Adenylate Energy Charge in Escherichia coli During Growth and Starvation

The value of the adenylate energy charge, [(adenosine triphosphate) + (1/2) (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types.     

Perforin Promotes Amyloid Beta Internalisation in Neurons

Studies on the mechanisms of neuronal amyloid-β (Aβ) internalisation are crucial for understanding the neuropathological progression of Alzheimer’s disease (AD). We here investigated how extracellular Aβ peptides are internalised and focused on three different pathways: (i) via endocytic mechanisms, (ii) via the receptor for advanced glycation end products (RAGE) and (iii) via the pore-forming protein perforin. Both Aβ₄₀ and Aβ₄₂ were internalised in retinoic acid differentiated neuroblastoma (RA-SH-SY5Y) cells. A higher concentration was required for Aβ₄₀ (250 nM) compared with Aβ₄₂ (100 nM). The internalised Aβ₄₀ showed a dot-like pattern of distribution whereas Aβ₄₂ accumulated in larger and distinct formations. By confocal microscopy, we showed that Aβ₄₀ and Aβ₄₂ co-localised with mitochondria, endoplasmic reticulum (ER) and lysosomes. Aβ treatment of human primary cortical neurons (hPCN) confirmed our findings in RA-SH-SY5Y cells, but hPCN were less sensitive to Aβ; therefore, a 20 (Aβ₄₀) and 50 (Aβ₄₂) times higher concentration was needed for inducing uptake. The blocking of endocytosis completely inhibited the internalisation of Aβ peptides in RA-SH-SY5Y cells and hPCN, indicating that this is a major pathway by which Aβ enters the cells. In addition, the internalisation of Aβ₄₂, but not Aβ₄₀, was reduced by 55 % by blocking RAGE. Finally, for the first time we showed that pore formation in cell membranes by perforin led to Aβ internalisation in hPCN. Understanding how Aβ is internalised sheds light on the pathological role of Aβ and provides further ideas of inhibitory strategies for preventing Aβ internalisation and the spreading of neurodegeneration in AD.     

Evaluation of the bony repair in rat cranial defect using near infrared reflectance spectroscopy and discriminant analysis

We set out to determine whether near infrared reflectance spectroscopy (NIRS) combined with principal component analysis–linear discriminant analysis (LDA) or, variable selection techniques employing successive projection algorithm or genetic algorithm (GA) could evaluate the bone repair in cranial critical‐size (5 mm) defect after stimulation with collagen sponge scaffold and/or infrared low‐level laser therapy directly on the local. Forty‐five Winstar rats were divided into nine groups of five each, namely: group H – healthy, n = 5 (without treatment and without cranial critical‐size defect), (GI positive control – n = 5, 21 days or n = 5, 30 days) without treatment and with cranial critical‐size defect; (GII‐n = 5, 21 days or n = 5, 30 days) cranial critical‐size defect filled with collagen sponge scaffold; (GIII–n = 5, 21 days or n = 5, 30 days) cranial critical‐size defect submitted to low‐level laser therapy; (GIV–n = 5, 21 days or n = 5, 30 days) cranial critical‐size defect submitted to combined collagen sponge scaffold + low‐level laser therapy treatment. In relation to the histological analysis, the collagen sponge scaffold + low‐level laser therapy treatment group (GIV) 30 days showed the best result with the presence of secondary bone, immature bone (osteoid) and newly formed connective tissue (periosteum). GA–LDA model also successfully classified control class of the others classes. Thus, the results provided by the good‐quality classification model revealed the feasibility of NIRS for application to evaluation of the wound healing in rat cranial defect, thanks to the short analysis time of a few seconds and nondestructive advantages of NIRS as an alternative approach for bone repair purposes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1160–1168, 2017     

Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.     

A randomised, blinded, placebo-controlled trial in dementia patients continuing or stopping neuroleptics (the DART-AD trial)

Background

There have been increasing concerns regarding the safety and efficacy of neuroleptics in people with dementia, but there are very few long-term trials to inform clinical practice. The aim of this study was to determine the impact of long-term treatment with neuroleptic agents upon global cognitive decline and neuropsychiatric symptoms in patients with Alzheimer disease.

Methods and Findings

Design: Randomised, blinded, placebo-controlled parallel two-group treatment discontinuation trial.Setting: Oxfordshire, Newcastle and Gateshead, London and Edinburgh, United Kingdom.Participants: Patients currently prescribed the neuroleptics thioridazine, chlorpromazine, haloperidol trifluoperazine or risperidone for behavioural or psychiatric disturbance in dementia for at least 3 mo.Interventions: Continue neuroleptic treatment for 12 mo or switch to an identical placebo.Outcome measures: Primary outcome was total Severe Impairment Battery (SIB) score. Neuropsychiatric symptoms were evaluated with the Neuropsychiatric Inventory (NPI).Results: 165 patients were randomised (83 to continue treatment and 82 to placebo, i.e., discontinue treatment), of whom 128 (78%) commenced treatment (64 continue/64 placebo). Of those, 26 were lost to follow-up (13 per arm), resulting in 51 patients per arm analysed for the primary outcome. There was no significant difference between the continue treatment and placebo groups in the estimated mean change in SIB scores between baseline and 6 mo; estimated mean difference in deterioration (favouring placebo) −0.4 (95% confidence interval [CI] −6.4 to 5.5), adjusted for baseline value (p = 0.9). For neuropsychiatric symptoms, there was no significant difference between the continue treatment and placebo groups (n = 56 and 53, respectively) in the estimated mean change in NPI scores between baseline and 6 mo; estimated mean difference in deterioration (favouring continue treatment) −2.4 (95% CI −8.2 to 3.5), adjusted for baseline value (p = 0.4). Both results became more pronounced at 12 mo. There was some evidence to suggest that those patients with initial NPI ≥ 15 benefited on neuropsychiatric symptoms from continuing treatment.

Conclusions

For most patients with AD, withdrawal of neuroleptics had no overall detrimental effect on functional and cognitive status. Neuroleptics may have some value in the maintenance treatment of more severe neuropsychiatric symptoms, but this benefit must be weighed against the side effects of therapy.Trial registration: Cochrane Central Registry of Controlled Trials/National Research Register (#ISRCTN33368770).     

Improving potency and selectivity of a new class of non-Zn-chelating MMP-13 inhibitors

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1′ pocket.