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1.
Org Lett ; 2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39046190

RESUMO

Herein, we present a highly efficient method for constructing the intricate 5-5-6 fused ring system commonly found in the polycyclic furanobutenolide-derived cembranoid and norcembranoid natural product family with remarkable diastereoselectivity, utilizing an intramolecular Diels-Alder reaction as the cornerstone. Notably, employing a propargyl ether tether as the dienophile yields significant enhancements in the transformation process compared to its propargyl ester counterpart, as demonstrated in our previous total synthesis of havellockate. This advancement holds promising implications for future investigations, offering a streamlined pathway for rapidly assembling the tricyclic core characteristic of this diverse family of natural products.

2.
J Am Chem Soc ; 144(44): 20232-20236, 2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36287147

RESUMO

The first total synthesis of the furanobutenolide-derived cembranoid diterpenoid havellockate is disclosed. Our convergent strategy employs a Julia-Kocienski olefination to join two enantioenriched fragments to produce a diene that is subsequently used in a propiolic acid esterification/Diels-Alder cascade. This sequence generates the fused carbocyclic core of the natural product in short order. A challenging Zn-mediated Barbier allylation then forges the final C-C bond and also establishes two vicinal stereogenic centers. Finally, a Cu-catalyzed aerobic oxidation facilitates the formation of the ß-hydroxybutanolide to complete the total synthesis.


Assuntos
Diterpenos , Estereoisomerismo
4.
Prenat Diagn ; 35(6): 534-43, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25641621

RESUMO

OBJECTIVE: To develop a single-tube multi-marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non-deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity. METHODS: We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha-globin gene cluster, and optimized a single-tube assay combining detection of α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application. RESULTS: Of 42 markers encompassing the α-globin gene cluster that were identified in silico, 9 were highly polymorphic (0.68 ≤ polymorphism information content ≤ 0.92; 0.66 ≤ Ho ≤ 0.90; 10 ≤ alleles ≤ 35) and optimized to co-amplify directly from a single cell. A validation analysis of 100 single lymphoblasts yielded 100% amplification success for all markers, and individual marker allele drop-out (ADO) rates of 0-5%. Clinical application of the assay in PGD for Hb Bart's (2 cases/cycles) resulted in a twin pregnancy and healthy live birth of two baby girls. CONCLUSIONS: This single-tube nonaplex microsatellite PCR panel can be applied directly to PGD of most deletional Hb Bart's without the need for deletion-specific customization, and to linkage-based PGD of non-deletional Hb Bart's.


Assuntos
Hemoglobinas Anormais/genética , Hidropisia Fetal/genética , Diagnóstico Pré-Implantação/métodos , Alelos , Sequência de Bases , Linhagem Celular , Simulação por Computador , Transferência Embrionária , Feminino , Fertilização in vitro , Haplótipos , Humanos , Hidropisia Fetal/diagnóstico , Recém-Nascido , Repetições de Microssatélites , Modelos Genéticos , Reação em Cadeia da Polimerase , Gravidez , Deleção de Sequência , Talassemia alfa/diagnóstico , Talassemia alfa/genética
5.
J Assist Reprod Genet ; 31(7): 921-5, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24752713

RESUMO

PURPOSE: Previous studies have shown that a modified one-step slow freezing method with higher sucrose concentration (0.2 M) can achieve higher embryo and blastomere survival rates that are comparable to vitrification. However, no study has evaluated the efficacy of a one-step method using commercial slow freezing kit without altering its composition. This retrospective study examines the effects of using 1.5 M PROH with 0.1 M sucrose (F2 medium) alone in a one-step slow freezing method compared to the conventional two-step method. METHODS: Cleavage stage embryos from 526 thaw cycles previously cryopreserved by either the conventional two-step slow freezing method or the modified one-step method were studied. The embryo and blastomere survival rates, cleavage rate, clinical pregnancy rate and live birth rate were compared between the two groups. RESULTS: The results showed that the embryo survival rate was significantly higher in the modified one-step method compared to the conventional two-step method (86.9 % and 83.1 %, respectively; p = 0.04). Total blastomere survival rate was also significantly increased as a result of the modification (81.0 % versus 76.5 %; p < 0.001). However, there was no statistical difference in the cleavage rates, clinical pregnancy rates (CPR/ET) and live birth rates between the two methods. CONCLUSIONS: Slow freezing using the one-step method is superior to the conventional two-step method in terms of embryo and blastomere survival rates without affecting cleavage rate and clinical outcomes. It can be routinely applied to cleavage stage embryo cryopreservation in IVF centres for greater workflow efficiency.


