Tuning plasmid DNA amounts for cost-effective transfections of mammalian cells: when less is more.

Transient gene expression (TGE) in mammalian cells is a well-known approach to the fast expression of recombinant proteins. The human cell line HEK (human embryonic kidney) 293F is widely used in this field, due to its adaptability to grow in suspension to high cell densities in serum-free media, amenability to transfection, and production of recombinant proteins in satisfactory quantities for functional and structural analysis. Amounts of plasmid DNA (pDNA) required in transfections for TGE remain high (usually 1 µg pDNA/mL, or even higher), representing a noticeable proportion of the overall cost. Thus, there is an economic need to reduce amounts of coding pDNA in TGE processes. In this work, amounts of both pDNA and transfecting agent used for TGE in HEK 293F cells have been explored in order to reduce them without compromising (or even improving) the productivity of the process in terms of protein yield. In our hands, minimal polyethyleneimine (PEI) cytotoxicity and optimum protein yields were obtained when transfecting at 0.5 µg pDNA/mL (equal to 0.5 µg pDNA/million cells) and a DNA-to-PEI ratio of 1:3, a trend confirmed for several unrelated recombinant proteins. Thus, carefully tuning pDNA and transfecting agent amounts not only reduces the economic costs but also results in higher recombinant protein yields. These results surely have a direct application and interest for the biopharmaceutical industry, always concerned in increasing productivity while decreasing economic costs. KEY POINTS: • Mammalian cells are widely used to produce recombinant proteins in short times. • Tuning DNA and transfecting agent are of great interest to optimize economic costs. • Reducing DNA and transfecting agent amounts result in higher protein yields.

Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol.

Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the "major vault protein" (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of "ready-to-use" loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs.

QSTR Modeling to Find Relevant DFT Descriptors Related to the Toxicity of Carbamates.

Compounds containing carbamate moieties and their derivatives can generate serious public health threats and environmental problems due their high potential toxicity. In this study, a quantitative structure-toxicity relationship (QSTR) model has been developed by using one hundred seventy-eight carbamate derivatives whose toxicities in rats (oral administration) have been evaluated. The QSRT model was rigorously validated by using either tested or untested compounds falling within the applicability domain of the model. A structure-based evaluation by docking from a series of carbamates with acetylcholinesterase (AChE) was carried out. The toxicity of carbamates was predicted using physicochemical, structural, and quantum molecular descriptors employing a DFT approach. A statistical treatment was developed; the QSRT model showed a determination coefficient (R2) and a leave-one-out coefficient (Q2LOO) of 0.6584 and 0.6289, respectively.

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method.

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease.

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.

Time-Course of CYP450 Genes Expression From Dendroctonus rhizophagus (Curculionidae: Scolytinae) During Early Hours of Drilling Bark and Settling Into the Host Tree.

Dendroctonus bark beetles (Scolytinae) are one of the most important disturbance agents of coniferous forests in North and Central America. These beetles spend their lives almost entirely under the tree bark, and their survival and reproductive success depend on their ability to overcome the toxic effect of the trees' oleoresin. The cytochromes P450 (CYPs) are associated with the detoxification process of xenobiotics, as well as other physiological processes. Different cytochromes (families 4, 6, and 9) in the Dendroctonus species have been expressed under several experimental conditions; nevertheless, the expression time-course of these genes is unknown. To explore the induction speed of CYPs, we evaluated the relative expression of the CYP6BW5, CYP6DG1, CYP6DJ2, CYP9Z18, and CYP9Z20 genes at the early hours of drilling and settling into a tree (1, 2, 4, 6, 8, 12, 18 h) both in females and males, solitary or paired, of the bark beetle Dendroctonus rhizophagus Thomas and Bright. Our findings show that the five genes were rapidly overexpressed in the early hours (1 to 6 h) in both sexes and in solitary and paired conditions, suggesting their participation in the detoxification process. Additionally, the CYPs expression shows up- and down-regulation patterns through these short times, suggesting their probable participation in other physiological processes as the biosynthesis of hormones, pheromones or compounds related to reproduction.

A new computational model for the prediction of toxicity of phosphonate derivatives using QSPR.

