Silver-nanoparticle-modified nanocellulose synthesized by pyroligneous acid: cytotoxicity towards HaCat cells.

In the present study, pyroligneous acid, also known as wood vinegar, has been employed as reducing and stabilizing agent in the synthesis of silver nanoparticles (AgNPs) anchored on nanocellulose (NC). The idea is to confer the latter bactericidal properties for its typical uses such as in cosmetics and food-packing. It has been demonstrated that AgNPs can be directly produced onto NC in one-pot fashion while dramatically enhancing the kinetics of AgNPs synthesis (2 h for reaction completion) in comparison to the NC-less counterpart (10 days for reaction completion). Furthermore, NC allowed for a narrower size distribution of AgNPs. NC-supported and non-supported AgNPs had sizes of 5.1 ± 1.6 nm and 16.7 ± 4.62 nm, respectively. Immortalized human keratinocytes (HaCat) cells were then employed as model to evaluate the cytotoxicity of the AgNPs-NC compound. The latter was found not to impact cell proliferation at any formulation, while decreasing the viability by only 6.8% after 72 h. This study contributes to the development of more environmentally benign routes to produce nanomaterials and to the understanding of their impact on cells.

Cellular toxicology and mechanism of the response to silver-based nanoparticle exposure in Ewing's sarcoma cells.

Ewing's sarcoma is the most aggressive connective tissue tumor, mainly affecting children and adolescents; the 5 year survival rate is only 50%. Current treatments have poor effectiveness, and more efficient treatments are being sought. Silver-based nanoparticles, such as silver chloride nanoparticles (AgCl-NPs) and silver/silver chloride (Ag/AgCl-NPs) nanoparticles, can be biologically produced and can release Ag+ ions into solution; however, their antitumor activity has been minimally investigated. The aim of this study was to evaluate the antitumor potential of AgCl-NPs and Ag/AgCl-NPs against Ewing's sarcoma cells. A673 cells (Ewing's sarcoma) were treated for 72 h with 0-12.5 µg ml-1 of Ag/AgCl-NPs or 0-40 µg ml-1 of AgCl-NPs. Human cells from the RPE-1 cell line (pigmented retinal epithelium) were used as a model of nontumor cells. The RPE-1 cells were less affected by the administration of AgCl-NPs or Ag/AgCl-NPs, with small reductions in the number of cells and viability and a small increase in apoptosis rates, while lysosomal damage, changes in reactive oxygen species (ROS) production, loss of mitochondrial membrane potential and alterations in microfilaments or cell areas were not observed. A673 tumor cells had significantly reduced number and viability levels when treated with AgCl-NPs, with reductions of 65.05% and 99.17%, respectively, whereas with Ag/AgCl-NP treatment, reductions of 65.53% and 92.51% were observed, respectively. When treated with silver-based nanoparticles, A673 cells also showed a significant increase in ROS production and loss of mitochondrial membrane potential, which culminated in an increase in the percentage of apoptosis among the population. Lysosomal damage was also observed when A673 cells were treated with the highest concentration of AgCl-NPs. In conclusion, the results showed that both AgCl-NPs and Ag/AgCl-NPs had some antitumor activity with minimal effects against healthy cells, which demonstrated the possibility of their use in cancer therapy.

Biomedical application of graphitic carbon nitrides: tissue depositionin vivo, induction of reactive oxygen species (ROS) and cell viability in tumor cells.

The urgency for new materials in oncology is immediate. In this study we have developed the g-C3N4, a graphitic-like structure formed by periodically linked tris-s-triazine units. The g-C3N4has been synthesized by a simple and fast thermal process. XRD has shown the formation of the crystalline sheet with a compacted structure. The graphite-like structure and the functional groups have been shown by Raman and FTIR spectroscopy. TEM image and AFM revealed the porous composed of five or six C-N layers stacked. DRS and Photoluminescence analyses confirmed the structure with band gap of 2.87 eV and emission band at 448 nm in different wavelengths excitation conditions. The biological results showed inhibitory effect on cancer cell lines and non-toxic effect in normal cell lines. To the best of our knowledge, this is the first work demonstrating the cytotoxic effects of 2D g-C3N4in a cancer cell line, without any external or synergistic influence. The biodistribution/tissue accumulation showed that g-C3N4present a tendency to accumulation on the lung in the first 2 h, but after 24 h the profile of the biodistribution change and it is found mainly in the liver. Thus, 2D-g-C3N4showed great potential for the treatment of several cancer types.

Ohmic heating increases inactivation and morphological changes of Salmonella sp. and the formation of bioactive compounds in infant formula.

The effect of ohmic heating (OH) (50, 55, and 60 °C, 6 V/cm) on the inactivation kinetics (Weibull model) and morphological changes (scanning electron microscopy and flow cytometry) of Salmonella spp. in infant formula (IF) was evaluated. In addition, thermal load indicators (hydroxymethylfurfural and whey protein nitrogen index, HMF, and WPNI) and bioactive compounds (DPPH, total phenolics, ACE, α-amylase, and α-glucosidase inhibitory activities) were also studied. OH presented a more intense inactivation rate than conventional heating, resulting in a reduction of about 5 log CFU per mL at 60 °C in only 2.91 min, being also noted a greater cell membrane deformation, higher formation of bioactive compounds, and lower values for the thermal load parameters. Overall, OH contributed to retaining the nutritional value and improve food safety in IF processing.

Impact of culture conditions on the chlorophyll content of microalgae for biotechnological applications.

Chlorophyll is a commercially important natural green pigment responsible for the absorption of light energy and its conversion into chemical energy via photosynthesis in plants and algae. This bioactive compound is widely used in the food, cosmetic, and pharmaceutical industries. Chlorophyll has been consumed for health benefits as a nutraceutical agent with antioxidant, anti-inflammatory, antimutagenic, and antimicrobial properties. Microalgae are photosynthesizing microorganisms which can be extracted for several high-value bioproducts in the biotechnology industry. These microorganisms are highly efficient at adapting to physicochemical variations in the local environment. This allows optimization of culture conditions for inducing microalgal growth and biomass production as well as for changing their biochemical composition. The modulation of microalgal culture under controlled conditions has been proposed to maximize chlorophyll accumulation. Strategies reported in the literature to promote the chlorophyll content in microalgae include variation in light intensity, culture agitation, and changes in temperature and nutrient availability. These factors affect chlorophyll concentration in a species-specific manner; therefore, optimization of culture conditions has become an essential requirement. This paper provides an overview of the current knowledge on the effects of key environmental factors on microalgal chlorophyll accumulation, focusing on small-scale laboratory experiments.

Cryptococcus neoformans capsular polysaccharides form branched and complex filamentous networks viewed by high-resolution microscopy.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. Its main virulence factor is an extracellular polysaccharide capsule whose structure, assembly and dynamics remain poorly understood. In this study, we apply improved protocols for sample preparation and recently-developed scanning microscopy techniques to visualize the ultrastructure of the C. neoformans capsule at high-resolution (up to 1 nm) and improved structural preservation. Although most capsule structures in nature consist of linear polymers, we show here that the C. neoformans capsule is a 'microgel-like' structure composed of branched polysaccharides. Moreover, we imaged the capsule-to-cell wall link, which is formed by thin fibers that branch out of thicker capsule filaments, and have one end firmly embedded in the cell wall structure. Together, our findings provide compelling ultrastructural evidence for a branched and complex capsule conformation, which may have important implications for the biological activity of the capsule as a virulence factor.

The Uptake of GABA in Trypanosoma cruzi.

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.

Nanoscopic Localization of Surface-Exposed Antigens of Borrelia burgdorferi.

Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, is transmitted to humans through the bite of infected Ixodes spp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means of the pathogen to escape the vertebrates' immune system. One strategy employed by Lyme disease spirochetes to evade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins that is vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterial surface antigens using different microscopy techniques, from fluorescent wide field to super-resolution and immunogold-scanning electron microscopy. A fluorescent strain of B. burgdorferi spirochetes was labeled with monoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is more evenly distributed over the surface than OspB and OspC that were present as punctate areas.

Assessment of silver nanoparticles' antitumor effects: Insights into cell number, viability, and morphology of glioblastoma and prostate cancer cells.

Silver nanoparticles (AgNPs) hold promise for cancer therapy. This study aimed to evaluate their impact on tumor and non-tumor cell number, viability, and morphology. Antitumor activity was tested on U-87MG (glioblastoma) and DU-145 (prostate cancer) cell lines. Treatment with AgNPs notably reached a reduction of U-87MG and DU-145 cell growth by 89.30% and 79.74%, respectively, resulting in slower growth rates. AgNPs induced DNA damage, evidenced by reduced nuclear area and DNA content via fluorescent image-based analyses. Conversely, HFF-1 non-tumor cells displayed no significant changes post-AgNPs exposure. Viability assays revealed substantial reductions in U-87MG and DU-145 cells (79% and 63% in MTT assays, 30% and 52.2% in high-content analyses), while HFF-1 cells exhibited lower sensitivity. Tumor cells had notably lower IC50 values than non-tumor cells, indicating selective susceptibility. Transmission electron microscopy (TEM) showed morphological changes post-AgNPs administration, including increased vacuoles, myelin figures, membrane ghosts, cellular extravasation, and membrane projections. The findings suggest the potential of AgNPs against glioblastoma and prostate cancer, necessitating further exploration across other cancer cell lines.

Thermosonication as an effective substitution for fusion in Brazilian cheese spread (Requeijão Cremoso) manufacturing: The effect of ultrasonic power on technological properties.

In this initial study, the impact of thermosonication as an alternative to the traditional fusion in Brazilian cheese spread (Requeijão Cremoso) manufacture was investigated. The effect of ultrasound (US) power was evaluated considering various aspects such as gross composition, microstructure, texture, rheology, color, fatty acid composition, and volatile compounds. A 13 mm US probe operating at 20 kHz was used. The experiment involved different US power levels (200, 400, and 600 W) at 85 °C for 1 min, and results were compared to the conventional process in the same conditions (85 °C for 1 min, control treatment). The texture became softer as ultrasound power increased from 200 to 600 W, which was attributed to structural changes within the protein and lipid matrix. The color of the cheese spread also underwent noticeable changes for all US treatments, and treatment at 600 W resulted in increased lightness but reduced color intensity. Moreover, the fatty acid composition of the cheese spread showed variations with different US power, with samples treated at 600 W showing lower concentrations of saturated and unsaturated fatty acids, as well as lower atherogenicity and thrombogenicity indexes, indicating a potentially healthier product. Volatile compounds were also influenced by US, with less compounds being identified at higher powers, especially at 600 W. This could indicate possible degradation, which should be evaluated in further studies regarding US treatment effects on consumer perception. Hence, this initial work demonstrated that thermosonication might be interesting in the manufacture of Brazilian cheese spread, since it can be used to manipulate the texture, color and aroma of the product in order to improve its quality parameters.

Applications of Silver Nanoparticles in Patent Research.

BACKGROUND: Silver nanoparticles (AgNPs) have been widely applied in research and industrial fields, finding applications in nanomedicine, drug delivery, biomedical devices, electronics, the energy sector, and environmental protection. Patents provide information on the industrial viability of product technologies, and the number of patent documents provides an estimate of the evolution of a specific technological field. AIMS: The present work aims to describe the current trends in AgNPs patent applications. In addition, a retrospective study of published patents in Brazil is presented. METHODS: Analyses of AgNPs-related patents were conducted using the free platform for patent search Lens® in 2010-2019 and articles published in same period using the Scholar® base. The patent applications and their evolution over time, major depositors and holders, and the main technological areas associated with AgNP applications have been described. RESULTS: China and United States are the major patent applicants for nanotechnologies. The worldwide distribution of publications of journal articles shows that China, India, and the United States are the leading countries in the total number of articles published, in that order. CONCLUSION: Our study of patent applications and published articles confirmed the growing global increase in new technologies involving NPs and AgNPs, particularly in the biotechnology area, in the fields of medicine and agriculture.

Impact of adding xylooligosaccharides encapsulated in butter: Microstructural, optical, rheological and sensory aspects.

This study investigated the microstructure, rheological properties, and sensory characteristics of butters produced with free and encapsulated xylooligosaccharides (XOS). Four formulations of butter were processed: BCONT: 0 % w/w XOS (control); BXOS: 20% w/w free XOS; BXOS-ALG: 20% w/w XOS microencapsulated with alginate (XOS-alginate ratio of 3:1 w/w); and BXOS-GEL: 20% w/w XOS microencapsulated with alginate-gelatin (XOS-alginate-gelatin ratio of 3:1:1.5 w/w). The microparticles showed a bimodal distribution, low size and low span values, demonstrating physical stability to be included in emulsions. The XOS-ALG presented surface weighted mean diameter (D3.2) of 90.24 µm, volume-weighted mean diameter (D4.3) of 131.8 µm, and Span of 2.14. In contrast, the XOS-GEL presented D3.2 of 82.80 µm, D4.3 of 141.0 µm, and a Span of 2.46. Products with XOS were characterized by higher creaminess, sweet taste, and lower salty taste than the control. However, the addition form significantly impacted the other evaluated parameters. The utilization of XOS in a free form (BXOS) resulted in smaller droplet sizes (1.26 µm) than encapsulated XOS and control (XOS-ALG = 1.32 µm / XOS-GEL = 1.58 µm, / BCONT = 1.59 µm), and changes in the rheological parameters (higher values of shear stress, viscosity, consistency index, rigidity (J0), and Newtonian viscosity (ηN) and lower elasticity (τ)). Furthermore, it changed the color parameters (more yellow and dark color, lower L* and higher b* values). On the other hand, the utilization of micropaticles of XOS (BXOS-ALG and BXOS-GEL) kept shear stress, viscosity, consistency index, rigidity (J0), and elasticity (τ) more similar to control. The products had a less intense yellow color (lower b* values) and was perceived with more consistency and butter taste. However, the presence of particles was perceived by consumers. The results suggest that consumers were more attentive to reporting flavor-related attributes than texture. In conclusion, adding microparticles of XOS could improve butter's rheological and sensory properties. In conclusion, adding microparticles of XOS could improve butter's rheological and sensory properties.

Adaptor protein-3 (AP-3) complex mediates the biogenesis of acidocalcisomes and is essential for growth and virulence of Trypanosoma brucei.

Acidocalcisomes are acidic calcium and polyphosphate storage organelles found in a diverse range of organisms. Here we present evidence that the biogenesis of acidocalcisomes in Trypanosoma brucei is linked to the expression of adaptor protein-3 (AP-3) complex. Localization studies in cell lines expressing ß3 and δ subunits of AP-3 fused to epitope tags revealed their partial co-localization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes, with the Golgi marker Golgi reassembly and stacking protein, and with antibodies against the small GTPase Rab11. Ablation of the ß3 subunit by RNA interference (RNAi) resulted in disappearance of acidocalcisomes from both procyclic and bloodstream form trypanosomes, as revealed by immmunofluorescence and electron microscopy assays, with no alterations in trafficking of different markers to lysosomes. Knockdown of the ß3 subunit resulted in lower acidic calcium, pyrophosphate, and polyphosphate content as well as defects in growth in culture, resistance to osmotic stress, and virulence in mice. Similar results were obtained by knocking down the expression of the δ subunit of AP-3. These results indicate that AP-3 is essential for the biogenesis of acidocalcisomes and for growth and virulence of T. brucei.

Hyperosmotic stress induces aquaporin-dependent cell shrinkage, polyphosphate synthesis, amino acid accumulation, and global gene expression changes in Trypanosoma cruzi.

The protist parasite Trypanosoma cruzi has evolved the ability to transit between completely different hosts and to replicate in adverse environments. In particular, the epimastigote form, the replicative stage inside the vector, is subjected to nutritional and osmotic stresses during its development. In this work, we describe the biochemical and global gene expression changes of epimastigotes under hyperosmotic conditions. Hyperosmotic stress resulted in cell shrinking within a few minutes. Depending on the medium osmolarity, this was followed by lack of volume recovery for at least 2 h or by slow recovery. Experiments with inhibitors, or with cells in which an aquaporin gene (TcAQP1) was knocked down or overexpressed, revealed its importance for the cellular response to hyperosmotic stress. Furthermore, the adaptation to this new environment was shown to involve the regulation of the polyphosphate polymerization state as well as changes in amino acid catabolism to generate compatible osmolytes. A genome-wide transcriptional analysis of stressed parasites revealed down-regulation of genes belonging to diverse functional categories and up-regulation of genes encoding trans-sialidase-like and ribosomal proteins. Several of these changes were confirmed by Northern blot analyses. Sequence analysis of the 3'UTRs of up- and down-regulated genes allowed the identification of conserved structural RNA motifs enriched in each group, suggesting that specific ribonucleoprotein complexes could be of great importance in the adaptation of this parasite to different environments through regulation of transcript abundance.

Dual action of pyroligneous acid in the eco-friendly synthesis of bactericidal silver nanoparticles.

In the present study, we demonstrate that pyroligneous acid (PA), also known as wood vinegar, functions efficiently as both reducing and stabilizing agent in the synthesis of silver nanoparticles (AgNPs). The synthesis and stabilization of AgNPs take place in the following fashion: 1) in alkaline environment, oxygenated species (phenols in the present case) contained in PA reduce silver ions to metallic silver; 2) acetic acid, abundantly present in PA, adsorb onto the AgNPs conferring electrostatic stabilization. This mechanism is supported by GC-MS and RAMAN analysis, with the former revealing the compounds lacking in PA after nanoparticle synthesis and the latter demonstrating acetic acid adsorbed on the nanoparticles. The AgNPs produced via this method were quite stable up to 150 days (zeta potential = -56 mV). The AgNPs were then found to inhibit the growth of Escherichia coli and Staphylococcus aureus. Concerning PA, we showed that it displays bactericidal properties only under acidic conditions. This study contributes to the development of more environmentally benign routes to produce nanomaterials.

Assessing the antiproliferative effect of biogenic silver chloride nanoparticles on glioblastoma cell lines by quantitative image-based analysis.

Glioblastoma is the most life-threatening tumour of the central nervous system. Temozolomide (TMZ) is the first-choice oral drug for the treatment of glioblastoma, although it shows low efficacy. Silver nanoparticles (AgNPs) have been shown to exhibit biocidal activity in a variety of microorganisms, including some pathogenic microorganisms. Herein, the antiproliferative effect of AgCl-NPs on glioblastoma cell lines (GBM02 and GBM11) and on astrocytes was evaluated through automated quantitative image-based analysis (HCA) of the cells. The cells were treated with 0.1-5.0 µg/ml AgCl-NPs or with 9.7-48.5 µg/ml TMZ. Cells that received combined treatment were also analysed. At a maximum tested concentration of AgCl-NPs, GBM02 and GBM11, the growth decreased by 93% and 40%, respectively, following 72 h of treatment. TMZ treatment decreased the proliferation of GBM02 and GBM11 cells by 58% and 34%, respectively. Combinations of AgCl-NPs and TMZ showed intermediate antiproliferative effects; the lowest concentrations caused an inhibition similar to that obtained with TMZ, and the highest concentrations caused inhibition similar to that obtained with AgCl-NPs alone. No significant changes in astrocyte proliferation were observed. The authors' findings showed that HCA is a fast and reliable approach that can be used to evaluate the antiproliferative effect of the nanoparticles at the single-cell level and that AgCl-NPs are promising agents for glioblastoma treatment.

Ohmic heating as a method of obtaining paraprobiotics: Impacts on cell structure and viability by flow cytometry.

This study aimed to evaluate the effects of ohmic heating (OH) on probiotic inactivation, cell viability and morphology of the probiotic strains Lactobacillus acidophilus LA 05 (LA), Lacticaseibacillus casei 01 (LC), and Bifidobacterium animalis Bb 12 (BA) to develop paraprobiotics. OH at different electric field magnitudes (4, 8, and 12 V/cm at 60 Hz) and conventional heat treatment (CONV) were performed to determine the most adequate condition for the obtainment of paraprobiotics. Analysis of culturability, flow cytometry (FC), and Scanning electron microscope (SEM) was carried out. The complete inactivation by CONV was achieved only in the following conditions: LA - 95 °C/5 min, LC and BA - 95 °C/7 min. The same temperature profile was used in OH treatments to study the OH electrical effects. The OH treatment (8 V/cm) caused lower damage to the cell membrane integrity compared to the CONV treatment (p < 0.05). The OH showed to be adequate technology for the efficient production of paraprobiotics.

Can sucrose-substitutes increase the antagonistic activity against foodborne pathogens, and improve the technological and functional properties of sheep milk kefir?

The effect of different types of sugar (sucrose, demerara, brown, fructose, coconut sugar, and honey) on sheep milk kefir was evaluated. Microbial counts (Lactobacillus, Lactococcus, Leuconostoc, yeast), antagonistic activity against foodborne pathogens, microstructure (scanning electron microscopy), and antiproliferative activity of cancer cells were evaluated. Furthermore, the antioxidant activity (DPPH), inhibitory activity of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase, lactose content, lactic and acetic acids and ethanol, fatty acid profile and volatile organic compounds were determined. The addition of sugars increased the Lactobacillus population (up to 2.24 log CFU/mL), metabolites concentration, antagonistic activity against pathogens, antioxidant activity (11.1 to 24.1%), ACE inhibitory activity (27.5 to 37.6%), α-amylase inhibition (18 to 37.4%), and anti-proliferative activity. Furthermore, it improved the fatty acid profile and volatile compounds. The results suggest that the replacement of sucrose with different types of sugar constitute an interesting option to be used in sheep milk kefir formulations.

Subcellular proteomics of Trypanosoma cruzi reservosomes.

Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC-MS/MS analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles.

Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain.

Trypanosoma cruzi relies on highly galactosylated molecules as virulence factors and the enzymes involved in sugar biosynthesis are potential therapeutic targets. The synthesis of UDP-galactose in T. cruzi requires the activity of phosphoglucomutase (PGM), the enzyme that catalyzes the interconversion of glucose-6-phosphate and glucose-1-phosphate. Several enzymes that participate in carbohydrate metabolism in trypanosomes are confined to specialized peroxisome-like organelles called glycosomes. The majority of glycosomal proteins contain peroxisome-targeting signals (PTS) at the COOH- or at the amino-terminus, which drive their transport to glycosomes. We had previously identified the T. cruzi PGM gene (TcPGM) and demonstrated that it encodes a functional enzyme. Here, we show that, in contrast to yeast and mammalian cells, TcPGM resides in glycosomes of the parasite. However, no classical PTS1 or PTS2 motif is present in its sequence. We investigated glycosomal targeting by generating T. cruzi cell lines expressing different domains of TcPGM fused to the green fluorescent protein (GFP). The analysis of the subcellular localization of fusion proteins revealed that an internal targeting signal of TcPGM, residing between amino acid residues 260 and 380, is capable of targeting GFP to glycosomes. These results demonstrate that, in T. cruzi, PGM import into glycosomes is mediated by a novel non-PTS domain that is located internally in the protein.