Neutrophil Recruitment to Lymph Nodes Limits Local Humoral Response to Staphylococcus aureus

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.     

Investigation of the physiological properties and synthesis of PUFAs from Thraustochytrids and its electrophoretic karyotypes

Physiological properties of organism, such as the number of chromosomes, genome size, fatty acid profile and the activities of desaturases and elongases were investigated for four differentThraustochytrium species. The investigation revealed thatThraustochytrium aureum could synthesize a significant level of polyunsaturated fatty acids (PUFAs), particularly docosa-hexaenoic acid (DHA), when compared to the other threeThraustochytrium species tested. A higher level of saturated fatty acids was observed byT. striatum followed byThraustochytrium sp. ATCC 26185. The PUFA accumulation rate was higher in the n-3 than in the n-6 pathway. A comparison of the activities for these desaturases and elongases of the four different species were also studied. Further, the electrophoretic karyotypes of Thraustochytrids were separated by pulsed-field gel electrophoresis (PFGE). The separation condition of PFGE was developed to obtain the different chromosomes from the variousThraustochytrium species. The number of chromosomes inT. aureum, T. striatum, Thraustochytrium sp. ATCC 20891 andThraustochytrium sp. ATCC 26185 were 13, 17, 10. 8, and the whole genome size of those species were estimated to be 12.9, 11.7, 11.3 and 9.93 Mbp, respectively.     

Ablation of sarcolipin results in atrial remodeling

Sarcolipin (SLN) is a key regulator of sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and its expression is altered in diseased atrial myocardium. To determine the precise role of SLN in atrial Ca(2+) homeostasis, we developed a SLN knockout (sln-/-) mouse model and demonstrated that ablation of SLN enhances atrial SERCA pump activity. The present study is designed to determine the long-term effects of enhanced SERCA activity on atrial remodeling in the sln-/- mice. Calcium transient measurements show an increase in atrial SR Ca(2+) load and twitch Ca(2+) transients. Patch-clamping experiments demonstrate activation of the forward mode of sodium/calcium exchanger, increased L-type Ca(2+) channel activity, and prolongation of action potential duration at 90% repolarization in the atrial myocytes of sln-/- mice. Spontaneous Ca(2+) waves, delayed afterdepolarization, and triggered activities are frequent in the atrial myocytes of sln-/- mice. Furthermore, loss of SLN in atria is associated with increased interstitial fibrosis and altered expression of genes encoding collagen and other extracellular matrix proteins. Our results also show that the sln-/- mice are susceptible to atrial arrhythmias upon aging. Together, these findings indicate that ablation of SLN results in increased SERCA activity and SR Ca(2+) load, which, in turn, could cause abnormal intracellular Ca(2+) handling and atrial remodeling.     

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

Al2O3 Nanoparticles Induce Mitochondria‐Mediated Cell Death and Upregulate the Expression of Signaling Genes in Human Mesenchymal Stem Cells

An increase in the broad usage of Al₂O₃ nanoparticles (ANPs) in the food and agricultural sectors may produce rare hazards for human health. The objective of this study was to assess the acute toxicity of ANPs in human mesenchymal stem cells (hMSCs) in vitro. Cell viability, cellular uptake, morphology, and gene expression using quantitative real‐time polymerase chain reaction (qRT‐PCR) were analyzed. The results indicate that ANPs have a significant and dose‐dependent effect on cytotoxicity. Control cells showed a characteristic, homogeneous nuclear staining pattern, whereas ANP‐exposed cells showed abnormal nuclear morphological changes such as condensation or fragmentation. An early characteristic of apoptosis was observed in ANP‐treated cells. Further confirmation of cell death in hMSCs was observed through increased expression of chosen signaling genes and also decreased expression of Bcl‐2 during mitochondria‐mediated cell death. Although they provide great advantages in food and agricultural products, the chronic and acute toxicity of ANPs still needs to be assessed carefully. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:469‐476, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21448     

EPHA2 polymorphisms and age-related cataract in India

Objective

We investigated whether previously reported single nucleotide polymorphisms (SNPs) of EPHA2 in European studies are associated with cataract in India.

Methods

We carried out a population-based genetic association study. We enumerated randomly sampled villages in two areas of north and south India to identify people aged 40 and over. Participants attended a clinical examination including lens photography and provided a blood sample for genotyping. Lens images were graded by the Lens Opacification Classification System (LOCS III). Cataract was defined as a LOCS III grade of nuclear ≥4, cortical ≥3, posterior sub-capsular (PSC) ≥2, or dense opacities or aphakia/pseudophakia in either eye. We genotyped SNPs rs3754334, rs7543472 and rs11260867 on genomic DNA extracted from peripheral blood leukocytes using TaqMan assays in an ABI 7900 real-time PCR. We used logistic regression with robust standard errors to examine the association between cataract and the EPHA2 SNPs, adjusting for age, sex and location.

Results

7418 participants had data on at least one of the SNPs investigated. Genotype frequencies of controls were in Hardy-Weinberg Equilibrium (p>0.05). There was no association of rs3754334 with cataract or type of cataract. Minor allele homozygous genotypes of rs7543472 and rs11260867 compared to the major homozygote genotype were associated with cortical cataract, Odds ratio (OR) = 1.8, 95% Confidence Interval (CI) (1.1, 3.1) p = 0.03 and 2.9 (1.2, 7.1) p = 0.01 respectively, and with PSC cataract, OR = 1.5 (1.1, 2.2) p = 0.02 and 1.8 (0.9, 3.6) p = 0.07 respectively. There was no consistent association of SNPs with nuclear cataract or a combined variable of any type of cataract including operated cataract.

Conclusions

Our results in the Indian population agree with previous studies of the association of EPHA2 variants with cortical cataracts. We report new findings for the association with PSC which is particularly prevalent in Indians.     

Presence of nanosilica (E551) in commercial food products: TNF-mediated oxidative stress and altered cell cycle progression in human lung fibroblast cells

Silica (E551) is commonly used as an anti-caking agent in food products. The morphology and the dimension of the added silica particles are not, however, usually stated on the food product label. The food industry has adapted nanotechnology using engineered nanoparticles to improve the quality of their products. However, there has been increased debate regarding the health and safety concerns related to the use of engineered nanoparticles in consumer products. In this study, we investigated the morphology and dimensions of silica (E551) particles in food. The silica content of commercial food products was determined using inductively coupled plasma optical emission spectrometry. The result indicates that 2.74–14. 45 μg/g silica was found in commercial food products; however, the daily dietary intake in increase causes adverse effects on human health. E551 was isolated from food products and the morphology, particle size, crystalline nature, and purity of the silica particles were analyzed using XRD, FTIR, TEM, EDX and DLS. The results of these analyses confirmed the presence of spherical silica nanoparticles (of amorphous nature) in food, approximately 10–50 nm in size. The effects of E551 on human lung fibroblast cell viability, intracellular ROS levels, cell cycle phase, and the expression levels of metabolic stress-responsive genes (CAT, GSTA4, TNF, CYP1A, POR, SOD1, GSTM3, GPX1, and GSR1) were studied. The results suggest that E551 induces a dose-dependent cytotoxicity and changes in ROS levels and alters the gene expression and cell cycle. Treatment with a high concentration of E551 caused significant cytotoxic effects on WI-38 cells. These findings have implications for the use of these nanoparticles in the food industry.     

Exercise Protects against Diet-Induced Insulin Resistance through Downregulation of Protein Kinase Cβ in Mice

Physical exercise is an important and effective therapy for diabetes. However, its underlying mechanism is not fully understood. Protein kinase Cβ (PKCβ) has been suggested to be involved in the pathogenesis of obesity and insulin resistance, but the role of PKCβ in exercise-induced improvements in insulin resistance is completely unknown. In this study, we evaluated the involvement of PKCβ in exercise-attenuated insulin resistance in high-fat diet (HFD)-fed mice. PKCβ^-/- and wild-type mice were fed a HFD with or without exercise training. PKC protein expression, body and tissue weight change, glucose and insulin tolerance, metabolic rate, mitochondria size and number, adipose inflammation, and AKT activation were determined to evaluate insulin sensitivity and metabolic changes after intervention. PKCβ expression decreased in both skeletal muscle and liver tissue after exercise. Exercise and PKCβ deficiency can alleviate HFD-induced insulin resistance, as evidenced by improved insulin tolerance. In addition, fat accumulation and mitochondrial dysfunction induced by HFD were also ameliorated by both exercise and PKCβ deficiency. On the other hand, exercise had little effect on PKCβ^-/- mice. Further, our data indicated improved activation of AKT, the downstream signal molecule of insulin, in skeletal muscle and liver of exercised mice, whereas PKCβ deficiency blunted the difference between sedentary and exercised mice. These results suggest that downregulation of PKCβ contributes to exercise-induced improvement of insulin resistance in HFD-fed mice.     

Identification of a vitamin D3-specific hydroxylase genes through actinomycetes genome mining

We previously completed whole-genome sequencing of a rare actinomycete named Sebekia benihana, and identified the complete S. benihana cytochrome P450 complement (CYPome), including 21 cytochrome P450 hydroxylase (CYP), seven ferredoxin (FD), and four ferredoxin reductase (FDR) genes. Through targeted CYPome disruption, a total of 32 S. benihana CYPome mutants were obtained. Subsequently, a novel cyclosporine A region-specific hydroxylase was successfully determined to be encoded by a CYP-sb21 gene by screening the S. benihana CYPome mutants. Here, we report that S. benihana is also able to mediate vitamin D₃ (VD₃) hydroxylation. Among the 32 S. benihana CYPome mutants tested, only a single S. benihana CYP mutant, ΔCYP-sb3a, failed to show regio-specific hydroxylation of VD₃ to 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃. Moreover, the VD₃ hydroxylation activity in the ΔCYP-sb3a mutant was restored by CYP-sb3a gene complementation. Since all S. benihana FD and FDR disruption mutants maintained VD₃ hydroxylation activity, we conclude that CYP-sb3a, a member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific VD₃ hydroxylation process in S. benihana. Expression of the CYP-sb3a gene exhibited VD₃ hydroxylation in the VD₃ non-hydroxylating Streptomyces coelicolor, implying that the regio-specific hydroxylation of VD₃ is carried out by a specific P450 hydroxylase in S. benihana.