Transformation of some 3α-substituted steroids by Aspergillus tamarii KITA reveals stereochemical restriction of steroid binding orientation in the minor hydroxylation pathway

Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17β-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11β-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3β-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3α-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11β-position (normal binding), indicating that the 3α-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1β-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11β-hydroxyl completely inhibited Baeyer–Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11β-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3α-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3α-acetate inhibited the Baeyer–Villiger monooxygenase with its activity being observed 24 h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17β-alcohol for the first time in this organism.     

Replanting of first‐cycle oil palm results in a second wave of biodiversity loss

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MEMS-Based Handheld Fourier Domain Doppler Optical Coherence Tomography for Intraoperative Microvascular Anastomosis Imaging

Purpose

To demonstrate the feasibility of a miniature handheld optical coherence tomography (OCT) imager for real time intraoperative vascular patency evaluation in the setting of super-microsurgical vessel anastomosis.

Methods

A novel handheld imager Fourier domain Doppler optical coherence tomography based on a 1.3-µm central wavelength swept source for extravascular imaging was developed. The imager was minimized through the adoption of a 2.4-mm diameter microelectromechanical systems (MEMS) scanning mirror, additionally a 12.7-mm diameter lens system was designed and combined with the MEMS mirror to achieve a small form factor that optimize functionality as a handheld extravascular OCT imager. To evaluate in-vivo applicability, super-microsurgical vessel anastomosis was performed in a mouse femoral vessel cut and repair model employing conventional interrupted suture technique as well as a novel non-suture cuff technique. Vascular anastomosis patency after clinically successful repair was evaluated using the novel handheld OCT imager.

Results

With an adjustable lateral image field of view up to 1.5 mm by 1.5 mm, high-resolution simultaneous structural and flow imaging of the blood vessels were successfully acquired for BALB/C mouse after orthotopic hind limb transplantation using a non-suture cuff technique and BALB/C mouse after femoral artery anastomosis using a suture technique. We experimentally quantify the axial and lateral resolution of the OCT to be 12.6 µm in air and 17.5 µm respectively. The OCT has a sensitivity of 84 dB and sensitivity roll-off of 5.7 dB/mm over an imaging range of 5 mm. Imaging with a frame rate of 36 Hz for an image size of 1000(lateral)×512(axial) pixels using a 50,000 A-lines per second swept source was achieved. Quantitative vessel lumen patency, lumen narrowing and thrombosis analysis were performed based on acquired structure and Doppler images.

Conclusions

A miniature handheld OCT imager that can be used for intraoperative evaluation of microvascular anastomosis was successfully demonstrated.     

Chemical and physical characteristics of carrageenan extracted from Eucheuma spinosum harvested from three different Indonesian coastal sea regions

Carrageenan extracted from Eucheuma spinosum harvested from three different coastal sea regions, where this alga has been mainly cultivated, were determined for their chemical and physical characteristics. The carrageenan was extracted from the seaweed using hot alkali followed by precipitation, drying, and milling. The carrageenan properties were determined in terms of yield, ash, mineral, sulfate content, functional group, molecular weight, and viscosity profile. Physical characteristics of carrageenan were evaluated by a texture analyzer for gel strength and a rapid visco analyzer for viscosity. The yield of carrageenan from Sumenep (34.81 ± 5.83%) and Takalar (37.16 ± 3.26%) was found to be relatively higher than that of Nusa Penida (25.81 ± 1.93%). The calcium content was higher than magnesium, potassium and sodium content, and no cadmium, lead, mercury, and arsenic detected in all carrageenan. The ash content was around 29%; while, the sulfate content was in the range of 30–32%, and those were not different in all carrageenan. The presence of sulfate content was identified by FTIR at absorption band of 1373 cm^?1. It was found that the molecular weight of carrageenan from Takalar were relatively higher and the gel strength of carrageenan from Takalar were significantly higher than that of carrageenan from Nusa Penida and Sumenep. Likewise, upon cooling from 80 to 20°C, the viscosity profile of carrageenan from Takalar characterized by higher viscosity compared to that of carrageenan from Sumenep and Nusa Penida. These results indicated that carrageenan from Nusa Penida, Sumenep, and Takalar were identified as iota‐carrageenan with similar physico‐chemical characteristics except for the gel strength, viscosity profile upon cooling from 80 to 20°C and the yield.     

Growth modulation of human cells in vitro by mild oxidative stress and 1,4-dihydropyridine derivative antioxidants

Reactive oxygen species and lipid peroxidation products are not only cytotoxic but may also modulate signal transduction in cells. Accordingly, antioxidants may be considered as modifiers of cellular redox signaling. Therefore, the effects of two novel synthetic antioxidants, analogues of 1,4-dihydropyridine derivatives, cerebrocrast and Z41-74 were analysed in vitro on human osteosarcoma cell line HOS, the growth of which can be modulated by lipid peroxidation. The cells were pretreated with either cerebrocrast or Z41-74 and afterwards exposed to mild, copper induced lipid peroxidation or to 4-hydroxynonenal (HNE), the end product of lipid peroxidation. The results obtained have shown that both antioxidants exert growth modulating effects interfering with the lipid peroxidation. Namely, cells treated with antioxidants showed increased metabolic rate and cell growth, thereby attenuating the effects of lipid peroxidation. Such biomodulating effects of cerebrocrast and Z41-74 resembled growth modulating effects of HNE, suggesting that the antioxidants could eventually promote cellular adaptation to oxidative stress interacting with redox signaling and hydroxynonenal HNE-signal transduction pathways. This may be of particular relevance for better understanding the beneficial role of hydroxynonenal HNE in cell growth control. Therefore, cerebrocrast and Z41-74 could be convenient to study further oxidative homeostasis involving lipid peroxidation.     

Quantifying killing of orangutans and human-orangutan conflict in Kalimantan, Indonesia

Human-orangutan conflict and hunting are thought to pose a serious threat to orangutan existence in Kalimantan, the Indonesian part of Borneo. No data existed prior to the present study to substantiate these threats. We investigated the rates, spatial distribution and causes of conflict and hunting through an interview-based survey in the orangutan's range in Kalimantan, Indonesia. Between April 2008 and September 2009, we interviewed 6983 respondents in 687 villages to obtain socio-economic information, assess knowledge of local wildlife in general and orangutan encounters specifically, and to query respondents about their knowledge on orangutan conflicts and killing, and relevant laws. This survey revealed estimated killing rates of between 750 and 1800 animals killed in the last year, and between 1950 and 3100 animals killed per year on average within the lifetime of the survey respondents. These killing rates are higher than previously thought and are high enough to pose a serious threat to the continued existence of orangutans in Kalimantan. Importantly, the study contributes to our understanding of the spatial variation in threats, and the underlying causes of those threats, which can be used to facilitate the development of targeted conservation management.     

Low serum MMP-1 in breast cancer: a negative prognostic factor?

In this study we investigated the prognostic significance of serum matrix metalloproteinase (MMP)-1 levels in early-stage breast cancer patients and correlated these levels with various clinicopathologic parameters. MMP-1 levels were determined by enzyme-linked immunosorbent assay. MMP-1 serum levels in patients (n = 60) were significantly lower than in healthy subjects (n = 20, p < 0.0001). We found significant negative correlation between serum levels of MMP-1 and several negative prognostic factors of breast cancer. Kaplan-Meier analysis showed significantly shorter 5-year survival in patients with lower values of MMP-1 compared to those with high levels of MMP-1 (p = 0.0147). Our results suggest a negative prognostic role of low serum MMP-1.     

An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17β side-chain.This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17β-acetoxy-5α-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17β-alcohol of testosterone.All steroidal metabolites were isolated by column chromatography and were identified by ¹H, ¹³C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.     

Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis,through Loss of Distinct kfi genes,Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health

Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.     

Comparative (meta)genomic analysis and ecological profiling of human gut-specific bacteriophage φB124-14

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome.