CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.     

Lateral position preference in grazing feral horses

Behavioural lateralisation is an effective way for animals to manage daily tasks by specialising behaviour to either side of the body. Many types of lateralisation are linked to the function of each brain hemisphere. Lateralisation of monitoring behaviour in mother–infant relationships occurs in a wide range of mammals, where infants frequently use their left eye to monitor their mother. However, few studies have focused on this type of spatial relationships among adults in daily life, such as during foraging. The present study focused on monitoring adult feral horse behaviour using quantitative analysis of spatial relationships, using drone technology. We found that horses form a localised spatial relationship with their nearest neighbour. Specifically, the nearest neighbour was located to the left rear of a target individual significantly more frequently than to the right rear. Furthermore, the nearest neighbour was less frequently located behind a target individual. We propose that this relationship is caused by a left-eye preference, because information via the left eye predominantly proceeds to the right hemisphere, which is dominant for social processing.     

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [¹⁴C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T₄ and the role of the integrin receptor in this event, and also to clarify whether the T₄ effect on MeAIB accumulation and on Ca²⁺ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T₄ by using ⁴⁵Ca²⁺ uptake and [⁴⁵C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T₄ appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T₄ increases extracellular Ca²⁺ uptake and Ca²⁺ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T₄. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T₄ integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T₄ may take a role in plasma membrane processes involved in the male reproductive system.     

Diplospory and obligate apomixis in Miconia albicans (Miconieae, Melastomataceae) and an embryological comparison with its sexual congener M. chamissois

Apomixis, or asexual reproduction through seeds, has been reported for species of the tribe Miconieae, Melastomataceae, but details of the process have yet to be described. We analyzed and compared sporogenesis and gametogenesis in the apomictic Miconia albicans and the sexual M. chamissois. The results point to some differences between species, which were related to the apomictic process. In M. albicans microsporogenesis, problems during meiosis and degeneration of its products led to total pollen sterility, while M. chamissois presented normal bicellular pollen grains in the mature anther. The absence or abnormality of meiosis in M. albicans megasporogenesis led to the formation of an unreduced embryo sac and also to egg cell parthenogenesis, which gave rise to the apomictic embryo. Embryo and endosperm development were autonomous, resulting in seeds and fruits independent of pollination and fertilization. Thus, in this species, apomixis can be classified as diplosporic and obligate. In contrast, meiosis was as expected in the sexual M. chamissois, and led to the development of a reduced embryo sac. Despite the divergent pathways, many embryological characteristics were similar between the studied species and other Melastomataceae and they seem to be conservative character states for the family.     

Antimicrobial effects of terpinen-4-ol against oral pathogens and its capacity for the modulation of gene expression

Abstract

This study evaluated the antibacterial activity of terpinen-4-ol against Streptococcus mutans and Lactobacillus acidophilus and its influence on gbpA (S. mutans) and slpA (L. acidophilus) gene expression. As measured by XTT assay, the concentrations of terpinen-4-ol that effectively inhibited the biofilm were 0.24% and 0.95% for S. mutans and L. acidophilus, respectively. Confocal microscopy revealed the presence of a biofilm attached to the enamel and dentin block surfaces with significant terpinen-4-ol effects against these microorganisms. The expression of the gbpA and slpA genes involved in adherence and biofilm formation was investigated using RT-PCR. Expression of these genes decreased after 15?min with 0.24% and 0.95% terpinen-4-ol in S. mutans and L. acidophilus, respectively. These findings demonstrate the antimicrobial activity of terpinen-4-ol and its ability to modulate the expression of gbpA and slpA genes, emphasizing the therapeutic capacity of terpinen-4-ol as an alternative to inhibit adherence in biofilm.     

Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.     

Wild plants eaten in childhood: a retrospective of Estonia in the 1970s–1990s

In this ethnobotanical study, the authors provide the first quantitative analysis of the use of wild edible plants in Estonia, describing the domains and assessing the food importance of different species. The information was collected using free‐listing written questionnaires and concerned plants used by the respondents in their childhood. As part of a major study, this article covers the responses of professionals with some botanical education at vocational or university level, to ensure the greatest possible reliability without using voucher specimens. Fifty‐eight respondents provided information on the use of 137 plant taxa, corresponding to approximately 6% of the native and naturalized vascular plants of Estonia. According to use frequency, the most typical wild food plant of Estonia is a fruit, eaten raw as a snack. The results clearly signal that the majority of famine and food shortage plants had already been forgotten by the end of the 20^th century, but new plants have been introduced as green vegetables for making salads. Despite changes in the nomenclature of the plants, the use of wild food plants in Estonia was still thriving at the turn of the 20^th century, covering many domains already forgotten in urbanized modern Europe. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 239–253.     

Activity of tocopherol oxidase in Phaseolus coccineus seedlings

In the present study, we have demonstrated that membrane-free extracts of etiolated shoots of Phaseolus coccineus seedlings show tocopherol oxidase activity. For this reaction, presence of membrane lipids, such as lecithin and mixture of plant lipids was required. The rate of the reaction was the highest for α-tocopherol and decreased in the order α ? β > γ > δ tocopherols. In the case of α-tocopherol, the main oxidation product was α-tocopherolquinone, while for the other tocopherol homologues the dominant products were other derivatives. When the enzyme activity was measured in leaves, hypocotyls and roots of etiolated seedlings of P. coccineus, the oxidase activity was the highest in extracts of leaves and decreased towards the roots where no activity was detected. The effect of hydrogen peroxide and of different inhibitors on the reaction suggest that tocopherol oxidase does not belong to peroxidases or flavin oxidases but rather to multi-copper oxidases, such as polyphenol oxidases or laccases. On the other hand, catechol, the well-known substrate of polyphenol oxidases and laccases, was not oxidized by the enzyme, indicating a high substrate specificity of the tocopherol oxidase.