Repositorio INDICASAT AIP
Not a member yet
227 research outputs found
Sort by
Amplified fragment length polymorphisms reveals high intraspecific variability in field isolates of Leishmania panamensis
No full textLeishmania parasites cause leishmaniasis, a potentially deadly re-emergent disease that affects millions throughout the world. In Panama, the disease is showing an increasing trend, with estimates of thousands of new cases every year. The main manifestations are the cutaneous and mucocutaneous forms. Genetic variability studies in Leishmania are extremely important to define key elements of the eco-epidemiology of the disease. However, few studies have addressed this issue in Panama, and these have been based mainly on kinetoplastid DNA RFLP. The amplified fragment length polymorphisms (AFLP) is a very efficient technique for rapid detection of genetic variability, particularly useful on organisms without sequenced genomes. Although this technique has been used successfully on many species, including several protozoa, its use for studying genetic variability in Leishmania parasites is just in its beginnings. We have optimized and used AFLP to address genetic diversity in Leishmania panamensis, a poorly studied member of the Viannia subgenus. We have found that this technique is able to generate high numbers of peaks when low selective EcoRI and MseI primers were used (+0, +1, +2 series). Additionally, we have found that an important proportion of those alleles, up to 57% for some primer combinations, are polymorphic. Some of these alleles are potentially useful to rapidly distinguish L. panamensis and L. guyanensis, the two most genetically similar species of the subgenus. The AFLP was an efficient technique to screen the Leishmania panamensis genome for polymorphisms, allowing the rapid detection of hundreds of polymorphic alleles.Leishmania parasites cause leishmaniasis, a potentially deadly re-emergent disease that affects millions throughout the world. In Panama, the disease is showing an increasing trend, with estimates of thousands of new cases every year. The main manifestations are the cutaneous and mucocutaneous forms. Genetic variability studies in Leishmania are extremely important to define key elements of the eco-epidemiology of the disease. However, few studies have addressed this issue in Panama, and these have been based mainly on kinetoplastid DNA RFLP. The amplified fragment length polymorphisms (AFLP) is a very efficient technique for rapid detection of genetic variability, particularly useful on organisms without sequenced genomes. Although this technique has been used successfully on many species, including several protozoa, its use for studying genetic variability in Leishmania parasites is just in its beginnings. We have optimized and used AFLP to address genetic diversity in Leishmania panamensis, a poorly studied member of the Viannia subgenus. We have found that this technique is able to generate high numbers of peaks when low selective EcoRI and MseI primers were used (+0, +1, +2 series). Additionally, we have found that an important proportion of those alleles, up to 57% for some primer combinations, are polymorphic. Some of these alleles are potentially useful to rapidly distinguish L. panamensis and L. guyanensis, the two most genetically similar species of the subgenus. The AFLP was an efficient technique to screen the Leishmania panamensis genome for polymorphisms, allowing the rapid detection of hundreds of polymorphic alleles
DNA Repair Mechanisms as Drug Targets in Prokaryotes
Get PDFNowadays, a great amount of pathogenic bacteria has been identified such as Mycobacterium sp. and Helicobacter pylori and have become a serious health problem around the world. These bacteria have developed several DNA repair mechanisms as a strategy to neutralize the effect of the exposure to endogenous and exogenous agents that will lead to two different kinds of DNA damage: single strand breaks (SSBs) and double strand breaks (DSBs). For SSBs repair, bacteria use the base excision repair (BER) and nucleotide excision repair (NER) mechanisms, which fix the damaged strand replacing the damaged base or nucleotide. DSBs repair in bacteria is performed by homologous recombination repair (HRR) and non-homologous end-joining (NHEJ). HRR uses the homologous sequence to fix the two damaged strand, while NHEJ repair does not require the use of its homologous sequence. The use of unspecific antibiotics to treat bacterial infections has caused a great deal of multiple resistant strains making less effective the current therapies with antibiotics. In this review, we emphasized the mechanisms mentioned above to identify molecular targets that can be used to develop novel and more efficient drugs in future.Nowadays, a great amount of pathogenic bacteria has been identified such as Mycobacterium sp. and Helicobacter pylori and have become a serious health problem around the world. These bacteria have developed several DNA repair mechanisms as a strategy to neutralize the effect of the exposure to endogenous and exogenous agents that will lead to two different kinds of DNA damage: single strand breaks (SSBs) and double strand breaks (DSBs). For SSBs repair, bacteria use the base excision repair (BER) and nucleotide excision repair (NER) mechanisms, which fix the damaged strand replacing the damaged base or nucleotide. DSBs repair in bacteria is performed by homologous recombination repair (HRR) and non-homologous end-joining (NHEJ). HRR uses the homologous sequence to fix the two damaged strand, while NHEJ repair does not require the use of its homologous sequence. The use of unspecific antibiotics to treat bacterial infections has caused a great deal of multiple resistant strains making less effective the current therapies with antibiotics. In this review, we emphasized the mechanisms mentioned above to identify molecular targets that can be used to develop novel and more efficient drugs in future
Occultocarpon, a new monotypic genus of Gnomoniaceae on Alnus nepalensis from China
Get PDFMicrofungi in the Gnomoniaceae (Diaporthales, Ascomycetes) comprise species commonly reported as pathogens and endophytes on trees and herbaceous hosts primarily from temperate forests of North America, Europe, and Japan. The diversity of Gnomoniaceae in China is poorly known, although several plant families that occur there specifically the Betulaceae are considered important hosts. An exploratory trip to Yunnan, China, resulted in the discovery of several members of the Gnomoniaceae. In this paper a new monotypic genus, Occultocarpon and its species, O. ailaoshanense, are described and illustrated. A phylogeny based on three genes (LSU, rpb2, tef1-α) reveals that O. ailaoshanense belongs to the Gnomoniaceae and forms a branch distinct from the currently known genera. Occultocarpon ailaoshanense is characterized by perithecia with thin, central to eccentric necks in groups embedded in a stroma and oblong elliptical-elongated, one-septate ascospores. Occultocarpon ailaoshanense occurs on the bark of branches of Alnus nepalensis (Betulaceae) in Yunnan, ChinaMicrofungi in the Gnomoniaceae (Diaporthales, Ascomycetes) comprise species commonly reported as pathogens and endophytes on trees and herbaceous hosts primarily from temperate forests of North America, Europe, and Japan. The diversity of Gnomoniaceae in China is poorly known, although several plant families that occur there specifically the Betulaceae are considered important hosts. An exploratory trip to Yunnan, China, resulted in the discovery of several members of the Gnomoniaceae. In this paper a new monotypic genus, Occultocarpon and its species, O. ailaoshanense, are described and illustrated. A phylogeny based on three genes (LSU, rpb2, tef1-α) reveals that O. ailaoshanense belongs to the Gnomoniaceae and forms a branch distinct from the currently known genera. Occultocarpon ailaoshanense is characterized by perithecia with thin, central to eccentric necks in groups embedded in a stroma and oblong elliptical-elongated, one-septate ascospores. Occultocarpon ailaoshanense occurs on the bark of branches of Alnus nepalensis (Betulaceae) in Yunnan, Chin
Comparison of the in vitro invasive capabilities of Plasmodium falciparum schizonts isolated by Percoll gradient or using magnetic based separation
Get PDFBackground: Percoll gradient centrifugation is often used for synchronization, enrichment, or isolation of a particular stage of Plasmodium falciparum. However, Percoll, a hyperosmotic agent, may have harmful effects on the parasites. Magnetic bead column (MBC) separation has been used as an alternative. This is a report of a headto-head comparison of the in vitro invasive capabilities of parasites isolated by either of the two methods. Methods: The P. falciparum laboratory strain isolate 7G8 was grown in vitro using standard procedures and synchronized using 5% sorbitol. On separate days when the schizont parasitaemia was >1%, the culture was split and half was processed by Percoll gradient centrifugation and the other half by magnetic bead column separation. Both processed parasites were placed back in culture and allowed to invade new uninfected erythrocytes. Results: In 10 paired assays, the mean efficiency of invasion of 7G8 parasites treated by Percoll gradient centrifugation was 35.8% that of those treated by magnetic bead column separation (95% CI, p = 0.00067) A paired t test with two tails was used for these comparisons. Conclusions: In this comparison, magnetic bead column separation of 7G8 schizonts resulted in higher viability and efficiency of invasion than utilizing Percoll gradient centrifugation.Background: Percoll gradient centrifugation is often used for synchronization, enrichment, or isolation of a particular stage of Plasmodium falciparum. However, Percoll, a hyperosmotic agent, may have harmful effects on the parasites. Magnetic bead column (MBC) separation has been used as an alternative. This is a report of a headto-head comparison of the in vitro invasive capabilities of parasites isolated by either of the two methods. Methods: The P. falciparum laboratory strain isolate 7G8 was grown in vitro using standard procedures and synchronized using 5% sorbitol. On separate days when the schizont parasitaemia was >1%, the culture was split and half was processed by Percoll gradient centrifugation and the other half by magnetic bead column separation. Both processed parasites were placed back in culture and allowed to invade new uninfected erythrocytes. Results: In 10 paired assays, the mean efficiency of invasion of 7G8 parasites treated by Percoll gradient centrifugation was 35.8% that of those treated by magnetic bead column separation (95% CI, p = 0.00067) A paired t test with two tails was used for these comparisons. Conclusions: In this comparison, magnetic bead column separation of 7G8 schizonts resulted in higher viability and efficiency of invasion than utilizing Percoll gradient centrifugation
Chemical Constituents of the New Endophytic Fungus Mycosphaerella sp. nov. and Their Anti-Parasitic Activity
Get PDFChemical investigation of a new endophytic fungus, Mycosphaerella sp. nov. strain F2140 associated with the foliage of the plant Psychotria horizontalis (Rubiaceae) in Panama, resulted in the isolation of cercosporin (1) and a new cercosporin analogue (3) as the major components. The structures of minor compounds in the extract were elucidated by detailed spectroscopic analysis as 2-(2-butyl)-3-hydroxy-6-ethyl-6-methylcyclohex-2-ene-1,5-dione (4), 3-(2-butyl)-6-ethyl-6methyl-5-hydroxy-2-methoxy-cyclohex-2-eneone (5), and an isomer of 5 (6). To study the influence of the hydroxy groups on the anti-parasitic activity of cercosporin, compound 1 was acetylated to obtain derivative 2. The isolated compounds 1–6 were tested in vitro to determine their anti-parasitic activity against the causal agents of malaria ( Plasmodium falciparum ), leishmaniasis ( Leishmania donovani ), and Chagas disease ( Trypanosoma cruzi ). Also, the cytotoxicity and potential anticancer activity of these compounds were evaluated using mammalian Vero cells and MCF7 cancer cell lines, respectively. Compounds 1 and 2 displayed high potency against L. donovani (IC50 0.46 and 0.64 μM), T. cruzi (IC50 1.08 and 0.78 μM), P. falciparum (IC50 1.03 and 2.99 μM), and MCF7 cancer cell lines (IC50 4.68 and 3.56 μM). Compounds 3–6 were not active in these assays at a concentration of 10 μg/mL.Chemical investigation of a new endophytic fungus, Mycosphaerella sp. nov. strain F2140 associated with the foliage of the plant Psychotria horizontalis (Rubiaceae) in Panama, resulted in the isolation of cercosporin (1) and a new cercosporin analogue (3) as the major components. The structures of minor compounds in the extract were elucidated by detailed spectroscopic analysis as 2-(2-butyl)-3-hydroxy-6-ethyl-6-methylcyclohex-2-ene-1,5-dione (4), 3-(2-butyl)-6-ethyl-6methyl-5-hydroxy-2-methoxy-cyclohex-2-eneone (5), and an isomer of 5 (6). To study the influence of the hydroxy groups on the anti-parasitic activity of cercosporin, compound 1 was acetylated to obtain derivative 2. The isolated compounds 1–6 were tested in vitro to determine their anti-parasitic activity against the causal agents of malaria ( Plasmodium falciparum ), leishmaniasis ( Leishmania donovani ), and Chagas disease ( Trypanosoma cruzi ). Also, the cytotoxicity and potential anticancer activity of these compounds were evaluated using mammalian Vero cells and MCF7 cancer cell lines, respectively. Compounds 1 and 2 displayed high potency against L. donovani (IC50 0.46 and 0.64 μM), T. cruzi (IC50 1.08 and 0.78 μM), P. falciparum (IC50 1.03 and 2.99 μM), and MCF7 cancer cell lines (IC50 4.68 and 3.56 μM). Compounds 3–6 were not active in these assays at a concentration of 10 μg/mL
Effects of structural properties of electrospun TiO2 nanofiber meshes on their osteogenic potential
Get PDFIdeal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the three-dimensional structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium implants, or more appropriately the TiO2 passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nanofiber meshes with different surface microroughness and nanofiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface microroughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nanofiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factorsIdeal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the three-dimensional structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium implants, or more appropriately the TiO2 passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nanofiber meshes with different surface microroughness and nanofiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface microroughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nanofiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factor
Automated Synchronization of P. falciparum using a Temperature Cycling Incubator
No full textAs malaria keeps affecting millions of lives every year, research based in culture of Plasmodium falciparum in vitro needs to be efficient and accurate. The development of better techniques and methodologies for the growth and maintenance of the parasites can save money, time, and lead to more trustable results. It has been observed, first in patients and then in the laboratory, that the malaria falciparum parasites growth is affected by high temperatures. This trait can be used with laboratory cultures to synchronize and maintain the parasites in the same stage of their cell cycle. This harmony of stages is very desirable for the purpose of conducting metabolomic, proteomic and transcriptome analysis as well as for drug screening. Most scientists in the field of malaria use chemicals (usually sorbitol) that kill certain stages of the parasite to obtain synchronization, but this latter method does not last long and the parasites thus treated should not be used for assays immediately after the treatment, due to the toxic effects that might have been infringed in the culture. A temperature cycling incubator (TCI) was acquired in our laboratory and it was used to test the synchronization of the multidrug resistant W2 and chloroquine resistant 7G8 strains, commonly used in our bioassays where they and their synchronization constitute essential tools for our drug discovery program. We followed the protocol designed by Haynes and Moch in 2002 and we made a comparison of the effectiveness of each of the two methods, chemical and temperature based. Our results show W2 synchronization by temperature cycling, with the help of an initial use of 0.3 M alanine, to last more than two months while a tight synchronization with the use of 5% sorbitol was lost as rapidly as in one week. Sorbitol could also be used with the TCI for synchronization with good results. However, 7G8 could not be efficiently synchronized with temperature cycling using the same program as that of W2.As malaria keeps affecting millions of lives every year, research based in culture of Plasmodium falciparum in vitro needs to be efficient and accurate. The development of better techniques and methodologies for the growth and maintenance of the parasites can save money, time, and lead to more trustable results. It has been observed, first in patients and then in the laboratory, that the malaria falciparum parasites growth is affected by high temperatures. This trait can be used with laboratory cultures to synchronize and maintain the parasites in the same stage of their cell cycle. This harmony of stages is very desirable for the purpose of conducting metabolomic, proteomic and transcriptome analysis as well as for drug screening. Most scientists in the field of malaria use chemicals (usually sorbitol) that kill certain stages of the parasite to obtain synchronization, but this latter method does not last long and the parasites thus treated should not be used for assays immediately after the treatment, due to the toxic effects that might have been infringed in the culture. A temperature cycling incubator (TCI) was acquired in our laboratory and it was used to test the synchronization of the multidrug resistant W2 and chloroquine resistant 7G8 strains, commonly used in our bioassays where they and their synchronization constitute essential tools for our drug discovery program. We followed the protocol designed by Haynes and Moch in 2002 and we made a comparison of the effectiveness of each of the two methods, chemical and temperature based. Our results show W2 synchronization by temperature cycling, with the help of an initial use of 0.3 M alanine, to last more than two months while a tight synchronization with the use of 5% sorbitol was lost as rapidly as in one week. Sorbitol could also be used with the TCI for synchronization with good results. However, 7G8 could not be efficiently synchronized with temperature cycling using the same program as that of W2
Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum
Get PDFPlasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine.Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine
In vitro and in vivo experimental models for drug screening and development for Chagas disease
Get PDFChagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease
Regulating in vivo calcification of alginate microbeads
Get PDFAlginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calciumcrosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1e6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mM bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcificationAlginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calciumcrosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1e6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mM bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcificatio