Fluorescence turn-off-on for highly selective detection of serum l-cysteine based on AuNCs-AuNPs ensembles.

The discriminative monitoring of biothiols in biological fluids is a great challenge. Herein, we developed a fluorescence "turn-off-on" probe based on AuNCs-AuNPs ensembles, which detected l-cysteine (l-Cys) with high selectivity. The strong blue fluorescence of l-hydroxyproline-capped gold nanoclusters (l-Hyp@AuNCs) at 465 nm was quenched by AuNPs via the inner filter effect. Interestingly, the fluorescence was recovered by interaction with l-Cys due to the electrostatic and specific Au-S interactions between l-Cys and AuNPs. The effect of the l-Cys amount on the fluorescence "off-on" efficiency showed good linearity in the range of 1.5-35.0 µM (R2 = 0.986) with a detection limit of 1.4 µM (3σ). The proposed method was successfully applied to test the serum l-Cys levels without the interference of glutathione, homocysteine and methionine. The AuNCs-AuNPs ensembles-based fluorescence "turn-off-on" protocol shows great potential in specific biothiol sensing.

Dissociation of biomolecules in liquid environments during fast heavy-ion irradiation.

The effect of aqueous environment on fast heavy-ion radiation damage of biomolecules was studied by comparative experiments using liquid- and gas-phase amino acid targets. Three types of amino acids with different chemical structures were used: glycine, proline, and hydroxyproline. Ion-induced reaction products were analyzed by time-of-flight secondary-ion mass spectrometry. The results showed that fragments from the amino acids resulting from the C-Cα bond cleavage were the major products for both types of targets. For liquid-phase targets, specific products originating from chemical reactions in solutions were observed. Interestingly, multiple dissociated atomic fragments were negligible for the liquid-phase targets. We found that the ratio of multifragment to total fragment ion yields was approximately half of that for gas-phase targets. This finding agreed with the results of other studies on biomolecular cluster targets. It is concluded that the suppression of molecular multifragmentation is caused by the energy dispersion to numerous water molecules surrounding the biomolecular solutes.

Differing energy densities with laser 670nm InGaP controls inflammation and collagen reorganization in burns.

PURPOSE: This study compared different energy densities of laser on second degrees burns in rats aiming to determine the most effective dosimetry in stimulation of the healing process. METHODS: Burns were induced in the dorsal skin of 54 animals divided into three groups (n: 18): 1-without treatment; 2-irradiated lesions by the Indium Gallium Phosphide (InGaP) 670nm (4.93J/cm2) laser; 3-irradiated lesions by the InGaP-670nm (9.86J/cm2) laser. Samples were collected on the 2, 10 and 18 days after injury for structural, morphometry, biochemical analysis and Western blotting. RESULTS: The energy densities examined were effective in significantly increasing the total number of fibroblasts and blood vessels and reduce the number of inflammatory cells particularly in irradiated lesions with 9.86J/cm2. This same energy density significantly increased the amount of GAGs (Glycosaminoglycans), decreased the TGF-ß1 (Transforming Growth Factor ß1) and increased the VEGF (Vascular and Endothelial Growth Factor) during the experimental period. This energy density also significantly increased the Collagen type I and decreased Collagen type III and the active isoform of metalloproteinase 9 (MMP-9). CONCLUSIONS: The energy density of 9.86J/cm2 was more effective in promoting cellular responses related to neoangiogenesis, decreasing inflammation and collagen fibers reorganization.

Effects of zoledronate on irradiated bone in vivo: analysis of the collagen types I, V and their cross-links lysylpyridinoline, hydroxylysylpyridinoline and hydroxyproline.

