Molecular Alterations in Core Subunits of Mitochondrial Complex I and Their Relation to Parkinson's Disease.

Among the myriad of neurodegenerative diseases, mitochondrial dysfunction represents a nexus regarding their pathogenic processes, in which Parkinson's disease (PD) is notable for inherent vulnerability of the dopaminergic pathway to energy deficits and oxidative stress. Underlying this dysfunction, the occurrence of defects in complex I (CI) derived from molecular alterations in its subunits has been described in the literature. However, the mechanistic understanding of the processes mediating the occurrence of mitochondrial dysfunction mediated by CI deficiency in PD remains uncertain and subject to some inconsistencies. Therefore, this review analyzed existing evidence that may explain the relationship between molecular alterations in the core subunits of CI, recognized for their direct contribution to its enzymatic performance, and the pathogenesis of PD. As a result, we discussed 47 genetic variants in the 14 core subunits of CI, which, despite some discordant results, were predominantly associated with varying degrees of deficiency in complex enzymatic activity, as well as defects in supercomplex biogenesis and CI itself. Finally, we hypothesized about the relationship of the described alterations with the pathogenesis of PD and offered some suggestions that may aid in the design of future studies aimed at elucidating the relationship between such alterations and PD.

STAT5 Is Necessary for the Metabolic Switch Induced by IL-2 in Cervical Cancer Cell Line SiHa.

The tumor cells reprogram their metabolism to cover their high bioenergetic demands for maintaining uncontrolled growth. This response can be mediated by cytokines such as IL-2, which binds to its receptor and activates the JAK/STAT pathway. Some reports show a correlation between the JAK/STAT pathway and cellular metabolism, since the constitutive activation of STAT proteins promotes glycolysis through the transcriptional activation of genes related to energetic metabolism. However, the role of STAT proteins in the metabolic switch induced by cytokines in cervical cancer remains poorly understood. In this study, we analyzed the effect of IL-2 on the metabolic switch and the role of STAT5 in this response. Our results show that IL-2 induces cervical cancer cell proliferation and the tyrosine phosphorylation of STAT5. Also, it induces an increase in lactate secretion and the ratio of NAD+/NADH, which suggest a metabolic reprogramming of their metabolism. When STAT5 was silenced, the lactate secretion and the NAD+/NADH ratio decreased. Also, the expression of HIF1α and GLUT1 decreased. These results indicate that STAT5 regulates IL-2-induced cell proliferation and the metabolic shift to aerobic glycolysis by regulating genes related to energy metabolism. Our results suggest that STAT proteins modulate the metabolic switch in cervical cancer cells to attend to their high demand of energy required for cell growth and proliferation.

Oxidase enzyme genes are differentially expressed during Acanthamoeba castellanii encystment.

Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A crucial survival strategy of A. castellanii involves the formation of highly resistant cysts during adverse conditions. This study delves into the cellular processes underpinning encystment, focusing on gene expression changes related to reactive oxygen species (ROS) balance, with a particular emphasis on mitochondrial processes. Our findings reveal a dynamic response within the mitochondria during encystment, with the downregulation of key enzymes involved in oxidative phosphorylation (COX, AOX, and NADHalt) during the initial 48 h, followed by their overexpression at 72 h. This orchestrated response likely creates a pro-oxidative environment, facilitating encystment. Analysis of other ROS processing enzymes across the cell reveals differential expression patterns. Notably, antioxidant enzymes, such as catalases, glutaredoxins, glutathione S-transferases, peroxiredoxins, and thioredoxins, mirror the mitochondrial trend of downregulation followed by upregulation. Additionally, glycolysis and gluconeogenesis are downregulated during the early stages in order to potentially balance the metabolic requirement of the cyst. Our study underscores the importance of ROS regulation in Acanthamoeba encystment. Understanding these mechanisms offers insights into infection control and identifies potential therapeutic targets. This work contributes to unraveling the complex biology of A. castellanii and may aid in combatting Acanthamoeba-related infections. Further research into ROS and oxidase enzymes is warranted, given the organism's remarkable respiratory versatility.

Evaluation of safety and effectiveness of NAD in different clinical conditions: a systematic review.

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide cofactor that is present in cells and in several important biological processes, including oxidative phosphorylation and production of adenosine triphosphate, DNA repair, calcium-dependent secondary messenger and gene expression. The purpose of this systematic review is to examine whether the coenzyme formulae NAD+ and NADH are safe and effective when acting as a supplement to humans. This systematic review of randomized clinical trials performed a search in six electronic databases: PubMed, MEDLINE (ovid), Embase, Cochrane CENTRAL (clinical trials), Web of Science, and Scopus. Secondary search included the databases (e.g., Clinical trials.gov, Rebec, Google Scholar - advance). Two reviewers assessed and extracted the studies independently. The risk of bias in studies was performed using version 2 of the Cochrane risk of bias tool for randomized trials. This review includes 10 studies, with a total of 489 participants. The studies included different clinical conditions, such as chronic fatigue syndrome (CFS), older adults, Parkinson's disease, overweight, postmenopausal prediabetes, and Alzheimer's disease. Based on studies, the supplementation with NADH and precursors was well tolerated and observed clinical results such as, a decrease in anxiety conditions and maximum heart rate was observed after a stress test, increased muscle insulin sensitivity, insulin signaling. Quality of life, fatigue intensity, and sleep quality among others were evaluated on patients with CFS. All studies showed some side effects, thus, the most common associated with NADs use are muscle pain, nervous disorders, fatigue, sleep disturbance, and headaches. All adverse events cataloged by the studies did not present a serious risk to the health of the participants. Overall, these findings support that the oral administration of NADH can be associated to an increase in general quality of life and improvement on health parameters (e.g., a decrease in anxiety, maximum heart rate, inflammatory cytokines in serum, and cerebrospinal fluid). NADH supplementation is safe and has a low incidence of side effects. Future investigations are needed to evidence the clinical benefits regarding specific diseases and doses administered.

Capsaicinoids and Their Effects on Cancer: The "Double-Edged Sword" Postulate from the Molecular Scale.

Capsaicinoids are a unique chemical species resulting from a particular biosynthesis pathway of hot chilies (Capsicum spp.) that gives rise to 22 analogous compounds, all of which are TRPV1 agonists and, therefore, responsible for the pungency of Capsicum fruits. In addition to their human consumption, numerous ethnopharmacological uses of chili have emerged throughout history. Today, more than 25 years of basic research accredit a multifaceted bioactivity mainly to capsaicin, highlighting its antitumor properties mediated by cytotoxicity and immunological adjuvancy against at least 74 varieties of cancer, while non-cancer cells tend to have greater tolerance. However, despite the progress regarding the understanding of its mechanisms of action, the benefit and safety of capsaicinoids' pharmacological use remain subjects of discussion, since CAP also promotes epithelial-mesenchymal transition, in an ambivalence that has been referred to as "the double-edge sword". Here, we update the comparative discussion of relevant reports about capsaicinoids' bioactivity in a plethora of experimental models of cancer in terms of selectivity, efficacy, and safety. Through an integration of the underlying mechanisms, as well as inherent aspects of cancer biology, we propose mechanistic models regarding the dichotomy of their effects. Finally, we discuss a selection of in vivo evidence concerning capsaicinoids' immunomodulatory properties against cancer.

Production of acetone, butanol, and ethanol by electro-fermentation with Clostridium saccharoperbutylacetonicum N1-4.

This manuscript describes the effect of altering the extracellular redox potential during the production of acetone, butanol, and ethanol on a dual chamber H-type microbial fuel cell by fermenting glucose with Clostridium saccharoperbutylacetonicum N1-4. Extracellular redox potential modification was achieved by either supplementing the microbial broth with the redox agent NADH or by poising the cathode potential at -600 mV vs. Ag/AgCl. The addition of NADH was found to foment the production of acetone via fermentation of glucose. The addition of 200 mM of NADH to the catholyte rendered the highest production of acetone (2.4 g L-1), thus outperforming the production of acetone by conventional fermentation means (control treatment) by a factor of 2.2. The experimental evidence gathered here, indicates that cathodic electro-fermentation of glucose favors the production of butanol. When poising the cathode potential at -600 mV vs Ag/AgCl (electro-fermentation), the largest production of butanol was achieved (5.8 g L-1), outperforming the control treatment by a factor of 1.5. The production of ABE solvents and the electrochemical measurements demonstrate the electroactive properties of C. saccharoperbutylacetonicum N1-4 and illustrates the usefulness of bio-electrochemical systems to improve conventional fermentative processes.

Imidazole and nitroimidazole derivatives as NADH-fumarate reductase inhibitors: Density functional theory studies, homology modeling, and molecular docking.

Chagas disease is caused by Trypanosoma cruzi. Benznidazole and nifurtimox are drugs used for its therapy; nevertheless, they have collateral effects. NADH-fumarate (FUM) reductase is a potential pharmacological target since it is essential for survival of parasite and is not found in humans. The objectives are to design and characterize the electronic structure of imidazole and nitroimidazole derivatives at DFT-M06-2X level in aqueous solution; also, to model the NADH-FUM reductase and analyze its intermolecular interactions by molecular docking. Quantum-chemical descriptors allowed to select the molecules with the best physicochemical properties and lowest toxicity. A high-quality three-dimensional structure of NADH-FUM reductase was obtained by homology modeling. Water molecules do not have influence in the interaction between FUM and NADH-FUM reductase. The main hydrogen-binding interactions for FUM were identified in NADH, Lys172, and Arg89; while hydrophobic interactions in Phe479, Thr174, Met63. The molecules S3-8, S2-8, and S1-8 could be inhibitors of NADH-FUM reductase.

Fumonisin-containing diets decrease the metabolic activity of myenteric neurons in rats.

Fumonisins are naturally occurring mycotoxins that contaminate food for human and animal consumption. They have neurotoxic effects, but the mechanisms by which these toxins affect the nervous system are not fully known. In the present study, male Wistar rats were fed between 21 and 63 days of age with diets that contained fumonisins B1+B2 at 0, 1, and 4 mg/kg. The following variables were assessed: food consumption, growth, body weight gain, and blood parameters. Morphoquantitave analyses of the most metabolically active myenteric neurons were performed, detected by NADH-diaphorase activity. Nitrergic neurons were detected by NADPH-diaphorase activity. The fumonisin-containing diets did not significantly alter food consumption or the body or plasma parameters. These diets decreased the metabolic activity of jejunal myenteric neurons, reducing neuronal density of the most metabolic active neurons by 30.8% and the cell body area by 4.3%. The diets also decreased the cell body area of nitrergic neurons by 22.1%. The effects of fumonisin B1 on the respiratory metabolism of isolated mitochondria in the brain and liver were also assessed. A decrease in oxygen consumption up to a 29% in the brain and 38% in the liver was observed in mitochondrial isolates to which 50 µM fumonisin B1 was added. The decrease in respiratory activity that was triggered by exposure to fumonisins was related to the lower metabolic activity of myenteric neurons, which had a negative impact on neuroplasticity of the enteric nervous system.

The fructose-dependent acceleration of ethanol metabolism.

The aim of the present study was to elucidate how fructose is able to increase the rate of ethanol metabolism in the liver, an observation previously termed the fructose effect. Previous studies suggest that an increase in ATP consumption driven by glucose synthesis from fructose stimulates the oxidation of NADH in the mitochondrial respiratory chain, allowing faster oxidation of ethanol by alcohol dehydrogenase; however, this idea has been frequently challenged. We tested the effects of fructose, sorbose and tagatose both in vitro and in vivo. Both ethanol and each sugar were either added to isolated hepatocytes or injected intraperitoneally in the rat. In the in vitro experiments, samples were taken from the hepatocyte suspension in a time-dependent manner and deproteinized with perchloric acid. In the in vivo experiments, blood samples were taken every 15 min and the metabolites were determined in the plasma. These metabolites include ethanol, glucose, glycerol, sorbitol, lactate, fructose and sorbose. Ethanol oxidation by rat hepatocytes was increased by more than 50% with the addition of fructose. The stimulation was accompanied by increased glucose, glycerol, lactate and sorbitol production. A similar effect was observed with sorbose, while tagatose had no effect. The same pattern was observed in the in vivo experiments. This effect was abolished by inhibiting alcohol dehydrogenase with 4-methylpyrazole, whereas inhibition of the respiratory chain with cyanide did not affect the fructose effect. In conclusion, present results provide evidence that, by reducing glyceraldehyde and glycerol and fructose to sorbitol, respectively, NADH is consumed, allowing an increase in the elimination of ethanol. Hence, this effect is not linked to a stimulation of mitochondrial re-oxidation of NADH driven by ATP consumption.

