A novel method for cloning of coding sequences of highly toxic proteins

Background

During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process.

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.     

Studies on solvatochromic properties of aminophenylstyryl-quinolinum dye, LDS 798, and its application in studying submicron lipid based structure

The styryl group of dyes has been used in cellular studies for over 20 years because of their solvatochromic and/or electrochromic properties. Here we report characterization of solubility and solvatochromic properties of a near infra-red styryl dye, styryl 11 or LDS 798. We have extended our studies to small unilamellar vesicles and lipid based nanoparticles and found that solvatochromic properties of this dye used in tandem with fluorescence correlation spectroscopy can be used to efficiently determine the diffusion coefficient and hence the size of the submicron lipid based particles. This technique has the potential to provide essential information about liposomal and vesicular structures and their movement in vitro and in situ.     

Optimal closed-loop deep brain stimulation using multiple independently controlled contacts

Deep brain stimulation (DBS) is a well-established treatment option for a variety of neurological disorders, including Parkinson’s disease and essential tremor. The symptoms of these disorders are known to be associated with pathological synchronous neural activity in the basal ganglia and thalamus. It is hypothesised that DBS acts to desynchronise this activity, leading to an overall reduction in symptoms. Electrodes with multiple independently controllable contacts are a recent development in DBS technology which have the potential to target one or more pathological regions with greater precision, reducing side effects and potentially increasing both the efficacy and efficiency of the treatment. The increased complexity of these systems, however, motivates the need to understand the effects of DBS when applied to multiple regions or neural populations within the brain. On the basis of a theoretical model, our paper addresses the question of how to best apply DBS to multiple neural populations to maximally desynchronise brain activity. Central to this are analytical expressions, which we derive, that predict how the symptom severity should change when stimulation is applied. Using these expressions, we construct a closed-loop DBS strategy describing how stimulation should be delivered to individual contacts using the phases and amplitudes of feedback signals. We simulate our method and compare it against two others found in the literature: coordinated reset and phase-locked stimulation. We also investigate the conditions for which our strategy is expected to yield the most benefit.     

Sequence-specific interaction between mitochondrial Fe-S scaffold protein Isu and Hsp70 Ssq1 is essential for their in vivo function

Isu, the scaffold for assembly of Fe-S clusters in the yeast mitochondrial matrix, is a substrate protein for the Hsp70 Ssq1 and the J-protein Jac1 in vitro. As expected for an Hsp70-substrate interaction, the formation of a stable complex between Isu and Ssq1 requires Jac1 in the presence of ATP. Here we report that a conserved tripeptide, PVK, of Isu is critical for interaction with Ssq1 because amino acid substitutions in this tripeptide inhibit both the formation of the Isu-Ssq1 complex and the ability of Isu to stimulate the ATPase activity of Ssq1. These biochemical defects correlate well with the growth defects of cells expressing mutant Isu proteins. We conclude that the Ssq1-Isu substrate interaction is critical for Fe-S cluster biogenesis in vivo. The ability of Jac1 and mutant Isu proteins to cooperatively stimulate the ATPase activity of Ssq1 was also measured. Increasing the concentration of Jac1 and mutant Isu together but not individually partially overcame the effect of the reduced affinity of the Isu mutant proteins for Ssq1. These results, along with the observation that overexpression of Jac1 was able to suppress the growth defect of an ISU mutant, support the hypothesis that Isu is "targeted" to Ssq1 by Jac1, with a preformed Jac1-Isu complex interacting with Ssq1.     

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmoniers algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.     

Bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E1

We evaluated in a double-blind study the bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E₁ (PGE₁-carbinol), described recently as a nonirritant bronchodilator in animals. Fifteen asthmatic patients received by inhalation single doses of 1, 10, and 30 μg PGE₁-carbinol, 55 μg PGE₂, and placebo (10% ethanol in normal saline, which was also used as diluent for the PGs). Such pulmonary function tests as forced expiratory volume in 1 second, forced vital capacity, and maximal expiratory flow were monitored during 2 hours following inhalation of each compound. 10 and 30 μg PGE₁-carbinol produced significant but short-acting bronchodilation, similar to that caused by 55 μg PGE₂. One-third of the patients reported mild cough and throat irritation during and shortly after inhalation of 30 μg PGE₁-carbinol or 55 μg PGE₂. Placebo and 1 μg PGE₁-carbinol produced minimal side effects, but neither agent caused bronchodilation. In an adjunctive, unblinded trial, the same patients received 400 μg fenoterol. Fenoterol caused greater bronchodilation 15 and 30 minutes after inhalation than did the PGs in the double-blind study.     

