Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues.

Most Departments of Pathology around the world have a considerable archive of formalin-fixed paraffin-embedded (FFPE) tissue suitable for molecular assessment. This article points out the potential DNA damage that may occur if basic steps are not followed during processing and storage of these samples. Furthermore, it hopes to establish parameters to optimize quality and quantity of DNA extracted from FFPE tissues.

Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues

Abstract Most Departments of Pathology around the world have a considerable archive of formalin-fixed paraffin-embedded (FFPE) tissue suitable for molecular assessment. This article points out the potential DNA damage that may occur if basic steps are not followed during processing and storage of these samples. Furthermore, it hopes to establish parameters to optimize quality and quantity of DNA extracted from FFPE tissues.

Resumo A maioria dos Departamentos de Patologia em todo o mundo têm um considerável acervo de tecidos embebidos em parafina e fixados em formalina, que são passíveis para análises moleculares. Este artigo apresenta os danos ao DNA que podem ocorrer se passos básicos não forem seguidos durante o processamento e armazenamento destas amostras. Além disso, procura estabelecer parâmetros para otimizar a qualidade e quantidade do DNA extraído de tecidos FFPE.

Controllable microwave three-wave mixing via a single three-level superconducting quantum circuit.

Three-wave mixing in second-order nonlinear optical processes cannot occur in atomic systems due to the electric-dipole selection rules. In contrast, we demonstrate that second-order nonlinear processes can occur in a superconducting quantum circuit (i.e., a superconducting artificial atom) when the inversion symmetry of the potential energy is broken by simply changing the applied magnetic flux. In particular, we show that difference- and sum-frequencies (and second harmonics) can be generated in the microwave regime in a controllable manner by using a single three-level superconducting flux quantum circuit (SFQC). For our proposed parameters, the frequency tunability of this circuit can be achieved in the range of about 17 GHz for the sum-frequency generation, and around 42 GHz (or 26 GHz) for the difference-frequency generation. Our proposal provides a simple method to generate second-order nonlinear processes within current experimental parameters of SFQCs.

[Expression of inhibin B betaB subunits in the testicular tissues of azoospermia patients with different pathological alterations].

OBJECTIVE: To investigate the expression of inhibin B betaB subunits in human testicular tissues. METHODS: Eighty-three cases of the azoospermia underwent testicular biopsy. In accordance with the pathological alterations of spermatogenesis, the samples were divided into four groups: Sertoli-cell-only syndrome (n = 21); hypospermatogenesis (n = 20), maturation arrest (n = 24) and almost normal spermatogenesis (n = 18). Immunohistochemical staining for inhibin B betaB subunits was conducted on the paraffin-embedded sections of different spermatogenesis to localize inhibin B betaB subunits in the seminiferous tubules. RESULTS: Immunohistochemically, positive products of inhibin B betaB subunits were found in both the seminiferous tubules and interstitial tissues of the testis as brown or yellow particles in the cytoplasm. Leydig cells and early intermediate spermatogenic cells showed a very strong positivity; Sertoli cells in the seminiferous tubules were mostly positive; peritubular myoid cells showed a weak positive staining; but no positive expression of inhibin B betaB subunits was found in late spermatids and mature sperm. CONCLUSION: Inhibin B may be produced by both Sertoli cells and early spermatogenic cells in the seminiferous tubules.

[Effects of Fructus lycii and Radix astragali on the function of sertoli cells in rat testes].

OBJECTIVE: To investigate the effects of the Chinese herbal medicines Fructus Lycii and Radix Astragali on the function of the Sertoli cells in the rat testis and their mechanisms. METHODS: Sertoli cells from the testes of the SD rats aged 18 - 22 days were isolated and cultured. The effects of Fructus Lycii, Radix Astragali and the combined administration of the two on the proliferation of Sertoli cells in vitro were detected by MTT assay, and their effects on the level of INHbetaB mRNA transcription in Sertoli cells in vitro were investigated in both normal environment and peroxide-damaging environment by RT-PCR. RESULTS: The proliferation of Sertoli cells was promoted by either Fructus Lycii or Radix Astragali at high concentration (P < 0.05), and significantly promoted by the combined administration at high concentration (P <0.01). Sertoli cell INHbetaB transcription was significantly up-regulated by Fructus Lycii, Radix Astragali and their combined administration in vitro (P < 0.01). When the level of INHbetaB mRNA in Sertoli cells significantly dropped (P < 0.01) in the presence of injury induced by peroxide (H2O2), it could be elevated by Radix Astragali (P < 0.05) and significantly up-regulated by Fructus Lycii or the combined administration in vitro (P < 0.01). CONCLUSION: Fructus Lycii, Radix Astragali and the combined administration of the two could promote and protect INHbetaB mRNA in Sertoli cells in vitro.

[Relationship between pathological alterations of spermatogenic impairment and serum inhibin B concentration in patients with azoospermia].

OBJECTIVE: To investigate the relationship between pathological alterations of spermatogenic impairment in seminiferous tubules and serum inhibin B concentration in patients with azoospermia and to verify the significance of INH B in evaluating spermatogenesis. METHODS: Eighty-three cases of azoospermia underwent testicular biopsy for the purpose of diagnosis. In accordance with the pathological alterations of spermatogenesis in seminiferous tubules, the samples were divided into four groups: Sertoli cell-only syndrome (n = 21); hypospermatogenesis (n = 20); maturation arrest (n = 24) and almost normal spermatogenesis (n = 18). Serum INHB and FSH, LH, T concentrations were tested before testicular biopsy for each patient respectively. RESULTS: The INHB levels were (20. 85 +/- 18.78) pg/ml, (67.25 +/- 40.98) pg/ml, (73.63 +/- 25.54) pg/ml and (149.48 +/- 27.92) pg/ml in the above four groups, respectively. There was no significant statistical difference in the level of serum INH B between maturation arrest and hypospermatogenesis groups (P > 0.05), and there was a very significant difference in almost normal spermatogenesis group and the other three groups, respectively (P < 0.001). There was no significant difference in the concentration of serum FSH when maturation arrest group compared with spermatogenesis group (P > 0.05), whereas between the other two groups and between each of them and maturation arrest or almost normal spermatogenesis there was a very significant difference in the level of serum FSH (P < 0.05); The concentrations of LH and T were not significantly different among the four groups (P > 0.05). CONCLUSION: Serum INHB concentration was decreased when spermatogenesis got impaired. It dropped the most markedly in Sertoli cell-only syndrome group. INH B reflects directly the spermatogenic function in seminiferous tubules of the testis. Therefore, it could be considered valuable for spermatogenesis and potential fertility in patients with azoospermia.

[Comparison of composite grafting of autoskin with acellular dermal matrix from different sources].

OBJECTIVE: To compare the composite grafts of acellular dermal matrix (ADM) from different sources with autoskin. METHODS: Six local white mini pigs were employed for the experiment. The pigs were randomly divided into four groups according to different skin grafts, i.e. A (human ADM with razor thin autoskin), B (porcine ADM with razor thin autoskin), C (razor thin autoskin only), and D (split thickness autoskin) as control. The survival rate, the contraction degree of the grafts, and the histological changes in grafting area were observed at 2, 4, 8, 12 and 24 hours after the operation. RESULTS: The grafted area in both A and B groups appeared smooth and elastic with satisfactory graft survival. The in growth of the host reparative cells such as fibroblast and vascular endothelium could be induced by composite grafts of different ADMs with skin grafting. The contraction areas in A and B groups seemed bigger than those in C and D groups. The tissue structure of grafting areas was similar to that of split thickness skin grafting area at 24 post-operation weeks. CONCLUSION: Combination of the homogenous and heterogeneous ADMs with autografts exhibited similar biological function during the observation period (24 weeks after operation). Xenogenous ADMs might have broader clinical applications.

[The establishment of testicular fibrosis model in Wistar rats].

OBJECTIVES: To establish the testicular fibrosis model in rats. METHODS: Wistar rats were divided into control group(n = 12) and model group(n = 40) randomly. Testicular fibrosis model was built with the classical method of establishing experimental autoimmune orchitis (EAO) combined with injecting Bacille Calmette-Guerin (BCG) into left testis. RESULTS: The incidence rate of EAO and the rate of testicular fibrosis were 100%, 11.1% and 100%, 81.5% at 80, 140 days after the first infection, respectively. CONCLUSIONS: The model of rat testicular fibrosis was established successfully.

Effect of mifepristone on estrogen and progesterone receptors in human endometrial and endometriotic cells in vitro.

OBJECTIVE: To investigate the expressions of estrogen (E) receptor and progesterone (P) receptor in human eutopic and ectopic endometrium and the effect of mifepristone (RU486) on them. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Twenty-two patients with ovarian endometriosis and 13 patients with uterine leiomyoma were recruited. INTERVENTION(S): Samples of ovarian endometrioma cyst tissue and endometrium were obtained from the 22 patients. A sample of endometrium was obtained from the 13 patients. MAIN OUTCOME MEASURE(S): Expressions of E and P receptors were determined using immunocytochemical method. RESULT(S): P receptor expression in endometrial epithelial cells with endometriosis was significantly higher than that without endometriosis in the early secretory phase. Estrogen receptor and epithelial P receptor expressions in endometriotic cells were significantly lower than those of endometrial cells during the proliferative phase, similar with the latter during the early secretory phase and significantly higher during the late secretory phase. RU486 down-regulated the expressions of E and P receptors in both the eutopic and the ectopic endometrial cells, and in some cases this down-regulating effect was more apparent when the concentration of RU486 was higher. CONCLUSION(S): Different steroid receptor expressions indicate different hormonal regulation between endometriotic and endometrial cells. The down-regulating effect on E and P receptors may be one of the therapeutic mechanisms of RU486 on endometriosis.

[The activation of vagus afferent in response to lipopolysaccharide the role of interleukin-1].

To study the possibility of activation of vagus afferent in response to lipopolysaccharide (LPS) through interleukin-1 (IL-1), Wistar rats were randomly divided into LPS group and saline (NS) group. The expression of c-Fos, CD14 and Mac-1 were detected by immunohistochemistry staining. IL-1 bioactivity was determined by L929 cell proliferation. The expression of IL-1R I mRNA was detected by in situ hybridization. Our results showed that there were some c-Fos protein expression positive neurons in the nodose ganglion in LPS group, but no c-Fos protein expression positive neurons in the nodose ganglion in control group. The number of macrophages (M phi) in the connective tissue surrounding the abdominal vagus increased significantly in response to LPS i.p. Forty-five minutes after the application of LPS, the IL-1 bioactivity in the supernatant of M phi was increased. Positive IL-1R mRNA neurons were also observed in the nodose ganglion in the LPS group. The results indicate that vagus afferent is activated in response to LPS and that IL-1 production might be involved in the activation of vagus afferent.