[Application of near infrared fluorescence imaging in detection of residual cancer in oral squamous cell carcinoma].

Objective: Local recurrence is the main cause of treatment failure in patients with oral squamous cell carcinoma (OSCC). This study was proposed to investigate the feasibility of near infrared fluorescence (NIF) via indocyanine green (ICG) for monitoring surgical marginal in operation for OSCC patients. Methods: In 35 patients with OSCC treated surgically in the Department of Oral and Maxillofacial Surgery, Nanjing University School of Medicine, from January 2019 to June 2020, ICG (0.75 mg/kg) was administered intravenously via elbow vein at (12±1) hours before surgery, and NIF was performed intraoperatively on the surgical field and the cut edge of the surgically excised specimen, and fluorescence intensity was measured for OSCC tissue and normal oral mucosa, abnormal fluorescence signals were taken and subjected to rapid cryopathological examination. Correlation between NIF tumor boundary grading and pathological tumor boundary grading was analyzed by Spearman correlation analysis. Results: Clear ICG NIF was obtained for tumor lesions in all 35 patients, with a positive rate of 100%. The fluorescence intensity of OSCC tissue was (412.73±146.56) au, which was higher than that of normal oral mucosa tissue [(279.38±82.56) au, P<0.01]. Abnormal fluorescence signals were detected at the tumor bed and the cut edge of the surgical resection specimen in 4 patients, of which 2 cases were pathologically confirmed as cancer cell residue and 2 cases as inflammatory cell infiltration. The rate of positive detection of cut margins using ICG NIF technique in OSCC was 5.7% (2/35). Twenty of the 35 OSCC patients had grade 1, 11 of grade 2, and 4 of grade 3 tumor borders revealed by NIF of surgical resection specimens, which was positively correlated with pathological tumor border (r=0.809, P<0.001). Conclusions: ICG NIF technique can effectively detect the residual cancer cells at the incision margin, which is of great clinical value in reducing local recurrence of OSCC after surgery due to intraoperative cancer residue.

Postoperative radiotherapy is dispensable for OSCC patients with micrometastases in lymph nodes.

Lymph node metastasis is a decisive factor for performing postoperative radiotherapy for oral squamous cell carcinoma (OSCC). However, whether OSCC patients with only micrometastasis need postoperative radiotherapy is unclear. In this study, OSCC patients (n = 311) with negative (n = 247), only micrometastasis (n = 44) and macrometastasis (n = 20) were detected and selected by HE staining. Micrometastasis was re-assessed using immunohistochemical staining of cytokeratin (CK) in HE-negative patients to find out the false negative cases. The results indicated that, among the negative lymph node cases (n = 247), the positive rate of CK was 4.94% (n = 12). Besides, the clinical features of the primary tumor in relation to the only micrometastatic status and the value of the postoperative radiotherapy on the only micrometastasis patients were evaluated. Patients with only micrometastasis had higher T stage and inferior worst pattern of invasion (WPOI) than patients without micrometastasis, but they had longer overall survival (OS), metastasis-free survival (MFS), and disease-free survival (DFS) than macrometastasis patients. However, the survival time of only micrometastasis patients with or without postoperative radiotherapy was comparable, even in patients with inferior WPOI. Radiotherapy, however, may only benefit patients with IV/V levels of micrometastasis. These data indicated that postoperative radiotherapy is dispensable for only micrometastasis OSCC patients.

[Evaluation and influencing factors of surgical margin status of oral squamous cell carcinoma].

Surgical resection with adequate margins is an essential component of the treatment for patients with oral squamous cell carcinoma (OSCC). A distance of 5 mm or more between healthy tissue to the tumor front is generally accepted as a safe margin. It is very important for surgeons to precisely evaluate the resection area of tumor both pre- and intra-operatively and try to achieve a safe margin, which will result in a decreased risk of local recurrence. The relationship of surgical margin status to patients' prognosis, and factors which will affect surgical margin distance demand are discussed in this paper. We recommend that adequate margins evaluation should take consideration of many factors such as anatomical location, depth of tumor invasion, pattern of tumor invasion, mucosal dysplasia grade and so on. With the development of molecular biology, surgical margin study at molecular level can give us a new strategy to evaluate its adequacy.