Radiotherapy can lead to a reduction of bone density with an increased risk of pathological fractures. Bisphosphonates may represent a preventive treatment option by increasing the density of anorganic bone mineral. Yet it is unknown how bisphosphonates act on irradiated collagen cross-links, which play an essential role for the mechanical stability of bone. The aim of this study was to evaluate the effects of zoledronate on bone collagens and their cross-links after irradiation. The right femur of 37 rats was irradiated with a single dose of 9.5 Gy at a high dose rate using an afterloading machine. Half of the rats (n=18) received additionally a single dose zoledronate (0.1 mg/kg body weight). Fourteen and 100 days after irradiation the femora were collected for histologic evaluation and determination of the collagen cross-links lysylpyridinoline, hydroxylysylpyridinoline, and hydroxyproline. The collagen types were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fourteen days after treatment the lysylpyridinoline levels of all treatment groups were significantly lower compared to the untreated control. After 100 days, in the combined radiotherapy+zoledronate group significantly lower lysylpyridinoline values were determined (p=0.009). Radiotherapy and/or zoledronate did not change significantly the level of hydroxylysylpyridinoline. The concentration of hydroxyproline was 14 days after irradiation significantly higher in the combined treatment group compared to the control. No significant differences were observed 100 days after treatment. Zoledronate does not have the ability to restore the physiological bone collagen cross-link levels after radiotherapy. However, this would be necessary for regaining the physiological mechanical stability of bone after irradiation and therefore to prevent effectively radiation-induced fractures.

Effects of preoperative pelvic irradiation on colonic anastomosis healing. An experimental study in rats.

PURPOSE: Colorectal anastomosis is a constant worry-issue among surgeons because of high rates of complications, specially the dehiscence. The preoperative irradiation on cancer surgeries might interfere in the healing process, leading to an unfavorable outcome. METHODS: In the present study, two groups of rats were irradiated previously to a colorectal anastomosis surgery, with intervals of 4 and 8 weeks between the procedures. Seven days after the surgery, healing process was evaluated for dehiscence presence and histologic inflammatory characteristics. Also, levels of hydroxyproline, metalloproteinases and vascular endothelial growth factor were measured. RESULTS: Our results showed a higher incidence of dehiscences on the animals submitted to irradiation, compared to controls, with a reduced inflammatory activity in the healing tissue. DISCUSSION: Comparing both irradiated groups, those irradiated 8 weeks before surgery showed higher levels of hydroxyproline and metalloproteinases, indicating higher efficiency of the healing process. In conclusion, preoperative irradiation interferes with intestinal anastomosis healing and a larger time interval between both procedures is safer in terms of the healing quality.

Effects of preoperative pelvic irradiation on colonic anastomosis healing: an experimental study in rats / Efeito da radioterapia pélvica pré-operatória na cicatrização de anastomoses colônicas: estudo experimental em ratos

PURPOSE: Colorectal anastomosis is a constant worry-issue among surgeons because of high rates of complications, specially the dehiscence. The preoperative irradiation on cancer surgeries might interfere in the healing process, leading to an unfavorable outcome. METHODS: In the present study, two groups of rats were irradiated previously to a colorectal anastomosis surgery, with intervals of 4 and 8 weeks between the procedures. Seven days after the surgery, healing process was evaluated for dehiscence presence and histologic inflammatory characteristics. Also, levels of hydroxyproline, metalloproteinases and vascular endothelial growth factor were measured. RESULTS: Our results showed a higher incidence of dehiscences on the animals submitted to irradiation, compared to controls, with a reduced inflammatory activity in the healing tissue. DISCUSSION: Comparing both irradiated groups, those irradiated 8 weeks before surgery showed higher levels of hydroxyproline and metalloproteinases, indicating higher efficiency of the healing process. In conclusion, preoperative irradiation interferes with intestinal anastomosis healing and a larger time interval between both procedures is safer in terms of the healing quality.

INTRODUÇÃO: As anastomoses colorretais são motivos constante de preocupação por parte dos cirurgiões, em virtude do alto índice de complicações, principalmente as deiscências. O uso da radioterapia previamente à cirurgia, nos casos de doença neoplásica, pode interferir no processo cicatricial das anastomoses, e levar a uma evolução desfavorável. MÉTODOS: Os autores estudaram dois grupos de ratos, submetidos a radioterapia e à confecção de uma anastomose no cólon, com intervalo de 04 e de 08 semanas entre os dois procedimentos, comparando com um grupo controle. Após 07 dias da cirurgia, estudaram-se vários aspectos do processo cicatricial: presença de deiscência, características inflamatórias do tecido, dosagem de hidroxiprolina, de mateloproteinase e de VEGF. RESULTADOS: Os autores detectaram maior índice de deiscência nos animais submetidos à radioterapia, com prejuízo da atividade inflamatória característica de um tecido em cicatrização. DISCUSSÃO: Dentre os dois grupos irradiados, aquele com intervalo de oito semanas entre a radioterapia e a confecção da anastomose teve dosagem mais alta de hidroxiprolina e metaloproteinase, demonstrando maior eficiência do processo cicatricial. CONCLUSÃO: A radioterapia prévia interfere no processo de cicatrização das anastomoses intestinais, e que um maior intervalo de tempo entre os dois procedimentos é melhor para garantia de uma cicatrização satisfatória.

Effects of two conventional preoperative radiation schedules on anastomotic healing in the rat colon.

BACKGROUND: Preoperative radiotherapy (RT) is an increasingly popular form of adjunct therapy for rectal cancer; however, little is known about its effects on matrix metalloproteinase (MMP) expression in colonic anastomotic healing. METHODS: Wistar rats were irradiated to a total dose of 25 or 40 Gy. Four days after the end of RT, an end-to-end colorectal anastomosis was performed. Animals were sacrificed at 1, 3, and 7 days after the anastomosis. A control group was studied similarly, but was not irradiated. RESULTS: No significant differences were found in peritonitis rate and anastomotic complications. The average bursting pressure and breaking strength were only reduced significantly in the rats irradiated with 40 Gy. However, the concentration and the content of hydroxyproline in anastomotic tissues were unchanged. In irradiated rats, MMP-2 and MMP-9 were significantly increased at 40 Gy, but not at 25 Gy. On the other hand, 25-Gy irradiation induced a smaller increase in the levels of the tissue inhibitors of metalloproteinase-1 compared with the controls. CONCLUSION: Anastomotic strength is adversely affected by high-dose fractionated preoperative RT. In contrast, preoperative RT at 25 Gy in five fractions over 5 days is safe with regard to the maintenance of wound strength in colorectal anastomosis.

Mechanical and biochemical effect of monopolar radiofrequency energy on human articular cartilage: an in vitro study.

BACKGROUND: There are growing concerns about thermal chondroplasty using radiofrequency energy to treat partial-thickness cartilage defects. However, most studies emphasize effects on chondrocyte viability, and other factors such as mechanical properties are less studied. HYPOTHESIS: Radiofrequency energy may cause significant effects on articular cartilage other than chondrocyte viability. STUDY DESIGN: Controlled laboratory study. METHODS: Human osteoarthritic cartilage samples were obtained from total knee arthroplasty, and monopolar radiofrequency energy was applied using commercially available equipment. Material properties (compressive stiffness, surface roughness, and thickness) just before and after thermal treatment were determined using ultrasound. A series of biochemical analyses were also performed after explant culture of the samples. RESULTS: The cartilage surface became smoother by radiofrequency energy, whereas cartilage stiffness or thickness was not altered significantly. Collagen fibrils, especially in the superficial layers, were converted to denatured form, whereas proteoglycan contents released in the media as well as retained in the tissue remained unchanged. The concentrations of matrix metalloproteinases (MMP-1 and MMP-2) were reduced remarkably. CONCLUSION: Radiofrequency energy is able to create a smooth cartilage surface and reduce catabolic enzymes at the cost of collagen denaturation and chondrocyte death in the superficial layers. The stiffness of the cartilage is not changed at time zero. CLINICAL RELEVANCE: Further animal as well as clinical studies will be necessary to fully evaluate the long-term effects of radiofrequency energy.

The effect of preoperative radiotherapy on systemic collagen deposition and postoperative infective complications in rectal cancer patients.

PURPOSE: Preoperative, high-dose radiotherapy for rectal cancer reduces local recurrence rates and improves overall survival. However, adverse effects in varying degrees include impaired wound healing and local infection. This study investigates the influence of preoperative, high-dose radiotherapy on subcutaneous accumulation of collagen in a primary rectal cancer group operated on with or without adjuvant radiotherapy. METHODS: Forty-two eligible patients who underwent total mesorectal excision surgery with or without radiotherapy were included in the study. Polytetrafluoroethylene tubings were implanted in the arm ten days before surgery (three days before the start of radiotherapy). Implants were extracted the day before surgery. New implants were inserted before surgery and were extracted ten days after surgery. The hydroxyproline and proline contents of the implants were measured and the hydroxyproline/proline ratio was calculated as a measure for deposited collagen relative to protein. Blood loss, postoperative complications, and blood levels of hemoglobin, leukocytes, and albumin were recorded. RESULTS: The two groups were similar in relation to Dukes stage, age, and body mass index. Infectious complications developed in 39 percent of patients after radiotherapy compared with 16 percent in the nonirradiated group. In the irradiated patients with infective complications we found a significant decrease in the hydroxyproline/proline ratio compared with that of irradiated patients without infections (P = 0.037). There was a significant decrease in the leukocyte count preoperatively and postoperatively in the irradiated group compared with surgery alone. CONCLUSIONS: High-dose, short-term radiotherapy does not have a systemic effect on collagen accumulation, but a significant reduction is manifested in infected patients. Radiotherapy also impairs leukocyte production and increases the postoperative infective complication rate.

A preliminary study of the effects of laser radiation on collagen metabolism in cell culture.

A low power Ga-As pulse laser was used to stimulate cultured human embryonic fibroblast cells. Energy fluencies varied from 0-1 J/cm2 over a period of 1-4 days. Fibroblast procollagen production was monitored by the synthesis of [3H] hydroxyproline, and DNA replication was assessed by [3H] thymidine incorporation. Following laser treatment, controlled pepsin digestion measured the increase in cell biostimulation. Maximum increase in collagen production and cell biostimulation occurred after 4 episodes of laser treatment at 24-hour intervals. Laser doses between 0.099 and 0.522 J/cm2 had the most significant stimulatory effects on fibroblast function. Clinical efficacy of the low power Ga-As pulse laser may be related to enhanced connective tissue repair.

[Changes in the levels of total and protein-bound hydroxyproline in the serum and urine of patients with cancer of the larynx after radiotherapy]. / Zmiany stezenia hydroksyproliny calkowitej i bialkowej w surowicy i moczu u chorych napromienianych z powodu raka krtani.

The serum concentration of total and protein-bound hydroxyproline as well as urinary hydroxyproline excretion were measured in 52 laryngeal cancer patients before, during and after irradiation. The modified colorimetric method described by Prockop and Udenfriend was used. After irradiation the increased total hydroxyproline concentration and excretion were observed, whereas no changes in serum protein-bound hydroxyproline were stated. Any correlation between the results of therapy and serum/urinary hydroxyproline levels were observed.

[Effect of early local post-irradiation reaction on total and protein-bound hydroxyproline levels in the blood and urine of patients with laryngeal cancer]. / Wplyw wczesnego miejscowego odczynu popromiennego na stezenie hydroksyproliny calkowitej i bialkowej w surowicy krwi i moczu u chorych na raka krtani.

In 52 laryngeal cancer patients treated by radiotherapy the serum and urine level of hydroxyproline was tested in connection with the intensity of local postirradiation reactions. The serum and urine level were tested by colorimetric method of Proctor and Udenfriend in Tomaszewski modification. In patients with second and third steps of local reaction intensity there was an elevated level of hydroxyproline only in urine. No correlation was observed between the serum level and local reaction.

Effects of ionizing radiations on human costal cartilage and exploration of the procedures to protect the tissue from radiation damage.

The chemical changes produced following 60Co-gamma-irradiation of human costal cartilage have been monitored using "critical electrolyte concentration" (CEC) measurements utilizing Alcian Blue--magnesium chloride and Toluidine Blue--sodium chloride systems. The decrease in the CEC with radiation can be related to the decrease in hexose and hexosamine contents of the cartilage. Such changes arise as a result of degradation of the glycosaminoglycan component of the tissue. The state of the collagen fibres after irradiation has been examined using the Van Giesson stain and by biochemical assay of the hydroxyproline moeity. Energy transfer methods have been utilized to protect the glycosaminoglycan component of the cartilage from radiation damage.