The topography of soft,adhesive diatom ‘trails’ as observed by Atomic Force Microscopy

Gliding diatoms foul surfaces by leaving behind ‘trails’ of secreted mucilage. Atomic force microscopy (AFM) used in ‘fluid tapping’ mode enabled the topography of the soft, adhesive trails in the natural hydrated state to be imaged, and without the artefacts resulting from fixation and/or dehydration. Diatom trails consist of a continuous, swollen ridge of material that dominates the trail, as well as a diffuse hydrated mucilage coating observed on either side of the main trail. The main trail material is evenly attached to the coverslip along its entire length, and appears to cure, or become less soft/adhesive, over time. Diatom trails observed with the scanning electron microscope were severely damaged by dehydration, while trails imaged by the AFM in ‘contact’ mode were damaged and/or removed by the action of the cantilever. The AFM used in ‘fluid tapping’ mode is an excellent tool for topographical studies of soft/adhesive biological molecules in the hydrated state, and will have great value for measuring their physical and mechanical properties when operated in ‘force modulation’ mode.     

Effects of Partial Replacement of Corn with Glycerin on Ruminal Fermentation in a Dual-Flow Continuous Culture System

The objective of this study was to evaluate the effects of partially replacing dry ground corn with glycerin on ruminal fermentation using a dual-flow continuous culture system. Six fermenters (1,223 ± 21 ml) were used in a replicated 3x3 Latin square arrangement with three periods of 10 d each, with 7 d for diet adaptation and 3 d for sample collections. All diets contained 75% concentrate and three dietary glycerin levels (0, 15, and 30% on dry matter basis), totaling six replicates per treatment. Fermenters were fed 72 g of dry matter/d equally divided in two meals/d, at 0800 and 2000 h. Solid and liquid dilution rates were adjusted daily to 5.5 and 11%/h, respectively. On d 8, 9, and 10, samples of 500 ml of solid and liquid digesta effluent were mixed, homogenized, and stored at -20°C. Subsamples of 10 ml were collected and preserved with 0.2 mL of a 50% H₂SO₄ solution for later determination of NH₃-N and volatile fatty acids. Microbial biomass was isolated from fermenters for chemical analysis at the end of each experimental period. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Glycerin levels did not affect apparent digestibility of DM (P _Lin. = 0.13; P _Quad. = 0.40), OM (P _Lin. = 0.72; P _Quad. = 0.15), NDF (P _Lin. = 0.38; P _Quad. = 0.50) and ADF (P _Lin. = 0.91; P _Quad. = 0.18). Also, glycerin inclusion did not affect true digestibility of DM (P _Lin. = 0.35; P _Quad. = 0.48), and OM (P _Lin. = 0.08; P _Quad. = 0.19). Concentrations of propionate (P < 0.01) and total volatile fatty acids (P < 0.01) increased linearly and concentrations of acetate (P < 0.01), butyrate (P = 0.01), iso-valerate (P < 0.01), and total branched-chain volatile fatty acids, as well as the acetate: propionate ratio (P < 0.01) decreased with glycerin inclusion. Linear increases on NH₃-N concentration in digesta effluent (P < 0.01) and on NH₃-N flow (P < 0.01) were observed due to glycerin inclusion in the diets. Crude protein digestibility (P = 0.04) and microbial N flow (P = 0.04) were greater in the control treatment compared with the other treatments and responded quadratically with glycerin inclusion. Furthermore, the inclusion of glycerin linearly decreased (P = 0.02) non-ammonia N flow. Glycerin levels did not affect the flows of total N (P _Lin. = 0.79; P _Quad. = 0.35), and dietary N (P _Lin. = 0.99; P _Quad. = 0.07), as well as microbial efficiency (P _Lin. = 0.09; P _Quad. = 0.07). These results suggest that partially replacing dry ground corn with glycerin may change ruminal fermentation, by increasing total volatile fatty acids, and propionate concentration without affecting microbial efficiency, which may improve glucogenic potential of beef cattle diets.     

Safe From Harm: Learned,Instructed, and Symbolic Generalization Pathways of Human Threat-Avoidance

Avoidance of threatening or unpleasant events is usually an adaptive behavioural strategy. Sometimes, however, avoidance can become chronic and lead to impaired daily functioning. Excessive threat-avoidance is a central diagnostic feature of anxiety disorders, yet little is known about whether avoidance acquired in the absence of a direct history of conditioning with a fearful event differs from directly learned avoidance. In the present study, we tested whether avoidance acquired indirectly via verbal instructions and symbolic generalization result in similar levels of avoidance behaviour and threat-beliefs to avoidance acquired after direct learning. Following fear conditioning in which one conditioned stimulus was paired with shock (CS+) and another was not (CS−), participants either learned or were instructed to make a response that cancelled impending shock. Three groups were then tested with a learned CS+ and CS− (learned group), instructed CS+ (instructed group), and generalized CS+ (derived group) presentations. Results showed similar levels of avoidance behaviour and threat-belief ratings about the likelihood of shock across each of the three pathways despite the different mechanisms by which they were acquired. Findings have implications for understanding the aetiology of clinical avoidance in anxiety.     

