Application of PolyPRep tools on HIV protease polyproteins using molecular docking

Abstract In recent years, the development of high-throughput technologies for obtaining sequence data leveraged the possibility of analysis of protein data in silico. However, when it comes to viral polyprotein interaction studies, there is a gap in the representation of those proteins, given their size and length. The prepare for studies using state-of-the-art techniques such as Machine Learning, a good representation of such proteins is a must. We present an alternative to this problem, implementing a fragmentation and modeling protocol to prepare those polyproteins in the form of peptide fragments. Such procedure is made by several scripts, implemented together on the workflow we call PolyPRep, a tool written in Python script and available in GitHub. This software is freely available only for noncommercial users.

Resumo Nos últimos anos, o desenvolvimento de tecnologias de alto rendimento para obtenção de dados sequenciais potencializou a possibilidade de análise de dados proteicos in silico. No entanto, quando se trata de estudos de interação de poliproteínas virais, existe uma lacuna na representação dessas proteínas, devido ao seu tamanho e comprimento. Para estudos utilizando técnicas de ponta como o Aprendizado de Máquina, uma boa representação dessas proteínas é imprescindível. Apresentamos uma alternativa para este problema, implementando um protocolo de fragmentação e modelagem para preparar essas poliproteínas na forma de fragmentos de peptídeos. Tal procedimento é feito por diversos scripts, implementados em conjunto no workflow que chamamos de PolyPRep, uma ferramenta escrita em script Python e disponível no GitHub. Este software está disponível gratuitamente apenas para usuários não comerciais.

Morphophysiological changes of Acca sellowiana (Myrtaceae: Myrtoideae) saplings under shade gradient

Abstract Understanding morphological and physiological changes under different light conditions in native fruit species in juveniles stage is important, as it indicate the appropriate environment to achieve vigorous saplings. We aimed to verify growth and morphophysiological changes under shade gradient in feijoa (Acca sellowiana (O. Berg) Burret) to achieve good quality saplings adequate to improve cultivation in orchards. The saplings were grown for twenty-one-month under four shading treatments (0%, 30%, 50%, and 80%). Growth, photosynthetic pigments, gas exchanges, chlorophyll fluorescence, and leaf anatomy parameters were evaluated. Saplings under full sun and 30% shade had higher height and diameter growth and dry mass accumulation due to higher photosynthesis rate. As main acclimatization mechanisms in feijoa saplings under 80% shade were developed larger leaf area, reduced leaf blade thickness, and enhanced quantum yield of photosystem II. Even so, the net CO2 assimilation and the electron transport rate was lower and, consequently, there was a restriction on the growth and dry mass in saplings under deep shade. Therefore, to obtain higher quality feijoa saplings, we recommend that it be carried out in full sun or up to 30% shade, to maximize the sapling vigor in nurseries and, later, this light environment can also be used in orchards for favor growth and fruit production.

Resumo A verificação de mudanças morfológicas e fisiológicas sob diferentes condições luminosas em espécies frutíferas nativas em estágio juvenil é importante, uma vez que indicam o ambiente adequado para a formação de mudas com alto vigor. Objetivou-se verificar o crescimento e as alterações morfofisiológicas sob gradiente de sombreamento em mudas de feijoa (Acca sellowiana (O. Berg) Burret) para obter mudas de boa qualidade, adequadas para fomentar os plantios da espécie em pomares. As mudas foram cultivadas por vinte e um meses sob quatro tratamentos de sombreamento (0%, 30%, 50% e 80%). Foram avaliados parâmetros de crescimento, pigmentos fotossintéticos, trocas gasosas, fluorescência da clorofila e anatomia foliar. Mudas a pleno sol e 30% de sombra apresentaram maior crescimento em altura, diâmetro e acúmulo de massa seca, devido à maior taxa de fotossíntese. Como principais mecanismos de aclimatação sob 80% de sombra, as mudas desenvolveram maior área foliar, redução da espessura do limbo foliar e aumento do rendimento quântico do fotossistema II. Mesmo assim, a assimilação líquida de CO2 e a taxa de transporte de elétrons foram menores e, consequentemente, houve restrição ao crescimento e acúmulo de massa seca das mudas no maior nível de sombreamento. Portanto, para a obtenção de mudas de feijoa de maior qualidade, recomendamos que seja realizada a pleno sol ou até 30% de sombra, para maximizar o vigor das mudas em viveiros e, posteriormente, este ambiente de luz também pode ser utilizado em pomares para favorecer o crescimento e a produção de frutos.

