Quantifying the reduction in potential health risks by determining the sensitivity of poliovirus type 1 chat strain and rotavirus SA-11 to electron beam irradiation of iceberg lettuce and spinach

Fresh produce, such as lettuce and spinach, serves as a route of food-borne illnesses. The U.S. FDA has approved the use of ionizing irradiation up to 4 kGy as a pathogen kill step for fresh-cut lettuce and spinach. The focus of this study was to determine the inactivation of poliovirus and rotavirus on lettuce and spinach when exposed to various doses of high-energy electron beam (E-beam) irradiation and to calculate the theoretical reduction in infection risks that can be achieved under different contamination scenarios and E-beam dose applications. The D(10) value (dose required to reduce virus titers by 90%) (standard error) of rotavirus on spinach and lettuce was 1.29 (± 0.64) kGy and 1.03 (± 0.05) kGy, respectively. The D(10) value (standard error) of poliovirus on spinach and lettuce was 2.35 (± 0.20) kGy and 2.32 (± 0.08) kGy, respectively. Risk assessment of data showed that if a serving (～14 g) of lettuce was contaminated with 10 PFU/g of poliovirus, E-beam irradiation at 3 kGy will reduce the risk of infection from >2 in 10 persons to approximately 6 in 100 persons. Similarly, if a serving size (～0.8 g) of spinach is contaminated with 10 PFU/g of rotavirus, E-beam irradiation at 3 kGy will reduce infection risks from >3 in 10 persons to approximately 5 in 100 persons. The results highlight the value of employing E-beam irradiation to reduce public health risks but also the critical importance of adhering to good agricultural practices that limit enteric virus contamination at the farm and in packing houses.     

Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.     

Phase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer

Background

Preclinical data support further investigation of ascorbic acid in pancreatic cancer. There are currently insufficient safety data in human subjects, particularly when ascorbic acid is combined with chemotherapy.

Methods and Findings

14 subjects with metastatic stage IV pancreatic cancer were recruited to receive an eight week cycle of intravenous ascorbic acid (three infusions per week), using a dose escalation design, along with standard treatment of gemcitabine and erlotinib. Of 14 recruited subjects enrolled, nine completed the study (three in each dosage tier). There were fifteen non-serious adverse events and eight serious adverse events, all likely related to progression of disease or treatment with gemcitabine or erlotinib. Applying RECIST 1.0 criteria, seven of the nine subjects had stable disease while the other two had progressive disease.

Conclusions

These initial safety data do not reveal increased toxicity with the addition of ascorbic acid to gemcitabine and erlotinib in pancreatic cancer patients. This, combined with the observed response to treatment, suggests the need for a phase II study of longer duration.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00954525","term_id":"NCT00954525"}}NCT00954525     

Sphingolipid Degradation in Leishmania (Leishmania) amazonensis

Background

Human leishmaniasis is caused by more than 20 Leishmania species and has a wide range of symptoms. Our recent studies have demonstrated the essential role of sphingolipid degradation in the virulence of Leishmania (Leishmania) major, a species responsible for localized cutaneous leishmaniasis in the Old World. In this study, we investigated the function of sphingolipid degradation in Leishmania (Leishmania) amazonensis, an etiological agent of localized and diffuse cutaneous leishmaniasis in South America.

Methodology/Principal Findings

First, we identified the enzyme LaISCL which is responsible for sphingolipid degradation in L. amazonensis. Primarily localized in the mitochondrion, LaISCL shows increased expression as promastigotes progress from replicative log phase to non-replicative stationary phase. To study its function, null mutants of LaISCL (Laiscl⁻) were generated by targeted gene deletion and complemented through episomal gene add-back. In culture, loss of LaISCL leads to hypersensitivity to acidic pH and poor survival in murine macrophages. In animals, Laiscl⁻ mutants exhibit severely attenuated virulence towards C57BL6 mice but are fully infective towards BALB/c mice. This is drastically different from wild type L. amazonensis which cause severe pathology in both BALB/c and C57BL 6 mice.

Conclusions/Significance

A single enzyme LaISCL is responsible for the turnover of sphingolipids in L. amazonensis. LaISCL exhibits similar expression profile and biochemical property as its ortholog in L. major. Deletion of LaISCL reduces the virulence of L. amazonensis and the outcome of Laiscl⁻-infection is highly dependent on the host''s genetic background. Therefore, compared to L. major, the role of sphingolipid degradation in virulence is substantially different in L. amazonensis. Future studies may reveal whether sphingolipid degradation is required for L. amazonensis to cause diffuse cutaneous infections in humans.     

