Fluctuation in contamination dynamics of L. monocytogenes in quargel (acid curd cheese) lots recalled during the multinational listeriosis outbreak 2009/2010

For the first time it has been possible to determine the contamination level of Listeria monocytogenes in the very cheese lots of acid curd cheese that caused a multinational outbreak between 2009/2010. The listeriosis outbreak accounted for 34 clinical cases and eight deaths. The cheese, which was distributed in Austria, Germany, the Czech Republic, Poland and Slovakia, was recalled on the 23rd January 2010. All recalled lots were immediately investigated after call back from the retail market. The company manufactured two different cheese types, (i) red smear ripened--and (ii) mold coated/white veined--acid curd cheese. Depending on the lot production dates, cheese samples (n=1045) were analyzed at three different time points: (i) beginning to mid shelf-life (lot nos. 15-18; production period 5.1.2010-13.1.2010); (ii) end of shelf-life (lot nos. 9-18; production period 21.12.2009-13.1.2010) and, (iii) ≤46days after the expiry date (lot nos. 1-18; production period 1.12.2009-13.1.2010). Qualitative and quantitative examinations of cheese samples were performed according to ISO 11290-1&2. Examination of the samples, according to ISO 11290-1, resulted in 16 L. monocytogenes positive (red smear type) and two negative lots (mold coated type). These results were confirmed by a combined enrichment/real-time PCR method. The contamination values obtained by quantitative ISO 11290-2 varied from ≤log 2 cell forming units (CFU)/g to log 8.1CFU/g. Three out of sixteen L. monocytogenes positive lots revealed a contamination level of ≤log 2CFU/g at the beginning of their shelf-life when stored at 4°C. Nevertheless, by increasing the storage life and/or the storage temperature (15, 22°C) the contamination level could be raised to between log 5 and log 6CFU/g. Our data indicate that 81.3% (13/16) of the recalled red smear quargel cheese lots were highly contaminated with L. monocytogenes. All this implies that the main contamination of the quargel cheese took place during the red smear process and that quargel cheese can easily support growth of L. monocytogenes.     

Microwave-assisted rapid synthesis of luminescent gold nanoclusters for sensing Hg2+ in living cells using fluorescence imaging

A microwave-assisted strategy for synthesizing dihydrolipoic acid (DHLA) capped fluorescent gold nanoclusters (AuNCs) has been developed. Irradiation with microwaves during synthesis enhanced the fluorescence quantum yield (QY) of AuNCs by about five-fold and shortened the reaction time from hours to several minutes. The as-synthesized DHLA-AuNCs possessed bright near-infrared fluorescence (QY: 2.9%), ultrasmall hydrodynamic diameter (3.3 nm), good colloidal stability over the physiologically relevant pH range of 5-10 as well as low cytotoxicity toward HeLa cells. Moreover, these DHLA-AuNCs were capable of sensing Hg(2+) through the specific interaction between Hg(2+) and Au(+) on the surface of AuNCs; the limit of detection (LOD) was 0.5 nM. A potential application in imaging intracellular Hg(2+) in HeLa cells was demonstrated by using spinning disc confocal microscopy.     

Natural and biomimetic materials for the detection of insulin

Microgravimetric sensors have been developed for detection of insulin by using quartz crystal microbalances as transducers, in combination with sensitive layers. Natural antibodies as coatings were compared with biomimetic materials to fabricate mass-sensitive sensors. For this purpose polyurethane was surface imprinted by insulin, which acts as a synthetic receptor for reversible analyte inclusion. The sensor responses for insulin give a pronounced concentration dependence, with a detection limit down to 1 μg/mL and below. Selectivity studies reveal that these structured polymers lead to differentiation between insulin and glargine. Moreover, antibody replicae were generated by a double imprinting process. Thus, biological recognition capabilities of immunoglobulins are transferred to synthetic polymers. In the first step, natural-immunoglobulin-imprinted nanoparticles were synthesized. Subsequently, these templated particles were utilized for creating positive images of natural antibodies on polymer layers. These synthetic coatings, which are more robust than natural analogues, can be produced in large amount. These biomimetic sensors are useful in the biotechnology of insulin monitoring.     

Method development for metaproteomic analyses of marine biofilms

The large-scale identification and quantitation of proteins via nanoliquid chromatography (LC)-tandem mass spectrometry (MS/MS) offers a unique opportunity to gain unprecedented insight into the microbial composition and biomolecular activity of true environmental samples. However, in order to realize this potential for marine biofilms, new methods of protein extraction must be developed as many compounds naturally present in biofilms are known to interfere with common proteomic manipulations and LC-MS/MS techniques. In this study, we used amino acid analyses (AAA) and LC-MS/MS to compare the efficacy of three sample preparation methods [6 M guanidine hydrochloride (GuHCl) protein extraction + in-solution digestion + 2D LC; sodium dodecyl sulfate (SDS) protein extraction + 1D gel LC; phenol protein extraction + 1D gel LC] for the metaproteomic analyses of an environmental marine biofilm. The AAA demonstrated that proteins constitute 1.24% of the biofilm wet weight and that the compared methods varied in their protein extraction efficiencies (0.85-15.15%). Subsequent LC-MS/MS analyses revealed that the GuHCl method resulted in the greatest number of proteins identified by one or more peptides whereas the phenol method provided the greatest sequence coverage of identified proteins. As expected, metagenomic sequencing of the same biofilm sample enabled the creation of a searchable database that increased the number of protein identifications by 48.7% (≥1 peptide) or 54.7% (≥2 peptides) when compared to SwissProt database identifications. Taken together, our results provide methods and evidence-based recommendations to consider for qualitative or quantitative biofilm metaproteome experimental design.     

Replacement of Highly Conserved E222 by the Photostable Non‐photoconvertible Histidine in GFP

The widely used green fluorescent protein (GFP) decarboxylates upon irradiation; this involves removal of the acidic function of the glutamic acid at position 222, thereby resulting in the irreversible photoconversion of GFP. To suppress this phenomenon, the photostable, non‐photoconvertible histidine was introduced at position 222 in GFP. The variant E222H shows negligible photodynamic processes and high expression yield. In addition, the stable and bright fluorescence over a wide pH range makes the E222H protein an alternative for GFP in fluorescence imaging and spectroscopy. Other fluorescent proteins are predicted to benefit from replacement of the catalytic glutamic acid by histidine.     

Study on the influence of electron beam irradiation on the thermal,mechanical, and rheological properties of ethylene‐octene copolymer with high comonomer content

Ethylene‐octene copolymer (EOC) was irradiated using electron beam irradiation at different dosages (30, 60, 90, and 120 kGy). Effect of irradiation dosage on thermal and mechanical properties was studied. When compared to low density polyethylene, EOC exhibited higher degree of crosslinking reflected in increased gel content, higher elastic modulus (G′), and lower tan δ obtained by rheology measurement at 150°C. Crosslinking caused improvement in high‐temperature creep and room temperature and also elevated temperature elastic properties. Differential scanning calorimetry revealed that e‐beam irradiation has caused a gradual reduction in crystallinity and a presence of a fraction with higher melting temperature. In the case of EOC, as the extent of crosslinking increased, stress at break showed an increasing trend whereas irradiation dosage had an inverse effect on elongation at break. Radiation dosage has positive effect on thermal stability estimated by thermogravimetric analysis. After 30 min of thermal degradation at 220°C, slightly higher C?O peak for crosslinked sample was found by Fourier transform infrared spectroscopy while for room temperature samples no C?O peak was detected. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013