Assuntos
Criopreservação/métodos , Técnicas de Cultura Embrionária , Fertilização in vitro , Congelamento , Fase de Clivagem do Zigoto , Transferência Embrionária/métodos , Feminino , Humanos , Gravidez , Taxa de Gravidez
7.
Singapore Med J ; 53(10): 648-54, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23112015

RESUMO

INTRODUCTION: We aimed to develop and implement a short tandem repeat (STR) polymerase chain reaction alternative to fluorescence in situ hybridisation (FISH) for the preimplantation genetic diagnosis (PGD) of chromosomal translocations. METHODS: Selected informative STRs located on translocated arms of relevant chromosomes were used to discriminate between normal and unbalanced chromosome states in each embryo. RESULTS: PGD cycles were performed on five couples where one spouse carried a balanced translocation. 27 embryos were analysed, of which 12 were normal/balanced, 12 were abnormal/unbalanced and three were indeterminate. Four PGD cycles proceeded to embryo transfer, of which two led to pregnancy. The first pregnancy showed a normal male karyotype, and a healthy baby was delivered at term. A second pregnancy unexpectedly miscarried in the second trimester from unknown causes. CONCLUSION: STR analysis is a simple and suitable alternative to FISH for detecting unbalanced chromosomal states in preimplantation embryos.


Assuntos
Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético/genética , Diagnóstico Pré-Implantação/métodos , Translocação Genética/genética , Feminino , Fertilização in vitro , Humanos , Masculino , Gravidez , Resultado da Gravidez
8.
Reprod Biomed Online ; 21(5): 642-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20864413

RESUMO

The high incidence of double-gene deletions in α-thalassaemia increases the risk of having pregnancies with homozygous α(0)-thalassaemia, the cause of the lethal haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. However, the current gap-PCR based PGD protocol for deletional α-thalassaemia requires specific primer design for each specific deletion. A universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome has been developed. Microsatellite markers 16PTEL05 and 16PTEL06 within the α-globin gene cluster were co-amplified with a third microsatellite marker outside the affected region in a multiplex-PCR reaction and analysed by capillary electrophoresis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. A total of 47 embryos were analysed. Three pregnancies were achieved from three couples, with the births of two healthy babies and one ongoing pregnancy. This work has successfully adapted an earlier protocol and developed a simple and reliable single-cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of type of deletion. Alpha-thalassaemia is one of the most common inheritable disorders worldwide. It is a blood disorder that, in its lethal form caused by deletion of all four copies of the α-globin gene, results in the demise of the affected fetus, a condition referred to as haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. Current PGD protocols for deletional α-thalassaemia utilize a strategy called gap-PCR, which requires the different assays for different deletion types. We have developed a universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome based on microsatellite marker analysis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. Forty-five embryos were analysed in total. Three pregnancies were achieved from three couples, with the births of two healthy babies and one pregnancy still ongoing. We have successfully adapted our earlier protocol and developed a simple and reliable single cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of the type of deletion.


Assuntos
Hemoglobinas Anormais/genética , Hidropisia Fetal/diagnóstico , Reação em Cadeia da Polimerase/métodos , Diagnóstico Pré-Implantação/métodos , Talassemia alfa/diagnóstico , Feminino , Humanos , Hidropisia Fetal/genética , Masculino , Repetições de Microssatélites , Gravidez , alfa-Globinas/genética , Talassemia alfa/genética
9.
J Mol Endocrinol ; 36(3): 425-33, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16720714

