The effect of age on protein synthesis in mouse liver

1. A system of microsomes and 105000g supernatant from livers of old mice is less able to promote the incorporation of [(14)C]phenylalanine into protein than a similar system from livers of young animals. 2. The decrease in [(14)C]phenylalanine incorporation is attributable to changes in microsomes from old animals rather than in the cell-sap fraction. 3. Decreased synthetic ability is found in various classes of microsomes from older animals, namely unfractionated, light and heavy microsomes, but not in detergent-washed ribonucleoprotein particles. 4. Deletions of certain detergent-soluble microsomal proteins accompany the decreased synthetic ability of microsomes from older animals. 5. Microsomes from old mice are less responsive to a synthetic messenger RNA, polyuridylic acid, and this is partly due to a higher rate of hydrolysis in the presence of cell sap from animals of extreme age. 6. Other more direct evidence, from the priming of a cell-free protein-synthesizing system from bacteria and the examination of ribonucleoprotein particles on sucrose density gradients, suggests that senescence is accompanied by a decrease in messenger RNA content.     

Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells

Metastatic colorectal cancer remains a serious health concern with poor patient survival. Although 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) is the standard therapy for colorectal cancer, it has met with limited success. Recurrence of the tumor after chemotherapy could partly be explained by the enrichment of the chemo-resistant sub-population of cancer stem cells (CSCs) that possess the ability for self-renewal and differentiation into different lineages in the tumor. Therefore development of therapeutic strategies that target CSCs for successful treatment of this malignancy is warranted. The current investigation was undertaken to examine the effectiveness of the combination therapy of dasatinib (a Src inhibitor) and curcumin (a dietary agent with pleiotropic effect) in inhibiting the growth and other properties of carcinogenesis of chemo-resistant colon cancer cells that are enriched in CSCs sub-population. Remnants of spontaneous adenomas from APC ^{Min +/-} mice treated with dasatinib and/or curcumin were analyzed for several cancer stem cell markers (ALDH, CD44, CD133 and CD166). Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant) and HT-29 (p53 mutant; K-ras wild type) were used to generate FOLFOX resistant (referred to as CR) cells. The effectiveness of the combination therapy in inhibiting growth, invasive potential and stemness was examined in colon cancer CR cells. The residual tumors from APC ^{Min +/-} mice treated with dasatinib and/or curcumin showed 80-90% decrease in the expression of the CSC markers ALDH, CD44, CD133, CD166. The colon cancer CR cells showed a higher expression of CSCs markers, cell invasion potential and ability to form colonospheres, compared to the corresponding parental cells. The combination therapy of dasatinib and curcumin demonstrated synergistic interactions in CR HCT-116 and CR HT-29 cells, as determined by Calcusyn analysis. The combinatorial therapy inhibited cellular growth, invasion and colonosphere formation and also reduced CSC population as evidenced by the decreased expression of CSC specific markers: CD133, CD44, CD166 and ALDH. Our data suggest that the combination therapy of dasatinib and curcumin may be a therapeutic strategy for re-emergence of chemo-resistant colon cancer by targeting CSC sub-population.     

Potential fields and barriers to diffusion in narrow cylindrical capillaries

In this paper, the wall potential along the center line of narrow solid capillaries has been derived. The potential barriers at the open end of such capillaries have been studied in detail. The influence of these potential barriers on the diffusion coefficients and their dependence on temperature and capillary radius have been evaluated. The implications of these energy barriers in the clarification of low pressure hysteresis phenomena have been pointed out.     

Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells

Introduction

Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.

Objectives

The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.

Methods

To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.

Results

Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.

Conclusion

Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.

     

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.     

Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs

Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the “distributed manufacturing” approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.     

Conformity to Bergmann's rule in birds depends on nest design and migration

Ecogeographical rules attempt to explain large‐scale spatial patterns in biological traits. One of the most enduring examples is Bergmann''s rule, which states that species should be larger in colder climates due to the thermoregulatory advantages of larger body size. Support for Bergmann''s rule, however, is not consistent across taxonomic groups, raising questions about what factors may moderate its effect. Behavior may play a crucial, yet so far underexplored, role in mediating the extent to which species are subject to environmental selection pressures in colder climates. Here, we tested the hypothesis that nest design and migration influence conformity to Bergmann''s rule in a phylogenetic comparative analysis of the birds of the Western Palearctic, a group encompassing dramatic variation in both climate and body mass. We predicted that migratory species and those with more protected nest designs would conform less to the rule than sedentary species and those with more exposed nests. We find that sedentary, but not short‐ or long‐distance migrating, species are larger in colder climates. Among sedentary species, conformity to Bergmann''s rule depends, further, on nest design: Species with open nests, in which parents and offspring are most exposed to adverse climatic conditions during breeding, conform most strongly to the rule. Our findings suggest that enclosed nests and migration enable small birds to breed in colder environments than their body size would otherwise allow. Therefore, we conclude that behavior can substantially modify species’ responses to environmental selection pressures.     

Cytophotometric evidence of variation in genome size of desmognathine salamanders

Summary The amount of DNA per haploid genome, the C-value, is often directly correlated with nuclear and cell volume, but inversely correlated with cell replication rate. Also, rates of cellular growth sometimes appear to be correlated with organismal developmental rates and life history patterns. Among vertebrates, salamanders exhibit the greatest variation in genome size. In the present study we have examined interspecific and intraspecific variation in blood cell DNA levels in the genus Desmognathus, which shows greater variation in life history traits than any other salamander genus. Specimens of Desmognathus quadramaculatus, D. Monticola, D. ochrophaeus and D. wrighti were collected from nature at two localities in the southern Appalachian Mountains. Estimates of genome size in pg of DNA were obtained from blood smears by DNA-Feulgen cytophotometry, using erythrocyte nuclei of Xenopus laevis as an internal reference standard of 6.35 pg DNA per cell. C-values of Desmognathus are the smallest in the order Caudata. Although significant variation in DNA levels was found among the four species, the differences were small, and do not support previously proposed relationships between C-value and life-history variation.