The role of dorsal root ganglia activation and brain-derived neurotrophic factor in multiple sclerosis

Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain-derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post-disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG-derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.     

Proteomic profiling of high glucose primed monocytes identifies cyclophilin A as a potential secretory marker of inflammation in type 2 diabetes

Hyperglycemia is widely recognized to be a potent stimulator of monocyte activity, which is a crucial event in the pathogenesis of atherosclerosis. We analyzed the monocyte proteome for potential markers that would enhance the ability to screen for early inflammatory status in Type 2 diabetes mellitus (T2DM), using proteomic technologies. Monocytic cells (THP-1) were primed with high glucose (HG), their protein profiles were analyzed using 2DE and the downregulated differentially expressed spots were identified using MALDI TOF/MS. We selected five proteins that were secretory in function with the help of bioinformatic programs. A predominantly downregulated protein identified as cyclophilin A (sequence coverage 98%) was further validated by immunoblotting experiments. The cellular mRNA levels of cyclophilin A in various HG-primed cells were studied using qRT-PCR assays and it was observed to decrease in a dose-dependent manner. LC-ESI-MS was used to identify this protein in the conditioned media of HG-primed cells and confirmed by Western blotting as well as ELISA. Cyclophilin A was also detected in the plasma of patients with diabetes. We conclude that cyclophilin A is secreted by monocytes in response to HG. Given the paracrine and autocrine actions of cyclophilin A, the secreted immunophilin could be significant for progression of atherosclerosis in type 2 diabetes. Our study also provides evidence that analysis of monocyte secretome is a viable strategy for identifying candidate plasma markers in diabetes.     

Resolvin E1 metabolome in local inactivation during inflammation-resolution

Resolvin E1 (RvE1; 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is a potent anti-inflammatory and proresolving mediator derived from the omega-3 eicosapentaenoic acid. In this study, we report the RvE1 metabolome, namely, the metabolic products derived from RvE1. RvE1 was converted to several novel products by human polymorphonuclear leukocytes and whole blood as well as in murine inflammatory exudates, spleen, kidney, and liver. The potential activity of each of the newly identified products was directly compared with that of RvE1. The new RvE1 products elucidated included 19-hydroxy-RvE1, 20-carboxy-RvE1, and 10,11-dihydro-RvE1. Metabolomic profiles of RvE1 were species-, tissue-, and cell type-specific. Direct comparisons of the bioactions between isolated RvE1 metabolic products indicated that 10,11-dihydro-RvE1, 18-oxo-RvE1, and 20-carboxy-RvE1 displayed reduced bioactivity in vivo. At concentrations as low as 1 nM, RvE1 enhanced macrophage phagocytosis, a proresolving activity that was reduced by metabolic inactivation. These results document novel metabolic products of RvE1 that impact its actions and that both omega-1 hydroxylation and reduction of conjugated double bonds in RvE1 are new pathways of four main routes of RvE1 metabolism in mammalian tissues. Together, these findings indicate that, during inflammation and its controlled resolution, specific tissues inactivate proresolving signals, i.e., RvE1, to permit the coordinated return to homeostasis. Moreover, the RvE1 metabolome may serve as a biomarker of these processes.     

The investigation of Protein A and Salmonella antibody adsorption onto biosensor surfaces by atomic force microscopy

The investigation of Protein A and antibody adsorption on surfaces in a biological environment is an important and fundamental step for increasing biosensor sensitivity and specificity. The atomic force microscope (AFM) is a powerful tool that is frequently used to characterize surfaces coated with a variety of molecules. We used AFM in conjunction with scanning electron microscopy to characterize the attachment of protein A and its subsequent binding to the antibody and Salmonella bacteria using a gold quartz crystal. The rms roughness of the base gold surface was determined to be approximately 1.30 nm. The average step height change between the solid gold and protein A layer was approximately 3.0 +/- 1.0 nm, while the average step height of the protein A with attached antibody was approximately 6.0 +/- 1.0 nm. We found that the antibodies did not completely cover the protein A layer, instead the attachment follows an island model. Salt crystals and water trapped under the protein A layer were also observed. The uneven adsorption of antibodies onto the biosensor surface might have led to a decrease in the sensitivity of the biosensor. The presence of salt crystals and water under the protein A layer may deteriorate the sensor specificity. In this report, we have discussed the application and characterization of protein A bound to antibodies which can be used to detect bacterial and viral pathogens.