Biocontrol of Listeria monocytogenes and Salmonella enterica on Fresh Strawberries with Lactic Acid Bacteria During Refrigerated Storage.

Small fruits such as strawberries have been increasingly implicated in outbreaks of foodborne illnesses. Salmonella enterica and Listeria monocytogenes may contaminate strawberries leading to potential public health concern. The objective of this study was to investigate the efficacy of a combined lactic acid bacteria (LAB) treatment of Lactobacillus plantarum and Pediococcus pentosaceus for controlling S. enterica and L. monocytogenes on fresh strawberries during storage at 4°C and 10°C. Strawberries purchased from a local grocery store were separately dip inoculated with Salmonella Newport, Salmonella Tennessee, Salmonella Thompson, or a three-strain cocktail of L. monocytogenes at ∼9 log colony-forming unit (CFU)/mL and allowed to air-dry for 1 h. Inoculated strawberries were then divided into three groups: (1) Control (pathogen alone), (2) Man, Rogosa, Sharpe (MRS) control (dipping in MRS broth), and (3) LAB treatment (dipping in a LAB cocktail of L. plantarum and P. pentosaceus). After treatment, strawberries were stored at 4°C or 10°C for 7 d in vented clamshell containers. Surviving Listeria, Salmonella, and LAB populations on strawberries were determined on 0, 1, 3, and 7 d post-treatment by plating on selective agars. At both 4°C and 10°C, LAB treatment significantly decreased Listeria populations by up to 2 log CFU/g compared to controls after 3 d of storage (p < 0.05). When strawberries were stored at 4°C, LAB treatment reduced ∼2.5 log, ∼2.7 log, and ∼2.9 log CFU/g in Salmonella Newport, Salmonella Tennessee, and Salmonella Thompson populations, respectively, compared to control on day 7. Similarly, ∼2.5 log CFU/g reductions of Salmonella populations were observed with LAB treatment at 10°C on day 7. LAB populations remained at ∼7.5 log CFU/g levels on strawberries at both temperatures throughout the entire study. Results of this study suggest that a combined LAB treatment can be potentially used as biocontrol agents against Salmonella and L. monocytogenes on strawberries at postharvest level.

Mealybug (Hemiptera: Pseudococcidae) Species Associated with Cacao Mild Mosaic Virus and Evidence of Virus Acquisition.

Theobroma cacao is affected by viruses on every continent where the crop is cultivated, with the most well-known ones belonging to the Badnavirus genus. One of these, cacao mild mosaic virus (CaMMV), is present in the Americas, and is transmitted by several species of Pseudococcidae (mealybugs). To determine which species are associated with virus-affected cacao plants in North America, and to assess their potential as vectors, mealybugs (n = 166) were collected from infected trees in Florida, and identified using COI, ITS2, and 28S markers. The species present were Pseudococcus jackbeardsleyi (38%; n = 63), Maconellicoccus hirsutus (34.3%; n = 57), Pseudococcus comstocki (15.7%; n = 26), and Ferrisia virgata (12%; n = 20). Virus acquisition was assessed by testing mealybug DNA (0.8 ng) using a nested PCR that amplified a 500 bp fragment of the movement protein-coat protein region of CaMMV. Virus sequences were obtained from 34.6 to 43.1% of the insects tested; however, acquisition did not differ among species, X2 (3, N = 166) = 0.56, p < 0.91. This study identified two new mealybug species, P. jackbeardsleyi and M. hirsutus, as potential vectors of CaMMV. This information is essential for understanding the infection cycle of CaMMV and developing effective management strategies.

Safety and feasibility of exoskeleton-assisted walking during acute/sub-acute SCI in an inpatient rehabilitation facility: A single-group preliminary study.

Context/objective: Information on the safety and feasibility of lower extremity powered exoskeletons for persons with acute/sub-acute spinal cord injury (SCI) is limited. Understanding the safety and feasibility of employing powered exoskeletons in acute/sub-acute (<6 months post injury) at a SCI acute inpatient rehabilitation (SCI-AIR) facility could guide clinical practice and provide a basis for larger clinical trials on efficacy and effectiveness. Design: Single group observational study. Setting: SCI-AIR. Participants: Participants (n = 12; age: 28-71 years; 58% AIS D; 58% male) with neurological levels of injuries ranging from C2 to L3. Interventions: Up to 90 min of exoskeleton-assisted locomotor training was provided up to three times per week during SCI-AIR. Outcome measures: Safety of device use during inpatient locomotor training was quantified as the number of adverse events (AE) per device exposure hour. Feasibility of device use was defined in terms of protocol compliance, intensity, and proficiency. Results: Concerning safety, symptomatic hypotension was the most common AE reported at 111-events/exoskeleton-hours. Protocol compliance had a mean (SD) of 54% (30%). For intensity, 77% of participants incorporated variable assistance into at least 1 walking session; 70% of participants' sessions were completed with a higher RPE than the physical therapist. In proficiency, 58% achieved at least minimal assistance when walking with the device. Conclusion: Exoskeleton training in SCI-AIR can be safe and feasible for newly injured individuals with SCI who have clinically defined ambulatory goals. Nonetheless, sufficient controls to minimize risks for AEs, such as hypotensive events, are required.

A U-Pb zircon age constraint on the oldest-recorded air-breathing land animal.

The oldest-known air-breathing land animal is the millipede Pneumodesmus newmani, found in the Cowie Harbour Fish Bed at Stonehaven, Scotland. Here we report the youngest, most concordant 238U-206Pb zircon age from ash below the fish bed of 413.7±4.4 Ma (±2σ), whereas the youngest age from a tuffaceous sandstone above the fish bed is statistically indistinguishable at 414.3±7.1 Ma. The Cowie Harbour Fish Bed thus appears to be lowermost Devonian (Lochkovian), contrary to the previously accepted mid-Silurian age based on palynomorphs from adjacent exposures. This has implications for the evolutionary timetable of land colonization, as the Cowie ages overlap late Lochkovian zircon ages reported elsewhere for andesite below the nearby (~50 mi) Rhynie Chert, which has more advanced terrestrial biota. The results postdate the possible late Silurian Ludford Lane locality in Shropshire, England. Pneumodesmus newmani is thus not the earliest air-breathing land animal, unless the Ludford Lane locality is younger than presently assigned.

Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory.

Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640-12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.

Real-time identification of bacteria and Candida species in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Delays in the identification of microorganisms are a barrier to the establishment of adequate empirical antibiotic therapy of bacteremia. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows the identification of microorganisms directly from colonies within minutes. In this study, we have adapted and tested this technology for use with blood culture broths, thus allowing identification in less than 30 min once the blood culture is detected as positive. Our method is based on the selective recovery of bacteria by adding a detergent that solubilizes blood cells but not microbial membranes. Microorganisms are then extracted by centrifugation and analyzed by MALDI-TOF-MS. This strategy was first tested by inoculating various bacterial and fungal species into negative blood culture bottles. We then tested positive patient blood or fluid samples grown in blood culture bottles, and the results obtained by MALDI-TOF-MS were compared with those obtained using conventional strategies. Three hundred twelve spiked bottles and 434 positive cultures from patients were analyzed. Among monomicrobial fluids, MALDI-TOF-MS allowed a reliable identification at the species, group, and genus/family level in 91%, 5%, and 2% of cases, respectively, in 20 min. In only 2% of these samples, MALDI-TOF MS did not yield any result. When blood cultures were multibacterial, identification was improved by using specific databases based on the Gram staining results. MALDI-TOF-MS is currently the fastest technique to accurately identify microorganisms grown in positive blood culture broths.

Recombinant human VEGF165b protein is an effective anti-cancer agent in mice.

Tumour growth is dependent on angiogenesis, the key mediator of which is vascular endothelial growth factor-A (VEGF-A). VEGF-A exists as two families of alternatively spliced isoforms - pro-angiogenic VEGF(xxx) generated by proximal, and anti-angiogenic VEGF(xxx)b by distal splicing of exon 8. VEGF(165)b inhibits angiogenesis and is downregulated in tumours. Here, we show for the first time that administration of recombinant human VEGF(165)b inhibits colon carcinoma tumour growth and tumour vessel density in nude mice, with a terminal plasma half-life of 6.2h and directly inhibited angiogenic parameters (endothelial sprouting, orientation and structure formation) in vitro. Intravenous injection of (125)I-VEGF(165)b demonstrated significant tumour uptake lasting at least 24h. No adverse effects on liver function or haemodynamics were observed. These results indicate that injected VEGF(165)b was taken up into the tumour as an effective anti-angiogenic cancer therapy, and provide proof of principle for the development of this anti-angiogenic growth factor splice isoform as a novel cancer therapy.

Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization.

Vascular endothelial growth factor (VEGF)-A regulates vascular development and angiogenesis. VEGF isoforms differ in ability to bind coreceptors heparan sulfate (HS) and neuropilin-1 (NRP1). We used VEGF-A165 (which binds HS and NRP1), VEGF-A121 (binds neither HS nor NRP1), and parapoxvirus VEGF-E-NZ2 (binds NRP1 but not HS) to investigate the role of NRP1 in organization of endothelial cells into vascular structures. All 3 ligands induced similar level of VEGFR-2 tyrosine phosphorylation in the presence of NRP1. In contrast, sprouting angiogenesis in differentiating embryonic stem cells (embryoid bodies), formation of branching pericyte-embedded vessels in subcutaneous matrigel plugs, and sprouting of intersegmental vessels in developing zebrafish were induced by VEGF-A165 and VEGF-E-NZ2 but not by VEGF-A121. Analyses of recombinant factors with NRP1-binding gain- and loss-of-function properties supported the conclusion that NRP1 is critical for VEGF-induced sprouting and branching of endothelial cells. Signal transduction antibody arrays implicated NRP1 in VEGF-induced activation of p38MAPK. Inclusion of the p38MAPK inhibitor SB203580 in VEGF-A165-containing matrigel plugs led to attenuated angiogenesis and poor association with pericytes. Our data strongly indicate that the ability of VEGF ligands to bind NRP1 influences p38MAPK activation, and formation of functional, pericyte-associated vessels.

Orf virus VEGF-E NZ2 promotes paracellular NRP-1/VEGFR-2 coreceptor assembly via the peptide RPPR.

Vascular endothelial growth factors (VEGFs) interact with the receptor tyrosine kinases (RTKs) VEGFR-1, -2, and -3; neuropilins (NRPs); and heparan sulfate (HS) proteoglycans. VEGF RTKs signal to downstream targets upon ligand-induced tyrosine phosphorylation, while NRPs and HS act as coreceptors that lack enzymatic activity yet modulate signal output by VEGF RTKs. VEGFs exist in various isoforms with distinct receptor specificity and biological activity. Here, a series of mammalian VEGF-A splice variants and orf virus VEGF-Es, as well as chimeric and mutant VEGF variants, were characterized to determine the motifs required for binding to NRP-1 in the absence (VEGF-E) or presence (VEGF-A(165)) of an HS-binding sequence. We identified the carboxyterminal peptides RPPR and DKPRR as the NRP-1 binding motifs of VEGF-E and VEGF-A, respectively. RPPR had significantly higher affinity for NRP-1 than DKPRR. VEGFs containing an RPPR motif promoted HS-independent coreceptor complex assembly between VEGFR-2 and NRP-1, independent of whether these receptors were expressed on the same or separate cells grown in cocultures. Functional studies showed that stable coreceptor assembly by VEGF correlated with its ability to promote vessel formation in an embryoid body angiogenesis assay.