Application of PolyPRep tools on HIV protease polyproteins using molecular docking / Aplicação da ferramenta PolyPRep em poliproteínas de protease de HIV usando docking molecular

Abstract In recent years, the development of high-throughput technologies for obtaining sequence data leveraged the possibility of analysis of protein data in silico. However, when it comes to viral polyprotein interaction studies, there is a gap in the representation of those proteins, given their size and length. The prepare for studies using state-of-the-art techniques such as Machine Learning, a good representation of such proteins is a must. We present an alternative to this problem, implementing a fragmentation and modeling protocol to prepare those polyproteins in the form of peptide fragments. Such procedure is made by several scripts, implemented together on the workflow we call PolyPRep, a tool written in Python script and available in GitHub. This software is freely available only for noncommercial users.

Resumo Nos últimos anos, o desenvolvimento de tecnologias de alto rendimento para obtenção de dados sequenciais potencializou a possibilidade de análise de dados proteicos in silico. No entanto, quando se trata de estudos de interação de poliproteínas virais, existe uma lacuna na representação dessas proteínas, devido ao seu tamanho e comprimento. Para estudos utilizando técnicas de ponta como o Aprendizado de Máquina, uma boa representação dessas proteínas é imprescindível. Apresentamos uma alternativa para este problema, implementando um protocolo de fragmentação e modelagem para preparar essas poliproteínas na forma de fragmentos de peptídeos. Tal procedimento é feito por diversos scripts, implementados em conjunto no workflow que chamamos de PolyPRep, uma ferramenta escrita em script Python e disponível no GitHub. Este software está disponível gratuitamente apenas para usuários não comerciais.

Morphophysiological changes of Acca sellowiana (Myrtaceae: Myrtoideae) saplings under shade gradient / Alterações morfofisiológicas em mudas de Acca sellowiana (Myrtaceae: Myrtoideae) sob gradiente de sombreamento

Abstract Understanding morphological and physiological changes under different light conditions in native fruit species in juveniles' stage is important, as it indicate the appropriate environment to achieve vigorous saplings. We aimed to verify growth and morphophysiological changes under shade gradient in feijoa (Acca sellowiana (O. Berg) Burret) to achieve good quality saplings adequate to improve cultivation in orchards. The saplings were grown for twenty-one-month under four shading treatments (0%, 30%, 50%, and 80%). Growth, photosynthetic pigments, gas exchanges, chlorophyll fluorescence, and leaf anatomy parameters were evaluated. Saplings under full sun and 30% shade had higher height and diameter growth and dry mass accumulation due to higher photosynthesis rate. As main acclimatization mechanisms in feijoa saplings under 80% shade were developed larger leaf area, reduced leaf blade thickness, and enhanced quantum yield of photosystem II. Even so, the net CO2 assimilation and the electron transport rate was lower and, consequently, there was a restriction on the growth and dry mass in saplings under deep shade. Therefore, to obtain higher quality feijoa saplings, we recommend that it be carried out in full sun or up to 30% shade, to maximize the sapling vigor in nurseries and, later, this light environment can also be used in orchards for favor growth and fruit production.

