[Deep neck space infections: a retrospective cohort study of surgical risk factors].

Objective: To explore risk factors affecting treatment for deep neck space infections (DNSIs) so as to provide guidance for appropriate early managements. Methods: A retrospective cohort study was conducted on inpatients with DNSIs admitted to the Department of Otolaryngology, Head and Neck Surgery, Affiliated Hospital of Qingdao University from March 2013 to February 2021. Patients were divided into surgical and non-surgical groups based on whether they had surgery or not. Information collected included demographic data, disease-related signs and symptoms, treatment history, systemic comorbidities, imaging data and laboratory indicators. Hypothesis testing, univariate Logistic regression and multivariate Logistic regression were used for data processing. Resuts A total of 61 patients were included, including 37 males and 24 females, aged 6-96 years. There were 35 cases (57.4%) in the surgical group and 26 cases (42.6%) in the non-surgical group. Multivariate analysis showed that risk factors for surgery as followings: neck dyskinesia (OR=0.03, 95%CI: 0.00-0.24), dysphagia (OR=0.10, 95%CI: 0.02-0.72), serum white blood cell count≥16.74×109/L (OR=1.18, 95%CI: 1.01-1.39) and interspace gas (OR=0.03, 95%CI: 0.00-0.30). Conclusion: Clinicians should be alert to these risk factors for surgery in the course of treatment and timely surgical treatment for patients who meet the conditions.

MiR-224-5p targets EGR2 to promote the development of papillary thyroid carcinoma.

OBJECTIVE: Various microRNAs (miRNAs) have been reported to be involved in the pathogenesis and development of human cancers, including papillary thyroid carcinoma (PTC). However, the role of miR-224-5p in PTC progression remains unclear. Therefore, the purpose of this study is to illuminate the function of miR-224-5p in PTC. PATIENTS AND METHODS: Expression of miR-224-5p and EGR2 was examined in PTC by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Transwell assay was used to detect cell migration and invasion. Western blot analysis was used to detect epithelial-mesenchymal transition (EMT). The relationship between miR-224-5p and EGR2 was confirmed by Dual-Luciferase assay. RESULTS: Upregulation of miR-224-5p and downregulation of EGR2 expression were detected in PTC tissues and cells. Upregulation of miR-224-5p was found to be associated with TNM stage and lymph node metastasis. Meanwhile, it also predicted poor prognosis in PTC patients. Functionally, upregulation of miR-224-5p promoted cell metastasis and EMT in PTC. In addition, miR-224-5p was detected to directly target EGR2. EGR2 expression was negatively correlated with EGR2 expression in PTC. Of note, overexpression of EGR2 attenuated the carcinogenic effects of miR-224-5p in PTC. CONCLUSIONS: MiR-224-5p promotes cell migration, invasion, and EMT in PTC by targeting EGR2.

Label-free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy.

Glioblastoma multiforme (GBM-WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10-12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.

Genome-wide identification, phylogeny and expression analysis of the lipoxygenase gene family in cucumber.

Plant lipoxygenase (LOX) is involved in growth and developmental control processes, through the biosynthesis of regulatory molecules and defense responses to pathogens, wounding and stress. To date, few LOX proteins and little tissue expression profiling have been reported in detail for cucumber (Cucumis sativus L.). Recent completion of the cucumber genome sequence now permits genome-wide analysis of the LOX gene family in cucumber as well as comparison with LOX in Arabidopsis and rice. We identified 23 candidate LOX genes in the cucumber genome; phylogenetic analysis indicated that these LOX members cluster into two groups, designated types 1 and 2, as expected from previous studies. Sequence analysis showed that five binding sites of iron, including two consensus histidines in the LOX domain, are highly conserved in the cucumber LOX proteins. Analysis of chromosomal localization and genome distribution suggested that tandem duplication and/or polyploidal duplication contributed to the expansion of the cucumber LOX gene family. Based on intron/exon structure analysis, only a few of the extant intron patterns existed in the ancestor of monocots and eudicots. Expression data showed widespread distribution of the cucumber LOX gene family within plant tissues, suggesting that they perform different functions in different tissues.

Identification of Peroxisome Membrane Proteins (PMPs) in Sunflower (Helianthus annuus L.) Cotyledons and Influence of Light on the PMP Developmental Pattern.

Boundary membranes were recovered from glyoxysomes, transition peroxisomes, and leaf-type peroxisomes purified from cotyledons of sunflower (Helianthus annuus L.) at three stages of postgerminative growth. After membranes were washed in 100 mM Na2CO3 (pH 11.5), integral peroxisome membrane proteins (PMPs) were solubilized in buffered aminocaproic acid/dodecyl maltoside (0.63 M/1.5%) and analyzed by nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Six prominent nondenatured PMP complexes and 10 prominent SDS-denatured polypeptides were identified in the membranes of the three types of peroxisomes. A nondenatured complex of approximately 140 kD, composed mainly of 24.5-kD polypeptides, decreased temporally, independently of seedling exposure to white, blue, or red light; only far-red light seemed to prevent its decrease. PMP complexes of approximately 120 and 70 kD, in contrast, were present at all stages and changed in polypeptide content. It remains to be determined whether these data reflect changes within in vivo complexes or within complexes formed following/during detergent solubilization. Conversion of glyoxysomes to leaf-type peroxisomes in white or red light after a 2-d dark period was accompanied by the appearance of three SDS-denatured PMPs: 27.5, 28, and 47 kD. The former two became part of the PMP120 and 70 complexes, as well as part of a new PMP130 complex that also possessed the PMP47. Growth of seedlings in blue or far-red light did not promote the appearance of PMPs 27.5 or 28. Blue light promoted the appearance of PMP47, and far-red light seemed to prevent its appearance. Chlorophyll likely is not the photoreceptor involved in accumulation of PMPs because the PMP composition is distinctly different in seedlings irradiated with red or blue light of comparable fluence rates. Several lines of evidence indicate that the synthesis and acquisition of membrane and all matrix proteins are not coupled. The data provide evidence for a change in PMP composition when sunflower or any other oilseed glyoxysomes are converted to leaf-type peroxisomes and suggest that the change is regulated by both photobiological and temporal mechanisms.

Low-density lipoprotein as a transporter of dolichol intermediates in the mammalian circulation.

The cholesteryl esters which make up the bulk of the core of the human low-density lipoprotein particle were removed by extraction into heptane and replaced with the fluorescent anthroyl or N-(7-nitrobenzyl-2-oxa-1,3-diazol-4-yl)aminohexanoyl esters of dolichol. The reconstituted low-density lipoproteins were efficiently internalized by normocholesterolaemic human fibroblasts but not by fibroblasts from patients lacking the low-density-lipoprotein receptor, or lacking the ability to internalize the receptor-lipoprotein complex. In normal fibroblasts, the reconstituted low-density lipoproteins were delivered to lysosomes after internalization. The results suggest that (i) dolichol intermediates in the human circulation are normally carried on low-density lipoproteins and (ii) that low-density lipoproteins are involved in the accumulation of dolichol intermediates in lysosomes during normal human aging and in certain diseases involving the lysosome. In addition, by incorporating these very hydrophobic probes into low-density lipoprotein, they can be presented to cells in culture at high concentration in a water-soluble form.

Uptake and transport of fluorescent derivatives of dolichol in human fibroblasts.

We are using fluorescent derivatives to visualize the endocytic transport of dolichol intermediates from the cell surface to the lysosome, and to estimate their rate of turnover within the lysosome. Anthroyl dolichol and anthroyl [1-14C]dolichol were synthesized and purified by chromatography on silica and C18 Sep-Paks followed by high-performance liquid chromatography on C18. The successful synthesis of anthroyl polyisoprenoid alcohols was confirmed by the use of uv-visible spectrometry and by fluorescence spectrometry. The purified esters were taken up into Ham's media containing 10-30% fetal calf serum or alternatively reconstituted into phospholipid liposomes for delivery to human fibroblasts in culture. The uptake of fluorescent dolichol esters into the cells and into lysosomes was demonstrated using fluorescence microscopy. The localization of anthroyl dolichol in lysosomes was further documented by simultaneously labeling fibroblasts with anthroyl dolichol and FITC-dextran a recognized lysosomal marker. Fibroblasts generally showed several groupings (domains) of lysosomes, some were dually labeled while others were labeled exclusively with either anthroyl dolichol or FITC-dextran. Labeling with anthroyl dolichol was very slow relative to labeling of the same fibroblasts with FITC-dextran suggesting that anthroyl dolichol acts as a labeling agent for intracellular membranes, particularly those of the lysosome while the dextran fluorescence is presumably of lysosolic origin. Several types of experiments were done with anthroyl [1-14C]dolichol to establish that the fluorescence seen in lysosomes represents anthroyl dolichol. Anthroyl dolichol appears to enter fibroblasts intact, since we were unable to recover any free [1-14C]dolichol from total lipid extracts of (i) media used for the uptake of anthroyl dolichol or (ii) the media removed from cells labelled for 42 h. In addition, attempts to hydrolyze anthroyl [1-14C]dolichol in vitro using whole fibroblast homogenates at pH 4.0 and 7.5 were unsuccessful, even though the fibroblasts expressed acid lipase activity using 4-methylumbelliferyl palmitate as substrate.

Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidermal growth factor (EGF) binding activity in both 3T3-1 and EGF receptor reconstituted NR-6 fibroblasts.

Measurements of nucleocytoplasmic transport of fluorescent-labeled macromolecules were performed in both an EGF-nonresponsive mutant fibroblast line (3T3-NR6) and in the same cell line reconstituted with active EGF receptors derived from rat hepatic membrane fraction. Immunolocalization studies of exogenously incorporated EGF receptors in reconstituted 3T3-NR6 fibroblasts demonstrated predominantly intracellular localization. The EGF receptor constructs also showed EGF-stimulated incorporation of [3H]thymidine, providing biochemical evidence for functional integration of the exogenously supplied EGF receptors into the reconstituted fibroblasts. Additional support for the functional incorporation of receptor may be inferred from the enhanced cellular accumulation of 125I-EGF in cells treated with chloroquine and leupeptin. 125I-EGF binding and transnuclear macromolecular transport measurements in mutant and reconstituted cells, in conjunction with such measurements on nuclei isolated from these cells, provide data consistent with a growth factor/nuclear signaling mechanism dependent on the nuclear acquisition of EGF binding activity from the plasma membrane.

Alkalinization of the lysosomes is correlated with ras transformation of murine and human fibroblasts.

The pH of the intralysosomal compartment of fibroblasts in culture was monitored by measuring the fluorescence emission intensity at 530 nm of fluid phase pinocytosed fluorescein-conjugated dextrans (FITC-dextrans) excited at 488 and 457 nm. Following the procedure of Ohkuma and Poole (Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. U. S. A. (1978) 75, 3327-3331), a relationship was established between the fluorescence emission intensity of the FITC-dextrans and pH. This correlation was used to determine the intralysosomal apparent pH (pHapp) of a series of fibroblast cultures. The mean intralysosomal pHapp values of nontransformed mouse 3T3 fibroblasts and an infinite life-span human fibroblast cell strain, designated MSU-1.1, was 5.0. In distinction that of 3T3 fibroblasts transformed to the malignant state by Kirsten murine sarcoma virus and MSU-1.1 cells transformed by transfection of the v-Ki-ras or T24 H-ras oncogene was 6.1. These measurements suggest that ras transformation results in a significant perturbation of lysosomal pH.

Dynamic continuity of cytoplasmic and membrane compartments between plant cells.

Fluorescence photobleaching was employed to examine the intercellular movement of fluorescein and carboxyfluorescein between contiguous soybean root cells (SB-1 cell line) growing in tissue culture. Results of these experiments demonstrated movement of these fluorescent probes between cytoplasmic (symplastic) compartments. This symplastic transport was inhibited with Ca2+ in the presence of ionophore A23187, and also with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Both of these agents have previously been demonstrated to inhibit gap junction-mediated cell-cell communication in animal cells. In a companion experiment, a fluorescent phospholipid analogue, N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylcholine (NBD-PC), was incorporated into soybean cell membranes to examine whether dynamic membrane continuity existed between contacting cells, a transport route not existing between animal cells. Photobleaching single soybean cells growing in a filamentous strand demonstrated that phospholipid did exchange between contiguous cells.

Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape.

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.

Epidermal growth factor and insulin stimulate nuclear pore-mediated macromolecular transport in isolated rat liver nuclei.

Fluorescence photobleaching was used to measure the effect of epidermal growth factor (EGF), insulin, and glucagon on the nuclear transport of fluorescent-labeled dextrans across the nuclear pore complex. EGF and insulin were found to stimulate transport approximately 200%, while boiling these polypeptide growth factors greatly diminished this enhancement activity. Glucagon demonstrated no enhancement effect. The nuclear transport enhancement effects were observed at EGF and insulin concentrations that elicit the various physiological responses, e.g., nanomolar range.

Fluorescence photobleaching analysis of nuclear transport: dynamic evidence for auxiliary channels in detergent-treated nuclei.

Nuclear transport experiments were performed on isolated rat liver nuclei to examine the permeability of membrane and detergent-free peripheral nuclear lamina. The transport of 64K molecular weight fluorescent-derivatized dextrans was measured by using the technique of fluorescence redistribution after photobleaching. Results of these experiments provide evidence for transport pathways that appear to be functionally distinct from nuclear pore complex channels. The suggestion is made that these supplemental pathways are embedded in the peripheral nuclear lamina and are normally masked by the inner nuclear membrane.

Nuclear actin and myosin as control elements in nucleocytoplasmic transport.

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.

Chemical factors that influence nucleocytoplasmic transport: a fluorescence photobleaching study.

The technique of fluorescence redistribution after photobleaching was used to measure the translocation rate of fluorescein-labeled dextrans across the nuclear pore complex in isolated rat liver nuclei. A transport assay system was established that could monitor the effect of biologically active molecules, e.g., ATP, GTP, cAMP on the translocation process. The results show that ATP, phosphoinositides, RNA, and insulin can enhance transport rates from 195 to 432%. It was further demonstrated that concanavalin A, but not wheat germ or soybean agglutinin, can block dextran transport completely. The effectors of dextran transport are similar to substances demonstrated to effect the efflux of RNA from isolated nuclei. A model for translocation through the nuclear pore is now presented that incorporates data from protein influx and RNA efflux experiments into a single pathway controlled by ATP.