Raman and Fluorescence Profiles Modifications of Muscular and Adipose Tissues Exposed to Low Energy X-ray Beams.

This work aims to investigate changes induced by low-energy radiation in adipose and muscular tissues employing autofluorescence and Raman spectroscopic techniques. X-ray beams expositions with 25 and 35 kV at 0.11, 1.1, and 2.1 Gy radiation dose levels were applied. Changes in Raman line intensities at specific bands assigned to collagen, proteins, and lipids were observed. Autofluorescent analysis exhibit variations in the collagen and nicotinamide adenine dinucleotide emission (NADH), resulting from the structural modifications, variations on the reduced/oxidized fluorophores equilibrium followed by radiation exposure. Results show that Raman and fluorescence spectroscopy are suitable techniques to evaluate radiation effects on biomolecules even at low radiation doses and energies.

Characteristics of microbial denitrification under different aeration intensities: Performance, mechanism, and co-occurrence network.

This study aimed to explore how dissolved oxygen (DO) affected the characteristics and mechanisms of denitrification in mixed bacterial consortia. We analyzed denitrification efficiency, intracellular nicotinamide adenine dinucleotide (NADH), relative expression of functional genes, and potential co-occurrence network of microorganisms. Results showed that the total nitrogen (TN) removal rates at different aeration intensities (0.00, 0.25, 0.63, and 1.25 L/(L·min)) were 0.93, 1.45, 0.86, and 0.53 mg/(L·min), respectively, which were higher than previously reported values for pure culture. The optimal aeration intensity was 0.25 L/(L·min), at which the maximum NADH accumulation rate and highest relative abundance of napA, nirK, and nosZ were achieved. With increased aeration intensity, the amount of electron flux to nitrate decreased and nitrate assimilation increased. On one hand, nitrate reduction was primarily inhibited by oxygen through competition for electron donors of a certain single strain. On the other hand, oxygen was consumed rapidly by bacteria by stimulating carbon metabolism to create an optimal denitrification niche for denitrifying microorganisms. Denitrification was performed via inter-genus cooperation (competitive interactions and symbiotic relationships) between keystone taxa (Azoarcus, Paracoccus, Thauera, Stappia, and Pseudomonas) and other heterotrophic bacteria (OHB) in aeration reactors. However, in the non-aeration case, which was primarily carried out based on intra-genus syntrophy within genus Propionivibrio, the co-occurrence network constructed the optimal niche contributing to the high TN removal efficiency. Overall, this study enhanced our knowledge about the molecular ecological mechanisms of aerobic denitrification in mixed bacterial consortia and has theoretical guiding significance for further practical application.

The southernmost record and an update of the geographical range of the Atlantic chupare, Styracura schmardae (Chondrichthyes: Myliobatiformes).

The present study established the southernmost distribution range for the Chupare stingray Styracura schmardae based on a specimen caught on the easternmost portion of Ceará State, northeastern Brazil. Identification was based on diagnostic morphological characters and molecular data (sequence of the mitochondrial DNA gene nd2; 1046 bp). This record expands the known distribution range for this species by more than 1200 km and confirms that S. schmardae is distributed in more than one biogeographical province. SIGNIFICANCE STATEMENT: There are few records of the Atlantic Chupare stingray for Brazil and most of them are associated with the Amazon River mouth. New records indicate that this species is also present in the South Atlantic portion of the Brazilian coast. Apparently having a low abundance throughout its range, these new records increase the range of occurrence of this species.

Chronic ingestion of deoxynivalenol-contaminated diet dose-dependently decreases the area of myenteric neurons and gliocytes of rats.

BACKGROUND: Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is commonly found in cereals ingested by humans and animals. Its ingestion is correlated with hepatic, hematologic, renal, splenic, cardiac, gastrointestinal, and neural damages, according to dose, duration of exposure and species. In this work, the effects of the ingestion of DON-contaminated diet at concentrations considered tolerable for human and animal intake were assessed. METHODS: Male Wistar rats aging 21 days were allotted to five groups that were given, for 42 days, diets contaminated with different concentrations of DON (0, 0.2, 0.75, 1.75, and 2 mg kg-1 of chow). Food ingestion, bodyweight, oxidative status and morphometric analyses of gliocytes, and neurons of jejunal myenteric ganglia were recorded. KEY RESULTS: At these concentrations, there was no food rejection, decrease in bodyweight gain, changes in oxidative status, or loss of either neurons or gliocytes. However, DON decreased gliocyte area, general neuronal population, nitrergic, cholinergic and NADH-diaphorase positive subpopulations and, as a result, ganglion area. CONCLUSIONS & INFERENCES: It was concluded that, even in the absence of visible effect, DON exposure reduces cell body area of gliocytes and neurons of the myenteric plexus of the rat jejunum.

Interplay between early-life malnutrition, epigenetic modulation of the immune function and liver diseases.

Early-life nutrition plays a critical role in fetal growth and development. Food intake absence and excess are the two main types of energy malnutrition that predispose to the appearance of diseases in adulthood, according to the hypothesis of 'developmental origins of health and disease'. Epidemiological data have shown an association between early-life malnutrition and the metabolic syndrome in later life. Evidence has also demonstrated that nutrition during this period of life can affect the development of the immune system through epigenetic mechanisms. Thus, epigenetics has an essential role in the complex interplay between environmental factors and genetics. Altogether, this leads to the inflammatory response that is commonly seen in non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome. In conjunction, DNA methylation, covalent modification of histones and the expression of non-coding RNA are the epigenetic phenomena that affect inflammatory processes in the context of NAFLD. Here, we highlight current understanding of the mechanisms underlying developmental programming of NAFLD linked to epigenetic modulation of the immune system and environmental factors, such as malnutrition.

High α-tocopherol dosing increases lipid metabolism by changing redox state in damaged rat gastric mucosa and liver after ethanol treatment.

Regeneration of ethanol-injured rat gastric mucosa must undergo changes in major metabolic pathways to achieve DNA replication and cell proliferation. These events are highly dependent on glucose utilization and inhibited by vitamin E (VE) (α-tocopherol) administration. Therefore, the present study aimed at assessing lipid metabolism in the gastric mucosa and ethanol-induced gastric damage and the effect of α-tocopherol administration. For this, rates of fatty acid ß-oxidation and lipogenesis were tested in gastric mucosa samples. Through histological analysis, we found loss of the mucosa's superficial epithelium, which became gradually normalized during the recovery period. Proliferation of gastric mucosa occurred with augmented formation of ß-oxidation by-products, diminished synthesis of triacylglycerols (TGs), as well as of phospholipids, and a reduced cytoplasmic NAD/NADH ratio, whereas the mitochondrial redox NAD/NADH ratio was much less affected. In addition, α-tocopherol increased palmitic acid utilization in the gastric mucosa, which was accompanied by the induction of 'mirror image' effects on the cell redox state, reflected in an inhibited cell gastric mucosa proliferation by the vitamin administration. In conclusion, the present study shows, for the first time, the role of lipid metabolism in the adaptive cell gastric mucosa changes that drive proliferation after a chronic insult. Moreover, α-tocopherol increased gastric mucosa utilization of palmitic acid associated with energy production. These events could be associated with its antioxidant properties in co-ordination with regulation of genes and cell pathways, including changes in the cell NAD/NADH redox state.

Actividad de enzimas antioxidantes eritrocitarias en una muestra de adultos jóvenes y mayores del Valle Central, Costa Rica / Erythrocyte antioxidant enzymes in Costa Rica

Resumen Introducción El daño oxidativo provocado por los radicales libres de oxígeno, está relacionado con el proceso de envejecimiento, con diversas patologías y con el estilo de vida de las personas pero, en el organismo, existen defensas enzimáticas antioxidantes que confieren una debida protección. Objetivo Determinar la actividad enzimática de la glucosa-6-fosfato deshidrogenasa (G6PD), la catalasa (CAT) y la NADH metahemoglobina reductasa (NADH-MR) en una población costarricense de adultos jóvenes y mayores, para establecer el intervalo de referencia y evaluar el efecto de algunas condiciones donde se presenta daño oxidativo. Materiales y métodos Se determinó la actividad enzimática en una población de 110 individuos de entre 19 y 95 años de edad, utilizando el método propuesto por Ernest Beutler. Se estableció el intervalo de referencia de la población y se evaluó el efecto de la edad, patologías de fondo y el fumado, mediante estadística descriptiva. Resultados Conforme avanza la edad, las enzimas NADH-MR y la CAT disminuyeron su actividad eritrocitaria y la G6PD no presentó cambios significativos. No se encontró evidencia de cambio significativo en la actividad enzimática con respecto al hábito de fumado y las condiciones patológicas estudiadas. Conclusiones Es necesario realizar más investigación en factores ambientales y estilo de vida que influyen en la actividad enzimática antioxidante. El tamaño de las poblaciones, la ausencia de métodos estandarizados y las condiciones del ensayo pueden afectar los resultados y su grado de significancia. Por lo tanto, deben estandarizarse las metodologías, de manera que en futuros proyectos se evalúen los resultados de acuerdo con estas condiciones.

Abstract Introduction The oxidative damage caused by free oxygen radicals is related to the aging process, some pathologies and people's lifestyle. However, in the human body there are antioxidant enzymatic defenses that give us an adequate protection. Objective To determine the enzyme activity of glucose-6-phosphate dehydrogenase (G6PD), catalase (CAT) and NADH methemoglobin reductase (NADH-MR), in a Costa Rican population of younger and older adults, in order to establish the reference range and to evaluate the effect of some conditions where oxidative damage occurs. Materials and methods The enzyme activity in a population of 110 individuals aged 19-95 years old was determined using the method proposed by Ernest Beutler. The population reference range was established and the effect of age, background pathologies and smoking were evaluated, using descriptive statistics. Results As age advances the enzyme NADH-MR and CAT decreased erythrocyte activity and G6PD presented no significant changes. Evidence of a significant change in the enzyme activity, with respect to the smoking habit and the pathological conditions, was not found. Conclusions It is necessary to carry out more research in terms of lifestyle and environmental factors, that influence the antioxidant enzyme activity. The size of populations, the absence of standardized methods and the conditions of the test can affect results and their degree of significance. For which methodologies should be standardized, so that in future projects the results can be evaluated according to these conditions.

Vanadium: History, chemistry, interactions with α-amino acids and potential therapeutic applications.

In the last 30 years, since the discovery that vanadium is a cofactor found in certain enzymes of tunicates and possibly in mammals, different vanadium-based drugs have been developed targeting to treat different pathologies. So far, the in vitro studies of the insulin mimetic, antitumor and antiparasitic activity of certain compounds of vanadium have resulted in a great boom of its inorganic and bioinorganic chemistry. Chemical speciation studies of vanadium with amino acids under controlled conditions or, even in blood plasma, are essential for the understanding of the biotransformation of e.g. vanadium antidiabetic complexes at the physiological level, providing clues of their mechanism of action. The present article carries out a bibliographical research emphaticizing the chemical speciation of the vanadium with different amino acids and reviewing also some other important aspects such as its chemistry and therapeutical applications of several vanadium complexes.

RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene.

The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100-200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways.