Morphology of defense glands of the opilionids (daddy longlegs) Leiobunum vittatum and L. flavum (Arachnida: Opiliones: Palpatores: Phalangiidae)

Opilionid defense glands consist of 0.5 × 0.9-mm sacs attached to the underside of low tubercles located on the dorsal side of the cephalothorax, posterior to the first pair of legs. Each gland opens via an elongated slit, located in the posterior floor of a crater that is situated at the summit of the tubercle. The center of the sac, called the reservoir, is lined by a cuticle consisting of epicuticle and endocuticle which is continuous through the slit with the exoskeleton. The layers of cuticle vary in thickness with different locations in the gland. A hemocoelomic (basement) membrane, 0.5–1, μ thick, forms the boundary between glandular cells and hemocoel. The gland has a nonsecretory portion consisting only of cuticle-supporting cells and a secretory portion consisting of secretory and cuticle-supporting cells. The cuticle lining the reservoir in the secretory area is broached by many cuticle-lined ductules, each of which drains an isolated intercellular space called the intercalated cistern. This in turn drains microvilli-lined canaliculi located between and extending into secretory cells. The cisterns are devoid of microvilli. Secretory cell cytoplasm contains a Golgi apparatus, many free ribosomes, rough endoplasmic reticulum (RER), two types of granules (speckled and dense), and mitochondria. Speckled granules are partially filled with fairly large particles and are found in association with the Golgi apparatus. They also surround canaliculi into which they empty. Dense granules are packed with very small particles, have a gray homogeneous appearance, and are scattered throughout the cytoplasm. Mitochondria containing matrix granules tend to scatter throughout the cytoplasm but are concentrated around canaliculi.     

Hyperthermic modification of cyclophosphamide metabolism in rat hepatic microsomes and liver slices

The ability of rat liver microsomes and liver slices to metabolize the antineoplastic compound cyclophosphamide was studied at 37° and at elevated temperatures comparable to those used for human systemic hyperthermic antineoplastic therapy. Temperatures above 40.5° and 41.8° inhibited cyclophospamide metabolism by microsomes and liver slices respectively. Therefore, cyclophosphamide may not be a suitable drug for combination with systemic hyperthermia in cancer therapy.     

The Normal Limits,Subclinical Significance,Related Metabolic Derangements and Distinct Biological Effects of Body Site-Specific Adiposity in Relatively Healthy Population

Background

The accumulation of visceral adipose tissue that occurs with normal aging is associated with increased cardiovascular risks. However, the clinical significance, biological effects, and related cardiometabolic derangements of body-site specific adiposity in a relatively healthy population have not been well characterized.

Materials and Methods

In this cross-sectional study, we consecutively enrolled 608 asymptomatic subjects (mean age: 47.3 years, 27% female) from 2050 subjects undergoing an annual health survey in Taiwan. We measured pericardial (PCF) and thoracic peri-aortic (TAT) adipose tissue volumes by 16-slice multi-detector computed tomography (MDCT) (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA) and related these to clinical characteristics, body fat composition (Tanita 305 Corporation, Tokyo, Japan), coronary calcium score (CCS), serum insulin, high-sensitivity C-reactive protein (Hs-CRP) level and circulating leukocytes count. Metabolic risk was scored by Adult Treatment Panel III guidelines.

Results

TAT, PCF, and total body fat composition all increased with aging and higher metabolic scores (all p<0.05). Only TAT, however, was associated with higher circulating leukocyte counts (ß-coef.:0.24, p<0.05), serum insulin (ß-coef.:0.17, p<0.05) and high sensitivity C-reactive protein (ß-coef.:0.24, p<0.05). These relationships persisted after adjustment in multivariable models (all p<0.05). A TAT volume of 8.29 ml yielded the largest area under the receiver operating characteristic curve (AUROC: 0.79, 95%CI: 0.74–0.83) to identify metabolic syndrome. TAT but not PCF correlated with higher coronary calcium score after adjustment for clinical variables (all p<0.05).

Conclusion

In our study, we observe that age-related body-site specific accumulation of adipose tissue may have distinct biological effects. Compared to other adiposity measures, peri-aortic adiposity is more tightly associated with cardiometabolic risk profiles and subclinical atherosclerosis in a relatively healthy population.     

Lineage and Genogroup-Defining Single Nucleotide Polymorphisms of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047–2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7.     

Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion

Ischemia-reperfusion injury is a microvascular event documented in numerous in vivo animal models. In animal models, prostaglandin and prostaglandin analogues have been found to ameliorate reperfusion injury. These studies were undertaken to evaluate human microvascular endothelial PGE(1) synthesis during in vitro ischemia followed by reperfusion. Human (neonatal) microvascular endothelial cell (MEC) cultures (n = 6) were subjected to sequential 2 h periods of normoxia (20% O(2)), ischemia (1.5% O(2)), and reperfusion (20% O(2)). Prostaglandin E(2) synthesis in conditioned media was determined by ELISA. Steady state levels of MEC prostaglandin H synthase (PGHS)-1 and -2 mRNA were assessed at the end of each 2-h period using RT-PCR and a quantitative mRNA ELISA. MEC PGHS protein levels were analyzed using an ELISA. PGE(1) release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE(1) release returned to normoxic levels at 30, 60, and 90 min, and exceeded normoxic levels at 120 min. PGHS-1 mRNA levels were undetectable during all experimental conditions. PGHS-2 mRNA levels were unchanged by ischemia, but were decreased by reperfusion. In contrast, PGHS-2 protein levels increased 3-fold during ischemia, and remained elevated during reperfusion. Human MEC do not express PGHS-1 mRNA in vitro. Prolonged ischemia decreases MEC PGE(1) synthesis, and stimulates increased PGHS-2 protein levels without altering the steady state levels of COX-2 mRNA. During reperfusion, increased PGHS-2 protein levels persist and are associated with stimulated PGE(2) secretion, despite relative decreases in PGHS-2 mRNA.     

Parameters affecting substance P measurement in heart, lung, and skin

Substance P (SP), a neuropeptide that is widely distributed both peripherally and centrally, mediates several pathophysiological processes. Among current assays for SP, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been most widely used. Several previous studies, mostly performed with nerve extracts or organ perfusates, determined that acidity of the extraction buffer as well as the number extractions performed constitute factors influencing accurate measurements. We used an ELISA protocol in this study to analyze methodological aspects of SP measurement in extracts from heart, skin, and lung. The extraction procedure had two steps, an acid extraction followed by a column extraction. We could effectively measure SP with extract from as little as 10 mg of tissue. For each tissue examined, different variables influenced the SP measured. For all tissues, the weight of tissue extracted was critical; the more tissue extracted, the lower the sensitivity of the assay. This problem could be overcome in skin by omitting the column extraction. When mechanical loses were considered (e.g., loss during extraction and SP retained by the column after elution), column extraction improved SP measurements only with lung tissue. The amount of SP remaining in the sample after the first extraction also varied among tissues. The first acid extraction effectively isolated 80% of total SP from skin. In contrast, the first extraction with lung tissue recovered only 58%. Because both acid and heat effectively release SP from nerve endings, this could reflect the presence of non-neuronal SP, especially in lung. High-dose capsaicin treatment, which depletes SP in nerve endings, caused 42% loss of SP in skin independent of amount of tissue extracted Our results suggest that a second acid extraction of tissue should be performed and that column extraction is clearly detrimental with skin samples.     

Coordination and speciation of cadmium in corn seedlings and its effects on macro- and micronutrients uptake

The effect of cadmium (Cd) on both the absorption of important nutrients and the synthesis of low molecular weight thiols (LMWTs) was investigated in corn plants. The inductively coupled plasma-optical emission spectroscopy results demonstrated that the concentration of Cd in tissues (mainly in roots) increased as the concentration in the medium increased. In addition, the concentration of phosphorus increased in roots of Cd treated plants but remained at normal concentration in shoots. On the other hand, the uptake of sulfur (S) followed a similar trend as the Cd uptake. The concentration of S and the production of LMWT were found to increase significantly upon exposure to Cd. The results of the X-ray absorption spectroscopy analyses indicated that Cd within tissues was bound to S ligands with interatomic distances of 2.51–2.52 Å. These results confirm a strong linkage between S uptake and the production of LMWT upon exposure to Cd.     

Macrophage-Tumor Cell Fusions from Peripheral Blood of Melanoma Patients

Background

While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.

Methods

We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.

Findings

Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.

Conclusions and Hypothesis

Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.