RESUMO

The steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) inhibits osteogenesis while stimulating adipogenesis in vitro. We hypothesized that 1,25(OH)(2)D(3) redirects the fate of osteoblast/adipocyte bipotential progenitors and other potential progenitors towards adipogenesis, a process possibly underlying the pathogenesis of osteopenic diseases such as osteoporosis. We therefore tested the global effects of 1,25(OH)(2)D(3) on the recruitment of mesenchymal progenitors including osteogenic, chondrogenic, adipogenic and myogenic lineages (colony forming cell (CFC)-osteoblast (CFC-O), CFC-chondrocyte (CFC-C), CFC-adipocyte (CFC-A), and CFC-myoblast (CFC-M) respectively) in rat calvaria (RC) cell populations using gene expression profiling of single cell-derived colonies. Based on expression of lineage specific transcripts, 86% of single cell-derived colonies in untreated cultures simultaneously co-expressed transcripts of two, three, or four of the mesenchymal lineages tested. The distribution of mesenchymal progenitors in 1,25(OH)(2)D(3)-treated cultures was significantly changed compared with the control group, i.e. CFC-O were reduced (from 6 to 0%) and CFC-O/A bipotential (0 to 8.2%), CFC-C (4 to 10.2%) and CFC-Fibroblast (CFC-F) (4 to 16%) were increased. 1,25(OH)(2)D(3) did not affect the frequency of tri- or tetra-lineage colonies. Single lineage CFC-A colonies were not detected in either the control or 1,25(OH)(2)D(3) treatment group under the conditions tested. Since the parietal bones used for cell isolation derive from neuroectoderm, we also analyzed for expression of the neural markers nestin and beta3 tubulin in these colonies. Surprisingly, 90% (45 of 50) of the colonies in the control group expressed neural markers, a frequency not changed by 1,25(OH)(2)D(3) treatment. The current studies demonstrate the global and developmental stage-specific effects of 1,25(OH)(2)D(3) on mesenchymal lineage progenitors, and suggest that the effects of 1,25(OH)(2)D(3) on osteogenesis and adipogenesis in RC populations are mediated, at least in part, by increased recruitment of CFC-O/A, but not CFC-A type precursors.


Assuntos
Calcitriol/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Crânio/citologia , Vitaminas/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/fisiologia , Animais , Biomarcadores/metabolismo , Calcitriol/farmacologia , Linhagem da Célula , Células Cultivadas , Perfilação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Ratos , Crânio/fisiologia , Vitaminas/farmacologia
10.
Bone ; 38(5): 662-70, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16503211

RESUMO

In some tissues, stem cells are enriched within the side population (SP) cells characterized by the efficient efflux of Hoechst 33342, but few data are yet available to address whether such is the case in bone tissue. When we Hoechst-stained and FACS-analyzed freshly isolated 20- or 21-day fetal rat calvaria (RC) cells, a small fraction of cells (0.15 +/- 0.05%) comprised of a distinct SP. When SP, non-SP and total/unfractionated (Total) RC cells were plated at a density of 30 cells per microtiter well, the percentage of wells containing bone-forming progenitors (CFU-O) was significantly higher in the SP compared to the non-SP or Total populations (13 +/- 4% vs. 1.8 +/- 0.4% and 0.7 +/- 0.4% respectively). The SP was also highly enriched for CFU-alkaline phosphatase (CFU-ALP) and CFU-fibroblast (CFU-F). While Dex increased the recruitment of CFU-O and CFU-F in the SP, it did not increase the frequency of CFU-ALP. Limiting dilution analysis showed a non-linear relationship between cell densities (1, 5, 10, 20 and 30 cells/microtiter well) and the frequency of readout CFU-O, CFU-ALP and CFU-F in all populations, suggesting a cell non-autonomous component to proliferation-differentiation of these progenitor types. When the developmental potential of SP cells for chondrocyte, adipocyte and neural lineages was assessed, SP cells were also found to be enriched for progenitors of all three lineages. These data demonstrate that Hoechst staining and SP sorting by flow cytometry are a useful strategy for the enrichment of CFU-O and possibly other precursors present in RC cell populations.


Assuntos
Osso e Ossos/citologia , Feto/citologia , Células-Tronco/citologia , Adipócitos/química , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Animais , Benzimidazóis/análise , Benzimidazóis/metabolismo , Cartilagem/citologia , Diferenciação Celular , Separação Celular , Condrócitos/química , Condrócitos/citologia , Condrócitos/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Osteócitos/química , Osteócitos/citologia , Osteócitos/metabolismo , Ratos , Crânio , Células-Tronco/química , Células-Tronco/metabolismo
11.
J Biotechnol ; 117(1): 99-109, 2005 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-15831251