Protease Activated Receptor-2 Contributes to Heart Failure

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.     

Ssq1, a mitochondrial Hsp70 involved in iron-sulfur (Fe/S) center biogenesis. Similarities to and differences from its bacterial counterpart

The results of in vivo and in organellar experiments indicate that the Hsp70 Ssq1 and the J-protein Jac1 function together to assist in the biogenesis of iron-sulfur (Fe/S) centers in the mitochondrial matrix. Here we present biochemical evidence supporting this idea. Isu, the proposed scaffold on which Fe/S centers are assembled, is a substrate for both Jac1 and Ssq1. Jac1 and Isu1 cooperatively stimulate the ATPase activity of Ssq1. In addition, Jac1 facilitates the interaction of Ssq1 with Isu1 in the presence of ATP. These findings are consistent with the role in Fe/S biogenesis previously proposed for the bacterial Hsp70 Hsc66 and J-protein Hsc20 that interact with the bacterial Isu homologue IscU. However, unlike the bacterial Hsp70, we found that Ssq1 has a high affinity for nucleotide, and shares a nucleotide exchange factor, Mge1, with a second mitochondrial Hsp70, Ssc1. Thus, whereas the bacterial and mitochondrial chaperone systems share critical features, they possess significant biochemical differences as well.     

Streptococcus pneumoniae Coinfection Is Correlated with the Severity of H1N1 Pandemic Influenza

Background

Initial reports in May 2009 of the novel influenza strain H1N1pdm estimated a case fatality rate (CFR) of 0.6%, similar to that of seasonal influenza. In July 2009, however, Argentina reported 3056 cases with 137 deaths, representing a CFR of 4.5%. Potential explanations for increased CFR included virus reassortment or genetic drift, or infection of a more vulnerable population. Virus genomic sequencing of 26 Argentinian samples representing both severe and mild disease indicated no evidence of reassortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence. Furthermore, no evidence was found for increased frequency of risk factors for H1N1pdm disease.

Methods/Principal Findings

We examined nasopharyngeal swab samples (NPS) from 199 cases of H1N1pdm infection from Argentina with MassTag PCR, testing for 33 additional microbial agents. The study population consisted of 199 H1N1pdm-infected subjects sampled between 23 June and 4 July 2009. Thirty-nine had severe disease defined as death (n = 20) or hospitalization (n = 19); 160 had mild disease. At least one additional agent of potential pathogenic importance was identified in 152 samples (76%), including Streptococcus pneumoniae (n = 62); Haemophilus influenzae (n = 104); human respiratory syncytial virus A (n = 11) and B (n = 1); human rhinovirus A (n = 1) and B (n = 4); human coronaviruses 229E (n = 1) and OC43 (n = 2); Klebsiella pneumoniae (n = 2); Acinetobacter baumannii (n = 2); Serratia marcescens (n = 1); and Staphylococcus aureus (n = 35) and methicillin-resistant S. aureus (MRSA, n = 6). The presence of S. pneumoniae was strongly correlated with severe disease. S. pneumoniae was present in 56.4% of severe cases versus 25% of mild cases; more than one-third of H1N1pdm NPS with S. pneumoniae were from subjects with severe disease (22 of 62 S. pneumoniae-positive NPS, p = 0.0004). In subjects 6 to 55 years of age, the adjusted odds ratio (OR) of severe disease in the presence of S. pneumoniae was 125.5 (95% confidence interval [CI], 16.95, 928.72; p<0.0001).

Conclusions/Significance

The association of S. pneumoniae with morbidity and mortality is established in the current and previous influenza pandemics. However, this study is the first to demonstrate the prognostic significance of non-invasive antemortem diagnosis of S. pneumoniae infection and may provide insights into clinical management.