Resumo A verificação de mudanças morfológicas e fisiológicas sob diferentes condições luminosas em espécies frutíferas nativas em estágio juvenil é importante, uma vez que indicam o ambiente adequado para a formação de mudas com alto vigor. Objetivou-se verificar o crescimento e as alterações morfofisiológicas sob gradiente de sombreamento em mudas de feijoa (Acca sellowiana (O. Berg) Burret) para obter mudas de boa qualidade, adequadas para fomentar os plantios da espécie em pomares. As mudas foram cultivadas por vinte e um meses sob quatro tratamentos de sombreamento (0%, 30%, 50% e 80%). Foram avaliados parâmetros de crescimento, pigmentos fotossintéticos, trocas gasosas, fluorescência da clorofila e anatomia foliar. Mudas a pleno sol e 30% de sombra apresentaram maior crescimento em altura, diâmetro e acúmulo de massa seca, devido à maior taxa de fotossíntese. Como principais mecanismos de aclimatação sob 80% de sombra, as mudas desenvolveram maior área foliar, redução da espessura do limbo foliar e aumento do rendimento quântico do fotossistema II. Mesmo assim, a assimilação líquida de CO2 e a taxa de transporte de elétrons foram menores e, consequentemente, houve restrição ao crescimento e acúmulo de massa seca das mudas no maior nível de sombreamento. Portanto, para a obtenção de mudas de feijoa de maior qualidade, recomendamos que seja realizada a pleno sol ou até 30% de sombra, para maximizar o vigor das mudas em viveiros e, posteriormente, este ambiente de luz também pode ser utilizado em pomares para favorecer o crescimento e a produção de frutos.

Morphophysiological changes of Acca sellowiana (Myrtaceae: Myrtoideae) saplings under shade gradient.

Understanding morphological and physiological changes under different light conditions in native fruit species in juveniles' stage is important, as it indicate the appropriate environment to achieve vigorous saplings. We aimed to verify growth and morphophysiological changes under shade gradient in feijoa (Acca sellowiana (O. Berg) Burret) to achieve good quality saplings adequate to improve cultivation in orchards. The saplings were grown for twenty-one-month under four shading treatments (0%, 30%, 50%, and 80%). Growth, photosynthetic pigments, gas exchanges, chlorophyll fluorescence, and leaf anatomy parameters were evaluated. Saplings under full sun and 30% shade had higher height and diameter growth and dry mass accumulation due to higher photosynthesis rate. As main acclimatization mechanisms in feijoa saplings under 80% shade were developed larger leaf area, reduced leaf blade thickness, and enhanced quantum yield of photosystem II. Even so, the net CO2 assimilation and the electron transport rate was lower and, consequently, there was a restriction on the growth and dry mass in saplings under deep shade. Therefore, to obtain higher quality feijoa saplings, we recommend that it be carried out in full sun or up to 30% shade, to maximize the sapling vigor in nurseries and, later, this light environment can also be used in orchards for favor growth and fruit production.

Application of PolyPRep tools on HIV protease polyproteins using molecular docking.

In recent years, the development of high-throughput technologies for obtaining sequence data leveraged the possibility of analysis of protein data in silico. However, when it comes to viral polyprotein interaction studies, there is a gap in the representation of those proteins, given their size and length. The prepare for studies using state-of-the-art techniques such as Machine Learning, a good representation of such proteins is a must. We present an alternative to this problem, implementing a fragmentation and modeling protocol to prepare those polyproteins in the form of peptide fragments. Such procedure is made by several scripts, implemented together on the workflow we call PolyPRep, a tool written in Python script and available in GitHub. This software is freely available only for noncommercial users.

Efficiency of lysine utilization by growing meat quail.

The objective of present study was to estimate the efficiency of lysine utilization by meat quail of 21 to 35 d of age. A total of 500 meat quails were distributed in a completely randomized design in a 2 × 5 factorial arrangement, with 2 sexes (male and female) and 5 digestible lysine levels (0.714, 0.816, 0.918, 1.020, and 1.122%) and 5 replications of 10 birds each. The variables studied were feather-free body weight (FFBW), feed intake (FI), lysine intake (LysI), feather-free body protein deposition (FFBPD), feather-free body lysine deposition (FFBLysD), feather-free body fat deposition (FFBFatD), feather weight (FW), feather protein deposition, feather lysine deposition, and feather fat deposition. The FFBW, FFBPD, FFBLysD, and FFBFatD were regressed as a function of LysI for each sex to estimate the efficiency of lysine utilization in the feather-free body (ELysFFB), and the individual equations were compared. In addition, a multiple regression without intercept was also used to estimate the ELysFFB and in feathers (ELysF) individually. To compare the ELysFFB obtained by the different methods, the t-statistic was used. There was no effect on sex × lysine level interactions for any variable. The females showed higher FFBW (5.07%) and FFBFatD (26.23%) than males. All variables increased with the level of dietary lysine, with the exception of FI, FW, and the deposition of nutrients within them. The ELysFFB values obtained by simple linear regression and multiple linear regression were 48.0 and 44.6%, respectively. As there was no difference in the efficiencies estimated by the different methodologies, the best estimate of ELysFFB was 46.3%, that is, the average. The best estimate of ELysF was 18.1%, obtained by multiple linear regression.

Galectin-3 orchestrates the histology of mesentery and protects liver during lupus-like syndrome induced by pristane.

Galectin-3 (Gal-3) controls intercellular and cell-extracellular matrix interactions during immunological responses. In chronic inflammation, Gal-3 is associated with fibrotic events, regulates B cell differentiation and delays lupus progression. Gal-3 deficient mice (Lgals3-/-) have intense germinal center formation and atypical plasma cell generation correlated to high levels IgG, IgE, and IgA. Here, we used pristane (2,6,10,14-tetramethylpentadecane) to induce lupus-like syndrome in Lgals3-/- and Lgals3+/+ BALB/c mice. Mesentery and peritoneal cells were monitored because promptly react to pristane injected in the peritoneal cavity. For the first time, mesenteric tissues have been associated to the pathogenesis of experimental lupus-like syndrome. In Lgals3+/+ pristane-induced mice, mesentery was hallmarked by intense fibrogranulomatous reaction restricted to submesothelial regions and organized niches containing macrophages and B lymphocytes and plasma cells. In contrast, Lgals3-/- pristane-treated mice had diffuse mesenteric fibrosis affecting submesothelium and peripheral tissues, atypical M1/M2 macrophage polarization and significant DLL1+ cells expansion, suggesting possible involvement of Notch/Delta pathways in the disease. Early inflammatory reaction to pristane was characterized by significant disturbances on monocyte recruitment, macrophage differentiation and dendritic cell (DC) responses in the peritoneal cavity of pristane-induced Lgals3-/- mice. A correlative analysis showed that mesenteric damages in the absence of Gal-3 were directly associated with severe portal inflammation and hepatitis. In conclusion, it has suggested that Gal-3 orchestrates histological organization in the mesentery and prevents lupoid hepatitis in experimental lupus-like syndrome by controlling macrophage polarization, Notch signaling pathways and DC differentiation in mesenteric structures.

Hypertonic saline solution (NaCl 7.2%) enhances renal excretion of acids in cattle with acute ruminal lactic acidosis.

The present study aimed to evaluate the efficiency of hypertonic saline solution (HSS) as a novel treatment of acute ruminal lactic acidosis (ARLA) in cattle, focusing on urinary excretion of acids. Twelve cannulated steers were submitted to experimentally induced ARLA by adminis- tering sucrose into the rumen. Twenty hours later, the cattle were randomly divided into two equal groups. The first group was treated with 7.5% HSS (5 mL/kg) over 15 min, and isotonic saline solution (ISS; 20 mL/kg) for the subsequent 165 minutes. The control group was administered ISS instead of HSS. Rumen and urine samples were collected at different times during the experiment from the baseline to 64 h post-induction. The induction caused a medium-to-moderate ruminal acidosis, and a moderate degree of systemic acidosis and dehydration. Steers treated with HSS increased by 50% its glomerular filtration rate (1.61 mL/min) compared to ISS group (1.06 mL/ min; p⟨0.03). The overall volume of urine excreted by HSS group was higher than that in ISS group (1.62 L vs 0.7 L; p⟨0.02). This increase in total volume of urine provided by HSS favored a greater excretion of H+ ions in urine, which was 3.39-fold higher in HSS group (64.3*10-7 vs 18.9*10-7 Mol) as well as lactate (241.7 vs 181.8 mMol) and P urinary excretion (3.8 vs 1.1 mMol) that reduced the urine pH (5.3 vs 5.7). Only the HSS group decreased significantly blood total lactic acid concentration (20.3 %) throughout the treatment. A positive relationship was found between the excretion of urinary phosphorus and urinary pH (r2=0.562). The results showed that this novel treatment with HSS enhanced renal excretion of acids and may be recommended as an additional treatment for cattle with lactic acidosis.

Genetic diversity of yacon accessions using ISSR markers.

Yacon cultivation has been intensified and the investigations of this crop have increased at the scientific, agricultural, and social levels because the roots of yacon show beneficial properties for human health, such as reducing cholesterol and glucose blood levels. Since the investigations involving yacon are very recent, there is little information available in terms of the genetic characterization of the cultivated genotypes. In view of the lack of information on the accessions cultivated in the State of Espírito Santo, Brazil, this pioneering study aimed to characterize 60 accessions cultivated in the state using ISSR yacon markers with emphasis on identifying the genetic diversity among the materials. The 20 ISSR primers used produced a total of 82 fragments, 39.6% of which presented polymorphism. The number of fragments per primer ranged from 1 to 10. The dissimilarity values ranged from 0 to 0.54 according to the Jaccard coefficient. A dendrogram was generated in which the accessions were divided into 3 groups; group 1 contained 58 individuals and groups 2 and 3 had only one individual in each group. The clustering of 58 accessions in a single group shows the low diversity in the materials examined. This low diversity indicates that new genotypes must be introduced in order to promote increased variability, which would minimize the adverse effects caused by biotic and abiotic factors.

Avaliação hemogasométrica, bioquímica e hematológica de ovinos suplementados com melão / Effects of sudden intake of melon on blood gas, biochemical and hematological parameters of sheep

O presente trabalho avaliou os efeitos da administração de duas diferentes quantidades de melão sobre variáveis hemogasométricas, bioquímicas e hematológicas de ovinos não adaptados. Foram utilizados 12 ovinos canulados, pesando 25kg de peso vivo, que nunca receberam ração concentrada. Os animais receberam dieta à base de feno (2,3% do peso vivo) e água à vontade. Os ovinos foram distribuídos aleatoriamente em dois grupos e receberam 25% ou 75% da matéria seca (MS) da dieta de melão triturado (G25% e G75%, respectivamente) diretamente no rúmen. Foram realizadas coletas de sangue e determinação do pH ruminal nos seguintes tempos: zero, 3, 6, 12, 18 e 24 horas após oferecimento do melão. Foi realizada análise hemogasométrica, do volume globular, determinação da concentração plasmática de lactato-L, glicose e osmolaridade sérica. No G25%, o pH sanguíneo variou entre 7,40 e 7,31, enquanto o G75% apresentou pH entre 7,38 e 7,26. Maiores concentrações de glicose plasmática foram detectadas no G75% no T3, T6 e T12 (P<0,05). Os ovinos que receberam 25% de melão mantiveram parâmetros sanguíneos dentro da normalidade, ao passo que, no G75%, os ovinos apresentaram discreta acidose metabólica sistêmica e hiperglicemia. A suplementação com 25% de melão pode ser uma alternativa segura na alimentação de ovinos.

This study evaluated the effects of two different amounts of melon on blood gas, biochemical and hematological variables of sheep not adapted. We used 12 cannulated sheep weighing 25 kg which never received concentrate. The animals received hay-based diet (2.3% of body weight) and water ad libitum. The sheep were randomly divided into two groups and received 25% or 75% of the dry matter (DM) of the diet of crushed melon (G25% and G75%, respectively) directly into the rumen. Blood collection and determination of ruminal pH were made at the following times: zero, three, six, 12, 18 and 24 hours after administration of the fruit. In whole blood was performed blood gas analysis and packed cell volume; in the plasma it was determined the concentrations of L-Lactate and glucose and in the serum the osmolarity. At G25% the blood pH ranged between 7.40 and 7.31, while G75% showed pH between 7.38 and 7.26. Higher concentrations of plasma glucose were detected in G75% after 3, 6 and 12 hours (P <0.05). Sheep receiving 25% of melon showed blood parameters within the normal range, while in the G75%, sheep had a mild systemic metabolic acidosis and hyperglycemia.

Pattern of inflammatory response to Loxosceles intermedia venom in distinct mouse strains: a key element to understand skin lesions and dermonecrosis by poisoning.

Envenomation caused by spiders Loxosceles induce intense dermonecrosis at the bite site and systemic disease. In this work we described the hyaluronidase and collagenase activities in vitro of the Loxosceles intermedia venom, but no phospholipase A2 activity. In vivo, we evaluated the effect of L. intermedia venom used different strain of mice, C57BL/6, BALB/c and Swiss. All mice developed paw edema after venom injection, persistent for 24 h in BALB/c and C57BL/6 mice. Histopathological analysis of the skin after venom injection revealed vascular congestion in Swiss mice and an inflammatory reaction in BALB/c and C57BL/6 mice. The mobilization of inflammatory cells from bone marrow, spleen and blood was investigated. Typical innate immune response with mobilization of myeloid cells and cytotoxic CD8 T lymphocytes was observed in C57BL/6 mice. In contrast, typical acquired/humoral immune response was observed in BALB/c mice, with preferential involvement of conventional B lymphocytes and CD4 T helper cells. The skin inflammation associated to mobilization of inflammatory cells indicated that mice models are strongly recommended to investigate specific cell types involved with immune response to the envenomation and mechanisms to inhibit skin lesions.

Peritoneal Submesothelial Stromal Cells Support Hematopoiesis and Differentiate into Osteogenic and Adipogenic Cell Lineages.

The peritoneum is a thin membrane that covers most of the abdominal organs, composed of a monolayer of mesothelial cells and subjacent submesothelial loose connective tissue. Cells from the peritoneal wall are correlated with peritoneal fibrosis and epithelial-to-mesenchymal transition. However, the distinct involvement of mesothelial or submesothelial cells in such phenomena is still not clear. Here, we propose a new strategy to obtain stromal cells from anterior peritoneal wall explant cultures. These cells migrated from peritoneal tissues and proliferated in vitro for 4 weeks as adherent fibroblast-like cells. Optical and electronic microscopy analyses of the fragments revealed a significant submesothelial disorganization. The obtained cells were characterized as cytokeratin- vimentin+ laminin+ α-smooth muscle actin+, suggesting a connective tissue origin. Moreover, at the third passage, these stromal cells were CD90+CD73+CD29+Flk-1+CD45-, a phenotype normally attributed to cells of mesenchymal origin. These cells were able to support hematopoiesis, expressing genes involved in myelopoiesis (SCF, G-CSF, GM-CSF, IL-7 and CXCL-12), and differentiated into osteogenic and adipogenic cell lineages. The methodology demonstrated in this work can be considered an excellent experimental model to understand the physiology of the peritoneal wall in healthy and pathological processes. Moreover, this work shows for the first time that submesothelial stromal cells have properties similar to those of mesenchymal cells from other origins.

Tris[2-(1,3-thiazol-2-yl-κN)-1H-benzimidazole-κN3]zinc(II) dinitrate ethanol hemisolvate monohydrate at 100 K.

The Zn(II) center in the dicationic complex of the title compound, [Zn(C(10)H(7)N(3)S)(3)](NO(3))(2)·0.5C(2)H(5)OH·H(2)O, is in a distorted octahedral environment with imperfect noncrystallographic C(3) symmetry. Each 2-(1,3-thiazol-2-yl)-1H-benzimidazole ligand coordinates in a bidentate manner, with the Zn-N(imidazole) bond lengths approximately 0.14 Å shorter than the Zn-N(thiazole) bond lengths. Charge-assisted hydrogen bonds connect cations, anions and water molecules. A lattice void is occupied by an ethanol solvent molecule disordered about a crystallographic inversion center and π-stacking is observed between one type of symmetry-related benzene rings.

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli.

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wild-type form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

(1S,2E,6R,7aR)-1,6-Dihy-droxy-2-(4-nitro-benzyl-idene)-2,3,5,6,7,7a-hexa-hydro-1H-pyrrolizin-3-one.

The crystal structure of the title compound, C(14)H(14)N(2)O(5), contains two distinct conformers in the asymmetric unit. The compound has three defined stereocenters, two of them contiguous, and a C=C double bond with an E conformation. The stereocenters exhibit the same chirality in both conformers, with significant differences in the conformation of the five-membered rings of the pyrrolizine unit (both either in a twist or in an envelope form) and in the dihedral angles between the corresponding mean planes and the benzene rings. A prominent feature is a change from almost coplanar rings in one conformer to a new conformation in the second conformer, in which the mean plane of a five-membered ring is almost perpendicular to the benzene ring, with a dihedral angle 87.19 (8)°; the corresponding angle in the first conformer is 14.02 (10)°. In the crystal, molecules are linked by O-H⋯O and C-H⋯O hydrogen bonds. Crystallographic data were essential to confirm the configuration of the double bond, which was unclear from the available two-dimensional NMR data. In addition, reliable Flack and Hooft parameters were obtained, allowing for the correct absolute structure to be determined.

(1S,2E,6R,7aR)-2-Benzyl-idene-1,6-dihy-droxy-2,3,5,6,7,7a-hexa-hydro-1H-pyrrolizin-3-one.

In the title compound, C(14)H(15)NO(3), the conformation of the double bond was determined to be E, confirming the result obtained from two-dimensional NMR data. The five-membered rings of the pyrrolizine unit exhibit C-envelope conformations, with C atoms displaced from the mean planes formed by the remaining rings atoms by 0.1468 (15) and 0.5405 (17) Å. The mean planes of these rings (through all ring atoms) have a dihedral angle of 49.03 (10)°. In the crystal, mol-ecules are linked by O-H⋯O hydrogen bonds. The absolute configuration of the mol-ecule was established, as judged by the, as judged by the obtained values for the Hooft and Flack parameters.

(1S,2S,6R,7aR)-2-Benzyl-1,6-dihy-droxy-hexa-hydro-pyrrolizin-3-one.

In the title compound, C(14)H(17)NO(3), the dihedral angles show that the H atoms at two stereocenters are in a trans-diaxial configuration. In the crystal, the molecules are linked by O-H⋯O hydrogen bonds. The absolute configuration of the molecule has been established on the basis of refinement of the Hooft and Flack parameters.

(1S,2R,6R,7aS)-1,2,6-Trihy-droxy-hexa-hydro-1H-pyrrolizin-3-one.

In the title compound, C(7)H(11)NO(4), prepared via a Morita-Baylis-Hillman adduct, the five-membered ring bearing three O atoms approximates to a twisted conformation, whereas the other ring is close to an envelope, with a C atom in the flap position. The dihedral angle between their mean planes (all atoms) is 23.11 (9)°. The new stereocenters are created in a trans-diaxial configuration. In the crystal, O-H⋯O and O-H⋯(O,O) hydrogen bonds link the mol-ecules, generating a three-dimensional network. A weak C-H⋯O inter-action also occurs.