Trapezius muscle innervation from the spinal accessory nerve and branches of the cervical plexus.

The aim of this study was to demonstrate the extent of motor innervation of the trapezius muscle from the accessory nerve and branches of the cervical plexus using intraoperative electroneurography and histochemical staining. In 34 patients during radical neck dissection the accessory nerve and C2-C4 branches running to the trapezius were identified and stimulated. Potentials were registered under three conditions: intact accessory nerve, section of superior part of communication between the nerve and the cervical branches, and complete section of the nerve. Projections that did not elicit responses were analyzed for acetylcholinesterase activity. Before cutting the accessory nerve, its stimulation led to a recordable contraction in all parts of the trapezius muscle in all patients. C2 contributions were seen in 15, C3 in 21 and C4 in 20 patients. After sectioning of the upper half of the nerve, the results were similar. After the nerve was completely cut, C2 contributions were seen in only 2 patients, but C3 were seen in 20 patients and C4 in 19 patients. Histochemical staining revealed that the branches with no responses contained both motor and sensory axons. The accessory nerve provides the main motor input to the trapezius muscle, but preservation of the C2-C4 branches to the muscle during modified neck dissection should improve outcomes.

Use of iodoacetylene as a dipolarphile in the synthesis of 5-iodoisoxazole derivatives.

Iodoacetylene 1 was prepared in situ from the reactions of ethynylmagnesium bromide or tributyl(ethynyl)tin with iodine. It was used as a dipolarphile in the [2 + 3] cyclization reaction with 1,3-dipolar nitrile oxide derivatives to produce 2-(5-iodoisoxazol-3-yl)pyridine 2 and 3-(4-fluorophenyl)-5-iodoisoxazole 8 in good yield (70-90%). Subsequently, several 5-substituted isoxazole derivatives 3 were obtained by Pd-catalyzed reactions. [reaction: see text]

Cerebellar granule cells acquire transferrin-free iron by a carrier-mediated process.

In this study, the mechanism of transferrin-free iron uptake by brain neuronal cells was investigated using the cultured cerebellar granule cells. Effects of incubation time, iron concentration, temperature and other divalent metals on the cellular uptake were determined. After five days of plating, the cells were incubated with different concentrations of transferrin-free iron in isotonic sucrose solution at different temperatures for a certain time. The cellular transferrin-free iron uptake was analysed by measuring the cellular radioactivity with a gamma-counter. The result showed that the cultured cerebellar granule cells had the capacity to acquire transferrin-free iron at pH 6.5, at which it was demonstrated that transferrin binds iron very poorly and only very little transferrin can be internalized by reticulocytes and HeLa cells. The iron uptake by cells increased with incubation time in a linear manner at a rate of 0.1076 pmol/microg protein/min within the first 10 min. The uptake was time- and temperature-dependent, iron concentration saturable, and inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+. These characteristics of transferrin-free iron uptake by the cultured cerebellar granule cells observed in this study, similar to those obtained from cells outside of the brain, implied that a carrier-mediated iron transport system might be present on the membrane of this type of brain neuronal cells. In addition, no significant difference in malondialdehyde measurement was found when the cells were incubated without or with the lower concentrations of iron (< 4 microM) for 20 min at 37 degrees C, demonstrating that this system was valid for studying membrane iron transport in this type of brain neuronal cell.

Effect of iron and lipid peroxidation on development of cerebellar granule cells in vitro.

The aim of this study was to investigate the effect of chelated ferric ion on neuronal development in vitro using cultured cerebellar granule cells of the rat. The cells were exposed to ferric nitrilotriacetate at varying concentrations for seven or 14 days. In addition to morphological studies, protein content determination and malondialdehyde measurement were performed. The study showed that cell development, with the addition of a lower concentration of chelated ferric ion (5 microM), could be kept in a normal condition, no significant changes in protein content and malondialdehyde production being found as compared with those of the controls, while the addition of higher concentrations of chelated ferric ion (> or = 10 microM) to the cultures demonstrated an adverse effect on development of cerebellar granule cell in vitro. Determination of protein content showed that the neuronal population decreased significantly, and the neuronal loss was inversely proportional to the iron concentrations added. Malondialdehyde measurement demonstrated that the extent of lipid peroxidation reaction increased remarkably with increasing iron concentration. A very close and highly significant correlation (gamma=0.985) between changes of malondialdehyde production and protein content was observed. These results suggest that the neuronal loss in the cultures with higher concentrations of iron was due to lipid peroxidation reaction induced by the addition of iron, and that iron overload might accelerate the process of ageing and death of cerebellar granule cells in vitro.