Structural and electronic properties of a series of 25 phosphonate derivatives were analyzed applying density functional theory, with the exchange-correlation functional PBEPBE in combination with the 6-311++G** basis set for all atoms. The chemical reactivity of these derivatives has been interpreted using quantum descriptors such as frontier molecular orbitals (HOMO, LUMO), Hirshfeld charges, molecular electrostatic potential, and the dual descriptor [[Formula: see text]]. These descriptors are directly related to experimental median lethal dose ([Formula: see text], expressed as its decimal logarithm [[Formula: see text]([Formula: see text]] through a multiple linear regression equation. The proposed model predicts the toxicity of phosphonates in function of the volume (V), the load of the most electronegative atom of the molecule (q), and the eigenvalue of the molecular orbital HOMO ([Formula: see text]. The obtained values in the internal validation of the model are: [Formula: see text]%, [Formula: see text]%, [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text]%. The toxicity of nine phosphonate derivatives used as test molecules was adequately predicted by the model. The theoretical results indicate that the oxygen atom of the O=P group plays an important role in the interaction mechanism between the phosphonate and the acetylcholinesterase enzyme, inhibiting the removal of the proton of the ser-200 residue by the his-440 residue.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.

Density Functional Theory and Electrochemical Studies: Structure-Efficiency Relationship on Corrosion Inhibition.

The relationship between structure and corrosion inhibition of a series of 30 imidazol, benzimidazol, and pyridine derivatives has been established through the investigation of quantum descriptors calculated with PBE/6-311++G**. A quantitative structure-property relationship model was obtained by examination of these descriptors using a genetic functional approximation method based on a multiple linear regression analysis. Our results indicate that the efficiency of corrosion inhibitors is strongly associated with aromaticity, electron donor ability, and molecular volume descriptors. In order to calibrate and validate the proposed model, we performed electrochemical impedance spectroscopy (EIS) studies on imidazole, 2-methylimidazole, benzimidazole, 2-chloromethylbenzimidazole, pyridine, and 2-aminopyridine compounds. The experimental values for efficiency of corrosion inhibition are in good agreement with the estimated values obtained by our model, thus confirming that our approach represents a promising and suitable tool to predict the inhibition of corrosion attributes of nitrogen containing heterocyclic compounds. The adsorption behavior of imidazole or benzimidazole heterocyclic molecules on the Fe(110) surface was also studied to elucidate the inhibition mechanism; the aromaticity played an important role in the adsorbate-surface complex.

A novel bio-functional material based on mammalian cell aggresomes.

Aggresomes are protein aggregates found in mammalian cells when the intracellular protein degradation machinery is over-titered. Despite that they abound in cells producing recombinant proteins of biomedical and biotechnological interest, the physiological roles of these protein clusters and the functional status of the embedded proteins remain basically unexplored. In this work, we have determined for the first time that, like in bacterial inclusion bodies, deposition of recombinant proteins into aggresomes does not imply functional inactivation. As a model, human α-galactosidase A (GLA) has been expressed in mammalian cells as enzymatically active, mechanically stable aggresomes showing higher thermal stability than the soluble GLA version. Since aggresomes are easily produced and purified, we propose these particles as novel functional biomaterials with potential as carrier-free, self-immobilized catalyzers in biotechnology and biomedicine.

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A.

Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

Auditory processing of communication calls in interacting bats.

There is strong evidence that social context plays a role in the processing of acoustic signals. Yet, the circuits and mechanisms that govern this process are still not fully understood. The insectivorous big brown bat, Eptesicus fuscus, emits a wide array of communication calls, including food-claiming calls, aggressive calls, and appeasement calls. We implemented a competitive foraging task to explore the influence of behavioral context on auditory midbrain responses to conspecific social calls. We recorded neural population responses from the inferior colliculus (IC) of freely interacting bats and analyzed data with respect to social context. Analysis of our neural recordings from the IC shows stronger population responses to individual calls during social events. For the first time, neural recordings from the IC of a copulating bat were obtained. Our results indicate that social context enhances neuronal population responses to social vocalizations in the bat IC.

Comparative metabarcoding and biodiversity of gut-associated fungal assemblages of Dendroctonus species (Curculionidae: Scolytinae).

