of RAA treatment is to prevent rupture of renal aneurysms, relieve secondary hypertension, and protect kidney function. However, the indications for RAA treatment remain controversial. Most surgeons generally believe that if the RAA is not ruptured, preventive surgical intervention is required if the following conditions exist: (1) large size (>2 cm), (2) symptoms, 3) refractory hypertension with significant renal artery stenosis or thromboembolism, 4) childbearing age (for women)  . The steFor patients with renal aneurysms undergoing treatment, it is recommended to complete a CTA, MRA, or DSA imaging examination before discharge , 33. TheIn summary, endovascular interventional treatment of renal aneurysms has the advantages of a high technical success rate, fewer complications, less trauma, a shorter hospital stay, and a lower reintervention rate. Its safety and efficacy are worth recognizing, and it has become the first choice of treatment for renal aneurysms. As technology matures, an increasing number of complex renal aneurysms can undergo endovascular interventional therapy. Long-term follow-up is warranted to rule out the possibility of aneurysm reperfusion.


T). By examining the extrapolated conductivity at the absolute zero temperature (T = 0), denoted as Our investigation focuses on the analysis of the conductive properties of high-mobility 2D-Si-MOSFETs as they approach the critical carrier density,  The four-terminal AC technique was employed, wherein lock-in amplifiers detected signals within the frequency range of 13 to 17 Hz.A two-dimensional 2D) percolation-type metal insulator transition (MIT) has been a topic of significant research interest in recent years. Several studies have explored the behavior of conductivity and the transition from a conducting state to an insulating state in 2D systems ,2,3. OneD percolaIn perature .In the present work, our investigation focuses on the behavior of the electrical conductivity T = 0) and To illustrate this linear The conductivity appears to be independent of temperatures below 2.1 K. The percolation model adopted is only valid for densities very close to the critical density . This aberration is incomprehensible and unexpected. We suspect the appearance of quantum phenomena at very low temperatures. That is why we only consider linear points.In In  studies ,26 have In previous works ,26, the The value of llations , where rr radius ,29.Theoretical investigations have also explored the linear relationship between eference , the autIn addition to this model of screening, the microemulsion model proposed in Refs. ,30 offerσ/dT~ns2 , which wσ/dT~ns2  demonstrNext, let us focus on the MIT observed in the Si-15 sample. The conductivity of the 2DES in this case can be described using the percolation model. According to this model, the conductivity follows a scaling function represented as ferences ,33. FiguIn previous studies ,34,35,36T* according to which the expression of the electrical resistivity can be represented by either one of the expressions mentioned below: (*T depends on carrier density We also showed a temperature The percolation transition occurs due to the existence of a disorder potential in the 2D gas, which possesses a characteristic length greater than the average interparticle distance. In high-density regimes, where the surface density Several authors have proposed phenomenological percolation models to describe the metal insulator transition. Meir’s non-interacting model  describeThe percolation model proposed by He and Xie describes a system consisting of a conductive liquid phase separated by regions of an insulating vapor phase . In the Shi et al.  proposedIt is worth noting that the model developed by Spivak  to interCMOS technology has been pivotal in revolutionizing the semiconductor industry and driving the digital revolution. However, with CMOS devices approaching their physical limits, researchers have focused on advancing CMOS technology to overcome challenges and expand its boundaries ,43,44,45Advancements in abridging CMOS technology have resulted in remarkable breakthroughs, addressing limitations and paving the way for future innovations. By exploring novel transistor architectures, leveraging advanced materials, exploring alternative computing paradigms, and embracing system-level integration, researchers are pushing CMOS technology boundaries. These advancements hold immense potential for achieving higher performance, improved energy efficiency, enhanced reliability, and unprecedented computational capabilities. Staying informed about the advancement enables researchers and industry professionals to leverage these advancements, driving further progress, and shaping the future of CMOS technology.In summary, the Si-MOSFETs’ sample exhibits a significant electron mobility near the MIT in a 2D system. The conductivity shows a linear variation with the temperature for behavior .