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry.

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.

Transferrin-bound iron uptake by the cultured cerebellar granule cells.

Excessive brain iron has been found in several neurodegenerative diseases. However, little information is available about mechanism of iron uptake by different types of brain cells including neurons. In this study, transferrin-bound iron (Tf-Fe) accumulation in the cultured cerebellar granule cell was investigated in vitro. After 5 days of culture, the cells were incubated with 1 microM of double-labelled transferrin (1251-Tf-59Fe) at 37 degrees C for 60 min. The cellular Tf-Fe and transferrin (Tf) uptake was analysed. The result showed (1) Tf uptake by the cells increased rapidly at the first 5 min, reaching its maximum after about 20 min of incubation; (2) Tf-Fe uptake kept increasing in a linear manner during the whole period of incubation; (3) the addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake; (4) trypsin and unlabelled Tf-Fe inhibited the uptake rate of Tf-Fe as well as Tf. The results suggested that Tf-Fe transport across the membrane of this type of neuron, much like other mammalian cells, was mediated by Tf-TfR endocytosis. Dysfunction of Tf or TfR would possibly lead to iron irregulation in the brain and consequently cause damage to neuronal functions.

Inhibitory mechanism of lead on transferrin-bound iron uptake by rabbit reticulocytes: a fractal analysis.

Experimental data of transferrin and transferrin-bound iron uptake by rabbit reticulocytes in the presence or absence of extracellular lead is analyzed by means of a fractal model. A highly significant correlation of fractal dimension (Df) of intracellular transferrin or transferrin-bound iron uptake with varying extracellular concentrations of lead (0 approximately 25 umol/L) was observed (Transferrin: r = 0.897, p = 0.015; transferrin-bound iron: r = 0.947, p = 0.004). The Df of membrane-bound transferrin (r = -0.618, p = 0.191) or transferrin-bound iron (r = 0.144, p = 0.786) did not appear to be markedly altered by lead. Further analysis shows that inhibitory degree of lead on intracellular iron uptake is higher than that on intracellular transferrin uptake. These results suggest that the inhibitory effect of lead on the iron uptake may occur in intracellular process rather than in membrane binding step, probably inhibiting translocation of iron across the endosomal membrane.

Quantification of heterocyclic amine carcinogens in cooked meats using isotope dilution liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

Heterocyclic aromatic amines (HAAs) are formed in cooked meats through pyrolysis reactions of different amino acids in the presence or absence of creatine/creatinine and sugars. HAAs are mutagens, colon/mammary gland carcinogens in rodents, and are suspected in the etiology of human cancers. In this study, cooked meats containing incurred HAAs as well as control (microwave) meat, were spiked with four labeled HAA internal standards (MeIQx, IQ, AAC and PhIP) and extracted using a liquid/liquid cleanup procedure. Isotope dilution measurements were made using on-line liquid chromatography atmosphere pressure chemical ionization tandem mass spectrometry with multiple reaction monitoring to provide the sensitivity and specificity needed for trace analysis in these complex matrices. The procedure was validated using control meat spiked with the four native HAAs at 0-50 ppb. The levels of HAAs found in cooked meats ranged from non-detectable (limit of detection 0.1-1.0 ppb) in microwave-cooked hamburger to 226 ppb PhIP and 104 ppb AAC in well-done grilled chicken. This methodology has the potential to provide accurate data on the consumption of HAAs in the diet for use in human cancer risk assessment.

Synthesis and antimalarial activities of several fluorinated artemisinin derivatives.

The carbonyl groups in several artemisinin derivatives were converted into geminal difluorinated compounds on treatment with diethylaminosulfur trifluoride. A number of other mono- and polyfluorinated artemisinin derivatives were prepared. Their in vitro antimalarial activities were all equal to or greater than the nonfluorinated analogs or precursors.

