Zirex: a Novel Zinc-Regulated Expression System for Lactococcus lactis

Here, we report a new zinc-inducible expression system for Lactococcus lactis, called Zirex, consisting of the pneumococcal repressor SczA and P_czcD. P_czcD tightly regulates the expression of green fluorescent protein in L. lactis. We show the applicability of Zirex together with the nisin-controlled expression system, enabling simultaneous but independent regulation of different genes.     

Soybean aeroallergen around the port of New Orleans: A potential cause of asthma

There have been reported epidemics of severe asthma in Barcelona, Spain, linked to a 10 kDa low molecular mass (LMM) allergen from soybean hulls that became airborne during unloading of ships. As a preliminary probe of the potential for dispersion of this allergen in USA cities, four automated air samplers were placed around a grain elevator in New Orleans and operated continuously from May to October 1990. The allergen was extracted from the filters and immunochemically assayed for soybean aeroallergen. On 31 separate days, the airborne allergen concentration in at least one of the samples was over 10000 U/m³ similar to those observed in Barcelona on some epidemic days. Areas North and East of the elevator were most affected. Serologie studies showed that of 50 asthmatics from New Orleans who were participants in an unrelated clinical study 4 or 8% demonstrated elevated titers of IgE antibody to LMM soybean allergen. Only 1 of 475 control sera (half of which were also asthmatic) obtained elsewhere in the US was positive for LMM soybean IgE antibody. Based on the findings in this study, there is a great possibility that on some days there is enough soybean allergen in the air and a sufficient frequency of soybean aeroallergen RAST positive asthmatics in New Orleans to warrant further investigation of the contribution of soybean aeroallergen to asthma around the port of New Orleans.Supported by NIAID # A121255. Mayo Clinic and Foundation and Minnesota Lung Association.     

Influence of ancient glacial periods on the Andean fauna: the case of the pampas cat (Leopardus colocolo)

Background  

While numerous studies revealed the major role of environmental changes of the Quaternary on the evolution of biodiversity, research on the influence of that period on current South-American fauna is scarce and have usually focused on lowland regions. In this study, the genetic structure of the pampas cat (Leopardus colocolo), a widely distributed felid, was determined and linked to ancient climate fluctuations on the Andean region.     

Quinolone action in Escherichia coli cells carrying gyrA and gyrB mutations

Abstract We have isolated spontaneous mutant strains of Escherichia coli KL16 showing different levels of nalidixic acid (NAL) resistance. From 40 independent mutants, 36 had gyrA and four had gyrB mutations. Most of the gyrA mutations (30/36) conferred high level NAL resistance. In contrast, the only gyrB mutation that conferred a relatively high level of NAL resistance also determined enhanced susceptibility to quinolones with a piperazinyl substituent at C7 position of the quinolone ring (amphoteric quinolones). This gyrB mutation (denoted gyrB1604 ), jointly with a gyrA mutation (denoted gyrA972 ) which confers a high level of quinolone resistance, were used to construct strain IC2476, carrying the two gyr mutant alleles. The susceptibility of this strain to amphoteric quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was similar to that of the gyrA972 single mutant. This result indicates that the change in GyrA subunit which determines a high level of quinolone-resistance has the capacity to mask the hypersusceptibility to amphoteric quinolones promoted by the GyrB1604 mutant subunit. This capacity was further confirmed by studying the effects of ciprofloxacin (CFX) on gyrase inhibition in the gyrA972 gyrB1604 strain.     

Safer one-pot synthesis of the ‘SHAPE’ reagent 1-methyl-7-nitroisatoic anhydride (1m7)

Estimating the reactivity of 2′-hydroxyl groups along an RNA chain of interest aids in the modeling of the folded RNA structure; flexible loops tend to be reactive, whereas duplex regions are generally not. Among the most useful reagents for probing 2′-hydroxyl reactivity is 1-methyl-7-nitroisatoic anhydride (1m7), but the absence of a reliable, inexpensive source has prevented widespread adoption. An existing protocol for the conversion of an inexpensive precursor 4-nitroisatoic anhydride (4NIA) recommends the use of NaH in dimethylformamide (DMF), a reagent combination that most molecular biology labs are not equipped to handle, and that does not scale safely in any case. Here we describe a safer, one-pot method for bulk conversion of 4NIA to 1m7 that reduces costs and bypasses the use of NaH. We show that 1m7 produced by this method is free of side products and can be used to probe RNA structure in vitro.     

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16^INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.