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Summary: In recent years, studies have shed light on the physiological role of plant glutamate receptor‐like channels (GLRs). However, the mechanism by which these channels are activated, and in particular, what is the physiological role of their binding to amino acids, remains elusive. The first direct biochemical demonstration that the Arabidopsis thaliana GLR3.3 isoform binds glutamate and other amino acids in a low micromolar range of concentrations was reported only recently. The first crystal structures of the ligand‐binding domains of AtGLR3.3 and AtGLR3.2 isoforms also have been released. We foresee that these new experimental pieces of evidence provide the basis for a better understanding of how GLRs are activated and modulated in different physiological responses. [ABSTRACT FROM AUTHOR]

Unilateral trade preferences are one of the most important instruments offered by developed countries to foster developing country exports. This paper analyzes the impact of unilateral trade preferences on developing countries by focusing on the experience of Mozambique. In this paper, we analyze whether unilateral preferences offered by the EU are "valuable" for Mozambican exporters based on the impact on preferential margins, utilization rates, and export prices. We use a detailed dataset with cif unit values at HS8-digits level covering the period 2000-2007. Our findings indicate that (i) for a large number of product lines, export margins are zero; (ii) utilization rates are generally high; however, (iii) this does not translate into a positive price margins captured by Mozambican exporters compared to MFN competitors. These findings cast doubts on the "value" of preferences and their potential impact on developing country exports. [ABSTRACT FROM AUTHOR]

Abstract: High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies. [Copyright &y& Elsevier]

Purpose  This study aims to evaluate the safety and efficacy of autologous aspirated and purified fat tissue injected percutaneously into the knee joint for the treatment of symptomatic osteoarthritis (OA). Methods  We reviewed 30 patients, who received an autologous percutaneous fat injection for the treatment of knee OA, from January 2012 to March 2015. Mean patients' age was 63.3 ± 5.3 years (range, 50-80 years). Body mass index was 25.1 ± 1.7. Clinical evaluation was based on pain visual analog scale (VAS) and WOMAC score for functional and subjective assessment. We also noted the adverse reactions and the consumption of nonsteroidal anti-inflammatory drugs in the posttreatment period. Results  All patients reported improvements with respect to pain: average VAS was 7.7 ± 1.2 at baseline, 5.2 ± 0.2 at 1-month follow-up, and 4.3 ± 1 at 3-month follow-up. A slight deterioration (5.0 ± 1.1) was evidenced at 1 year. Total WOMAC score was 89.9 ± 1.7 at baseline, 66.3 ± 1 at 1 month, 68.6 ± 1.7 at 3 months, and 73.2 ± 1.8 at 12 months of follow-up. Conclusion  Our preliminary findings suggest that autologous percutaneous fat injections are a valid treatment option for knee OA. Level of Evidence  Level IV, therapeutic case series.

Polarized epithelia form by oriented cell divisions in which the mitotic spindle aligns parallel to the epithelial plane. To orient the mitotic spindle, cortical cues trigger the recruitment of NuMA-dynein-based motors, which pull on astral microtubules via the protein LGN. We demonstrate that the junctional protein Afadin is required for spindle orientation and correct epithelial morphogenesis of Caco-2 cysts. Molecularly, Afadin binds directly and concomitantly to F-actin and to LGN. We determined the crystallographic structure of human Afadin in complex with LGN and show that it resembles the LGN-NuMA complex. In mitosis, Afadin is necessary for cortical accumulation of LGN and NuMA above the spindle poles, in an F-actin-dependent manner. Collectively, our results depict Afadin as a molecular hub governing the enrichment of LGN and NuMA at the cortex. To our knowledge, Afadin is the first-described mechanical anchor between dynein and cortical F-actin.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a central role in the regulation of cardiac and neuronal firing rate, and these channels can be dually activated by membrane hyperpolarization and by binding of cyclic nucleotides. cAMP has been shown to directly bind HCN channels and modulate their activity. Despite this, while there are selective inhibitors that block the activation potential of the HCN channels, regulation by cAMP analogs has not been well investigated. A comprehensive screen of 47 cyclic nucleotides with modifications in the nucleobase, ribose moiety, and cyclic phosphate was tested on the three isoforms HCN1, HCN2, and HCN4. 7-CH-cAMP was identified to be a high affinity binder for HCN channels and crosschecked for its ability to act on other cAMP receptor proteins. While 7-CH-cAMP is a general activator for cAMP- and cGMP-dependent protein kinases as well as for the guanine nucleotide exchange factors Epac1 and Epac2, it displays the highest affinity to HCN channels. The molecular basis of the high affinity was investigated by determining the crystal structure of 7-CH-cAMP in complex with the cyclic nucleotide binding domain of HCN4. Electrophysiological studies demonstrate a strong activation potential of 7-CH-cAMP for the HCN4 channel in vivo. So, this makes 7-CH-cAMP a promising activator of the HCN channels in vitro whose functionality can be translated in living cells.