Cerebellar interpositus nucleus modulates neuronal activity of lateral hypothalamic area.

Effects of stimulating the cerebellar interpositus nucleus (IN) on the neuronal activity of lateral hypothalamic area (LHA) were first observed in the cat. The results showed that (1) IN stimulation could elicit inhibitory, excitatory, inhibitory-excitatory and excitatory-inhibitory responses from LHA neurones, with a majority of inhibitory responses (46.9%); (2) the responsive latencies of LHA neurones to IN stimulation ranged from 5 to 45 ms, while most (83.6%) showed a short latency of < 15 ms; (3) of 67 LHA neurones which responded to the IN stimulation, 42 (62.7%) cells were identified to be glucose-sensitive neurones. These results suggest that IN may be involved in the role of LHA modulating food intake behaviour, as well as other non-somatic functions.

Synthesis and antimalarial activities of 12 beta-allyldeoxoartemisinin and its derivatives.

Synthesis of 12 beta-allyldeoxoartemisinin from dihydroartemisinin and subsequent transformations to other 12 beta-alkyldeoxoartemisinins are described. All compounds were tested in vitro versus two drug-resistant strains (Plasmodium falciparum) of malaria. The in vivo activity and toxicity of the most active compound, 12 beta-propyldeoxoartemisinin, were comparable to that of arteether.

Reaction of antimalarial endoperoxides with specific parasite proteins.

The endoperoxides are a new class of antimalarial agents, of which artemisinin (qinghaosu) is the prototype. We have previously shown that artemisinin is capable of alkylating proteins in model reactions. In the present study, we showed that when Plasmodium falciparum-infected erythrocytes are treated with a radiolabeled antimalarial endoperoxide, either arteether, dihydroartemisinin, or Ro 42-1611 (arteflene), the radioactivity is largely coverted into a form which can be extracted with sodium dodecyl sulfate (SDS). Autoradiograms of SDS-polyacrylamide gels showed that six malarial proteins are radioactively labeled by the three endoperoxides. This labeling occurs at physiological concentrations of drug and is not stage nor strain specific. The labeled proteins were not the most abundant proteins seen on Coomassie-stained gels. No proteins were labeled when uninfected erythrocytes were treated with these drugs, nor when infected erythrocytes were treated with the inactive analog deoxyarteether. Thus, the antimalarial endoperoxides appear to react with specific malarial proteins.

Effect of volatile anesthetics on the circular dichroism of bilirubin bound to human serum albumin.

The characteristic circular dichroism of bilirubin bound to human serum albumin undergoes a remarkable sign inversion on addition of halothane, chloroform and other volatile anesthetics. This sign inversion, which is completely reversed by removal of the anesthetic, reflects a pronounced conformational change of the bound ligand; probably a complete inversion of chirality. The observation suggests that association of volatile anesthetics with proteins can markedly alter the internal topography of receptor sites and potentially influence the stereoselectivity of ligand binding.

Circular dichroism of bilirubin-amine association complexes: insights into bilirubin-albumin binding.

Bichromophoric (4Z, 15Z)-bilirubin-IX alpha, the yellow-orange cytotoxic pigment of jaundice, adopts either of two intramolecularly hydrogen-bonded enantiomeric conformations that are in dynamic equilibrium in solution. The addition of optically active amines induces the pigment solutions to exhibit intense bisignate circular dichroism in the region of the bilirubin long wavelength uv-visible absorption band. The most intense circular dichroism Cotton effects, (delta epsilon) approximately equal to 130, are induced by beta-arylamines and are comparable to those exhibited by bilirubin complexes with serum albumin and other proteins. Like serum albumin and other proteins, the optically active base acts as a chiral complexation agent to induce an asymmetric transformation of bilirubin, whose induced bisignate circular dichroism Cotton effect is characteristic of exciton splitting of the component pyrromethenone chromophores. The amines thus serve as chiral templates for molecular recognition, and the complementary action of the amine complexation sites provides insight into the binding forces important in protein-bilirubin heteroassociation.