RESUMO

Encapsulating cells by polyelectrolyte complex coacervation can be accomplished at physiological temperature and buffer conditions. One of the oppositely charged polyelectrolytes in the microcapsule core can be collagen or any other natural extra-cellular matrices suitable for cellular support while the other polyelectrolyte forms the ultra-thin shell to ensure efficient mass transfer. These microcapsules with ultra-thin shell are difficult to produce in large quantities due to their fragility. In this study, electrostatic spraying technique was used to achieve a scalable production of one such type of microcapsules formed by complex coacervation between the cationic methylated collagen and anionic terpolymer of hydroxylethyl methacrylate, methyl methacrylate and methylacrylic acid (HEMA-MMA-MAA). It was found that the microcapsule sizes were dependent on several important operational parameters, such as the diameter of the spraying needle, the flow rate of the hepatocytes-collagen mixture and the voltage of the electrical field. The microcapsules with diameters of 200-800 microm and a narrow size distribution (standard deviation of 5-28%) were successfully produced. The above parameters also influenced the hepatocyte viability and functions. With a practical encapsulation rate of up to 55 ml/h per orifice required in bio-artificial liver-assisted device applications, we have produced large quantities of microcapsules maintaining comparable cell viability (>87%), mechanical stability and bio-functions to the manually extruded microcapsules.


Assuntos
Hepatócitos/fisiologia , Animais , Sobrevivência Celular , Masculino , Ratos , Ratos Wistar , Eletricidade Estática
12.
Biomaterials ; 25(17): 3531-40, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15020127

RESUMO

New anionic polyelectrolyte tetra-copolymers with photo-crosslinkable 4-(4-methoxycinnamoyl)phenyl methacrylate monomer in addition to a HEMA-MMA-MAA ter-copolymer system were synthesized. The tetra-copolymers were used to form photo-crosslinkable microcapsules with modified collagen by complex coacervation for rat hepatocytes encapsulation. The hepatocytes were encapsulated within a two-layered membrane comprising of modified collagen as the inner core and an outer photo-crosslinkable copolymer shell. Upon photo-crosslinking of the microcapsules with UV-Vis light irradiation, the mechanical strength and chemical stability of the microcapsules, and the cellular functions of the encapsulated hepatocytes were enhanced. Particularly, the mechanical stability of the microcapsules was dramatically strengthened. The new photo-crosslinkable tetra-copolymer formulation described in this article has opened a way to the development of hepatocyte microencapsulation technology for bioartifical liver assist device.


Assuntos
Técnicas de Cultura de Células/métodos , Colágeno/química , Hepatócitos/citologia , Hepatócitos/fisiologia , Metacrilatos/química , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/efeitos da radiação , Transplante de Células/instrumentação , Transplante de Células/métodos , Células Cultivadas , Reagentes de Ligações Cruzadas/efeitos da radiação , Eletrólitos/química , Hepatócitos/transplante , Luz , Masculino , Teste de Materiais , Metacrilatos/efeitos da radiação , Microesferas , Peso Molecular , Fotoquímica/métodos , Porosidade , Ratos , Ratos Wistar , Ureia/metabolismo
13.
Tissue Eng ; 9 Suppl 1: S65-75, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14511471

RESUMO

We previously encapsulated hepatocytes in ultrathin shell microcapsules and showed them to have enhanced differentiated functions over cells cultured in monolayer. Here we have used these microencapsulated hepatocytes in a bioartificial liver-assisted device (BLAD) with a rat hepatectomy model. Primary rat hepatocytes were encapsulated in 150- to 200-microm microcapsules, using an electrostatic droplet generator. The microencapsulated hepatocytes exhibited good in vitro urea synthesis activity in plasma from rats with fulminant hepatic failure (FHF). The ex vivo hemoperfusion was conducted in FHF rats by perfusing plasma at a rate of 1-2 mL/min through 1.5-2 x 10(8) encapsulated hepatocytes packed into a packed-bed bioreactor. Hemoperfusion with the bioreactor was initiated 5 h after operative induction of liver failure and continued for 7 h. The BLAD-treated rats showed improvements over the control groups in survival time and metabolic indicators, including ammonia and total bilirubin levels. Furthermore, expanded bed adsorption (EBA) detoxification technology using Streamline-SP resin was explored to complement the bioreactor with microencapsulated hepatocytes. In vitro experiments indicated that serum ammonia could be specifically removed in dose-dependent manner, whereas the total serum proteins were unaffected by the resin. In ex vivo experiments, hemoperfusion with the resin was initiated 3 h after operative induction of liver failure and continued for 7 h. The resin-treated rats showed obvious serum ammonia removal with no observable total blood protein and blood cell adsorption. Therefore, Streamline-SP resin can potentially be integrated into a BLAD for improved efficacy.


Assuntos
Cápsulas , Hepatócitos/metabolismo , Engenharia Tecidual , Amônia/metabolismo , Animais , Bilirrubina/metabolismo , Falência Hepática/terapia , Masculino , Ratos , Ratos Wistar , Fatores de Tempo
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