Subclinical Thyroid Dysfunction and Cognitive Decline in Old Age

Background

Subclinical thyroid dysfunction has been implicated as a risk factor for cognitive decline in old age, but results are inconsistent. We investigated the association between subclinical thyroid dysfunction and cognitive decline in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER).

Methods

Prospective longitudinal study of men and women aged 70–82 years with pre-existing vascular disease or more than one risk factor to develop this condition (N = 5,154). Participants taking antithyroid medications, thyroid hormone supplementation and/or amiodarone were excluded. Thyroid function was measured at baseline: subclinical hyper- and hypothyroidism were defined as thyroid stimulating hormones (TSH) <0.45 mU/L or >4.50 mU/L respectively, with normal levels of free thyroxine (FT4). Cognitive performance was tested at baseline and at four subsequent time points during a mean follow-up of 3 years, using five neuropsychological performance tests.

Results

Subclinical hyperthyroidism and hypothyroidism were found in 65 and 161 participants, respectively. We found no consistent association of subclinical hyper- or hypothyroidism with altered cognitive performance compared to euthyroid participants on the individual cognitive tests. Similarly, there was no association with rate of cognitive decline during follow-up.

Conclusion

We found no consistent evidence that subclinical hyper- or hypothyroidism contribute to cognitive impairment or decline in old age. Although our data are not in support of treatment of subclinical thyroid dysfunction to prevent cognitive dysfunction in later life, only large randomized controlled trials can provide definitive evidence.     

An accurate definition of the status of inactive hepatitis B virus carrier by a combination of biomarkers (FibroTest-ActiTest) and viral load

Background

The combination of transaminases (ALT), biopsy, HBeAg and viral load have classically defined the inactive status of carriers of chronic hepatitis B. The use of FibroTest (FT) and ActiTest (AT), biomarkers of fibrosis and necroinflammatory activity, has been previously validated as alternatives to biopsy. We compared the 4-year prognostic value of combining FT-AT and viral load for a better definition of the inactive carrier status.

Methods and Findings

1,300 consecutive CHB patients who had been prospectively followed since 2001 were pre-included. The main endpoint was the absence of liver-related complications, transplantation or death. We used the manufacturers'' definitions of normal FT (< = 0.27), normal AT (< = 0.29) and 3 standard classes for viral load. The adjustment factors were age, sex, HBeAg, ethnic origin, alcohol consumption, HIV-Delta-HCV co-infections and treatment.

Results

1,074 patients with baseline FT-AT and viral load were included: 41 years old, 47% African, 27% Asian, 26% Caucasian. At 4 years follow-up, 50 complications occurred (survival without complications 93.4%), 36 deaths occurred (survival 95.0%), including 27 related to HBV (survival 96.1%). The prognostic value of FT was higher than those of viral load or ALT when compared using area under the ROC curves [0.89 (95%CI 0.84–0.93) vs 0.64 (0.55–0.71) vs 0.53 (0.46–0.60) all P<0.001], survival curves and multivariate Cox model [regression coefficient 5.2 (3.5–6.9; P<0.001) vs 0.53 (0.15–0.92; P = 0.007) vs −0.001 (−0.003−0.000;P = 0.052)] respectively. A new definition of inactive carriers was proposed with an algorithm combining “zero” scores for FT-AT (F0 and A0) and viral load classes. This new algorithm provides a 100% negative predictive value for the prediction of liver related complications or death. Among the 275 patients with the classic definition of inactive carrier, 62 (23%) had fibrosis presumed with FT, and 3 died or had complications at 4 year.

Conclusion

In patients with chronic hepatitis B, a combination of FibroTest-ActiTest and viral load testing accurately defined the prognosis and the inactive carrier status.     

Systemic arteriopathy in SIV-infected rhesus macaques (Macaca mulatta)

BACKGROUND: Severe disseminated vasculopathy was observed in two simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). These animals developed clinical signs of AIDS, including lymphadenopathy, weight loss, diarrhea and collapse. RESULTS AND DISCUSSION: Grossly, both animals showed emaciation, lymphadenopathy, vegetations on the mitral valve, renal infarcts and a dilated intestine; one animal had multifocal hemorrhages in multiple organs. Histologically, both cases had disseminated arteriopathy characterized by intimal thickening and fibrosis with varying degrees of vasculitis. The lesion was prominent in the kidney, intestine, pancreas, liver, heart, lymph nodes, spleen and testis. Occasional venules had intimal thickening. Both cases had cytomegalovirus (CMV) infection with intranuclear inclusions, CMV antigen and nucleic acid; some inclusions were observed in endothelial cells within some of the vascular lesions in one of the two. These data suggest that CMV caused the unusual lesions.     

Molecular profile of the NF‐κB signalling pathway in human colorectal cancer

The development and progression of colorectal cancer (CRC) have been associated with inflammation processes that involve the overactivation of the NF‐κB signalling pathway. The characterization of the NF‐κB expression profile in CRC is an important topic since the suppression of NF‐κB represents a potential therapeutic approach. In this study, we assessed the expression levels of 84 NF‐κB‐related genes in paired tumoral (T) and peritumoral (PT) tissues from 18 CRC patients and 18 normal colonic mucosae, and the expression levels of three miRNAs targeting the most dysregulated genes revealed by the case–control analysis. Comparing the gene expression profile of T and controls, 60 genes were dysregulated. The comparison of T and PT revealed 17 dysregulated genes in the tumoral tissues, with IL1B, CXCL8, IL1A, and CSF2 being the most upregulated. Notably, through a bioinformatics analysis, the differential gene expression of 11 out of the 17 genes was validated on a larger cohort of 308 CRC patients compared with 41 controls. Moreover, a decrease in the levels of RELA, NOD1, CASP8, BCL2L1, ELK1, and IKBKB was identified in poorly differentiated tumours compared to moderately differentiated tumours. The analysis of the three miRNAs targeting IL1B, CXCL8, IL1A, and CSF2 showed that miR‐182‐5p was upregulated in T compared with PT, whereas miR‐10b‐5p was downregulated in T compared with PT and control tissues. Our results may contribute to the design of new experimental therapeutic strategies based on endogenous molecules, such as miRNAs, to target the genetic key players of the NF‐ κB pathway.     

Discovery of cell-active phenyl-imidazole Pin1 inhibitors by structure-guided fragment evolution

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-μM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.     

Optimisation of an oviposition protocol employing human chorionic and pregnant mare serum gonadotropins in the Barred Frog Mixophyes fasciolatus (Myobatrachidae)

ABSTRACT: BACKGROUND: Protocols for the hormonal induction of ovulation and oviposition are essential tools for managing threatened amphibians with assisted reproduction, but responses vary greatly between species and even broad taxon groups. Consequently, it is necessary to assess effectiveness of such protocols in representative species when new taxa become targets for induction. The threatened genus Mixophyes (family Myobatrachidae) has amongst the highest proportion of endangered species of all the Australian amphibians. This study developed and optimised the induction of oviposition in a non-threatened member of this taxon, the great barred frog (Mixophyes fasciolatus). METHODS: Gravid female M. fasciolatus were induced to oviposit on one or more occasions by administration of human chorionic gonadotropin (hCG) with or without priming with pregnant mare serum gonadotropin (PMSG). Treatments involved variations in hormone doses and combinations (administered via injection into the dorsal lymph sacs), and timing of administration. Pituitary homogenates from an unrelated bufonid species (Rhinella marina) were also examined with hCG. RESULTS: When injected alone, hCG (900 to 1400 IU) induced oviposition. However, priming with two time dependent doses of PMSG (50 IU, 25 IU) increased responses, with lower doses of hCG (200 IU). Priming increased response rates in females from around 30% (hCG alone) to more than 50% (p = 0.035), and up to 67%. Increasing the interval between the first PMSG dose and first hCG dose from 3 to 6 days also produced significant improvement (p<0.001). Heterologous pituitary extracts administered with hCG were no more effective than hCG alone (p = 0.628). CONCLUSIONS: This study found that M. fasciolatus is amongst the few amphibian species (including Xenopus (Silurana) and some bufonids) that respond well to the induction of ovulation utilising mammalian gonadotropins (hCG). The optimal protocol for M. fasciolatus involved two priming doses of PMSG (50 IU and 25 IU) administered at 6 and 4 days respectively, prior to two doses of hCG (100 IU), 24 hours apart. This study is also the first to demonstrate in an amphibian species that responds to mammalian gonadotropins that an increase in the ovulation rate occurs after priming with a gonadotropin (PMSG) with FSH activity.     

Characterisation of Ilomastat for Prolonged Ocular Drug Release

We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.