The genus Dendroctonus is a Holarctic taxon composed of 21 nominal species; some of these species are well known in the world as disturbance agents of forest ecosystems. Under the bark of the host tree, these insects are involved in complex and dynamic associations with phoretic ectosymbiotic and endosymbiotic communities. Unlike filamentous fungi and bacteria, the ecological role of yeasts in the bark beetle holobiont is poorly understood, though yeasts were the first group to be recorded as microbial symbionts of these beetles. Our aim was characterize and compare the gut fungal assemblages associated to 14 species of Dendroctonus using the internal transcribed spacer 2 (ITS2) region. A total of 615,542 sequences were recovered yielding 248 fungal amplicon sequence variants (ASVs). The fungal diversity was represented by 4 phyla, 16 classes, 34 orders, 54 families, and 71 genera with different relative abundances among Dendroctonus species. The α-diversity consisted of 32 genera of yeasts and 39 genera of filamentous fungi. An analysis of ß-diversity indicated differences in the composition of the gut fungal assemblages among bark beetle species, with differences in species and phylogenetic diversity. A common core mycobiome was recognized at the genus level, integrated mainly by Candida present in all bark beetles, Nakazawaea, Cladosporium, Ogataea, and Yamadazyma. The bipartite networks confirmed that these fungal genera showed a strong association between beetle species and dominant fungi, which are key to maintaining the structure and stability of the fungal community. The functional variation in the trophic structure was identified among libraries and species, with pathotroph-saprotroph-symbiotroph represented at the highest frequency, followed by saprotroph-symbiotroph, and saprotroph only. The overall network suggested that yeast and fungal ASVs in the gut of these beetles showed positive and negative associations among them. This study outlines a mycobiome associated with Dendroctonus nutrition and provides a starting point for future in vitro and omics approaches addressing potential ecological functions and interactions among fungal assemblages and beetle hosts.

Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

Evaluation of the Hybrid Membrane of ZnO Particles Supported in Cellulose Acetate for the Removal of Lead.

Water polluted by discarded heavy metals such as lead is creating a global pollution problem. In this work, adsorption of Pb(II) was realized in batch studies by a hybrid membrane of cellulose acetate with ZnO particles. First, ZnO particles were prepared by precipitation and immobilized on the membrane. The hybrid membrane was elaborated by interfacial polymerization. The structure and surface were characterized based on Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM). Batch experiments were carried out under different conditions where the number of particles of ZnO present in the membrane and the pH of the aqueous solution were varied. The Langmuir and Freundlich isotherm models were evaluated in the best adsorption conditions. Data fitted well with a Langmuir model with a maximum adsorption capacity of 15.55 mg·g-1, which was similar for this type of materials. Thermodynamic parameters such as Gibbs free energy, enthalpy, and entropy showed that the process was spontaneous and favorable. The hybrid membrane was evaluated in simulated wastewater of the battery industry with a superior efficiency of up to 97%; without the medium, it did not generate interference. These results suggest that Pb(II) removal by hybrid membrane is possible.

Varroa Destructor Classification Using Legendre-Fourier Moments with Different Color Spaces.

Bees play a critical role in pollination and food production, so their preservation is essential, particularly highlighting the importance of detecting diseases in bees early. The Varroa destructor mite is the primary factor contributing to increased viral infections that can lead to hive mortality. This study presents an innovative method for identifying Varroa destructors in honey bees using multichannel Legendre-Fourier moments. The descriptors derived from this approach possess distinctive characteristics, such as rotation and scale invariance, and noise resistance, allowing the representation of digital images with minimal descriptors. This characteristic is advantageous when analyzing images of living organisms that are not in a static posture. The proposal evaluates the algorithm's efficiency using different color models, and to enhance its capacity, a subdivision of the VarroaDataset is used. This enhancement allows the algorithm to process additional information about the color and shape of the bee's legs, wings, eyes, and mouth. To demonstrate the advantages of our approach, we compare it with other deep learning methods, in semantic segmentation techniques, such as DeepLabV3, and object detection techniques, such as YOLOv5. The results suggest that our proposal offers a promising means for the early detection of the Varroa destructor mite, which could be an essential pillar in the preservation of bees and, therefore, in food production.

Conceptual DFT, machine learning and molecular docking as tools for predicting LD50 toxicity of organothiophosphates.

CONTEXT: Several descriptors from conceptual density functional theory (cDFT) and the quantum theory of atoms in molecules (QTAIM) were utilized in Random Forest (RF), LASSO, Ridge, Elastic Net (EN), and Support Vector Machines (SVM) methods to predict the toxicity (LD50) of sixty-two organothiophosphate compounds. The A-RF-G1 and A-RF-G2 models were obtained using the RF method, yielding statistically significant parameters with good performance, as indicated by R2 values for the training set (R2Train) and R2 values for the test set (R2Test), around 0.90. METHODS: The molecular structure of all organothiophosphates was optimized via the range-separated hybrid functional ωB97XD with the 6-311 + + G** basis set. Seven hundred and eighty-seven descriptors have been processed using a variety of machine learning algorithms: RF LASSO, Ridge, EN and SVM to generate a predictive model. The properties were obtained with Multiwfn, AIMALL and VMD programs. Docking simulations were performed by using AutoDock 4.2 and LigPlot + programs. All the calculations in this work are carried out in Gaussian 16 program package.

Diabetic neuropathy: Past, present, and future.

Background: A sedentary lifestyle and an unhealthy diet have considerably increased the incidence of diabetes mellitus worldwide in recent decades, which has generated a high rate of associated chronic complications. Methods: A narrative review was performed in MEDLINE, EMBASES and SciELO databases, including 162 articles. Results: Diabetic neuropathy (DN) is the most common of these complications, mainly producing two types of involvement: sensorimotor neuropathy, whose most common form is symmetric distal polyneuropathy, and autonomic neuropathies, affecting the cardiovascular, gastrointestinal, and urogenital system. Although hyperglycemia is the main metabolic alteration involved in its genesis, the presents of obesity, dyslipidemia, arterial hypertension, and smoking, play an additional role in its appearance. In the pathophysiology, three main phenomena stand out: oxidative stress, the formation of advanced glycosylation end-products, and microvasculature damage. Diagnosis is clinical, and it is recommended to use a 10 g monofilament and a 128 Hz tuning fork as screening tools. Glycemic control and non-pharmacological interventions constitute the mainstay of DN treatment, although there are currently investigations in antioxidant therapies, in addition to pain management. Conclusions: Diabetes mellitus causes damage to peripheral nerves, being the most common form of this, distal symmetric polyneuropathy. Control of glycemia and comorbidities contribute to prevent, postpone, and reduce its severity. Pharmacological interventions are intended to relieve pain.

A polyphasic taxonomy analysis reveals the presence of an ecotype of Rahnella contaminans associated with the gut of Dendroctonus-bark beetles.

Species belonging to the genus Rahnella are dominant members of the core gut bacteriome of Dendroctonus-bark beetles, a group of insects that includes the most destructive agents of pine forest in North and Central America, and Eurasia. From 300 isolates recovered from the gut of these beetles, 10 were selected to describe an ecotype of Rahnella contaminans. The polyphasic approach conducted with these isolates included phenotypic characteristics, fatty acid analysis, 16S rRNA gene, multilocus sequence analyses (gyrB, rpoB, infB, and atpD genes), and complete genome sequencing of two isolates, ChDrAdgB13 and JaDmexAd06, representative of the studied set. Phenotypic characterization, chemotaxonomic analysis, phylogenetic analyses of the 16S rRNA gene, and multilocus sequence analysis showed that these isolates belonged to Rahnella contaminans. The G + C content of the genome of ChDrAdgB13 (52.8%) and JaDmexAd06 (52.9%) was similar to those from other Rahnella species. The ANI between ChdrAdgB13 and JaDmexAd06 and Rahnella species including R. contaminans, varied from 84.02 to 99.18%. The phylogenomic analysis showed that both strains integrated a consistent and well-defined cluster, together with R. contaminans. A noteworthy observation is the presence of peritrichous flagella and fimbriae in the strains ChDrAdgB13 and JaDmexAd06. The in silico analysis of genes encoding the flagellar system of these strains and Rahnella species showed the presence of flag-1 primary system encoding peritrichous flagella, as well as fimbriae genes from the families type 1, α, ß and σ mainly encoding chaperone/usher fimbriae and other uncharacterized families. All this evidence indicates that isolates from the gut of Dendroctonus-bark beetles are an ecotype of R. contaminans, which is dominant and persistent in all developmental stages of these bark beetles and one of the main members of their core gut bacteriome.

Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis.

Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods.