Anethole supplementation during in vitro maturation increases in vitro goat embryo production in a concentration-dependent manner.

During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 µg/mL (AN30); 300 µg/mL (AN300) or 2000 µg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 µg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 µg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 µg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 µg/mL is recommended.

[Clinical analysis of retropharyngeal lymph node metastasis in patients with hypopharyngeal squamous cell carcinoma].

Objective: To analyze the incidence and the related risk factors of retropharyngeal lymph node metastasis in patients with hypopharyngeal squamous cell carcinoma, evaluate the accuracy of preoperative enhanced CT in judging retropharyngeal lymph node metastasis, and investigate the impact of retropharyngeal lymph node metastasis on the prognosis. Methods: Retrospective analyses were made on 398 patients with hypopharyngeal squamous cell carcinoma who underwent surgery as the primary therapy and accepted retropharyngeal lymph node exploration and clearance during surgery in Shandong Provincial ENT Hospital from January 2014 to December 2019. Multivariate logistic regression analysis was used to clarify the related risk factors of retropharyngeal lymph node metastasis. Multivariate Cox regression analysis was used to investigate the impact of retropharyngeal lymph node metastasis on prognosis. The retropharyngeal lymph nodes of 218 cases with available preoperative enhanced CT images were evaluated by two experienced radiologists and compared with postoperative pathological results. Results: Retropharyngeal lymph node metastasis were confirmed in 54 of 398 (13.6%) cases according to postoperative pathology. The sensitivity and specificity of preoperative enhanced CT in the diagnosis of retropharyngeal lymph node metastasis were 34.6% and 91.1%, respectively, and the overall accuracy was 84.4%. Multivariate logistic regression analysis showed that the site of the primary lesion and pathological N stage were independent risk factors for retropharyngeal lymph node metastasis in hypopharyngeal squamous cell carcinoma. Patients with primary lesion located in the posterior wall of hypopharynx (OR=4.83, 95% CI: 1.27-18.40), N2 stage (OR=6.30, 95% CI: 2.25-17.67), and N3 stage (OR=26.89, 95% CI: 5.76-125.58) were prone to retropharyngeal lymph node metastasis. The 5-year overall survival rate of the 398 patients was 50.4%, and the 5-year disease-free survival rate was 48.3%. Multivariate Cox regression analysis showed that T stage, N stage, retropharyngeal lymph node metastasis, and radiotherapy were independent influencing factors for overall survival (T stage: HR=1.28, 95% CI: 1.06-1.54; N stage: HR=1.26, 95% CI: 1.14-1.40; retropharyngeal lymph node metastasis: HR=2.13, 95% CI: 1.47-3.08; radiotherapy: HR=0.54, 95% CI: 0.38-0.76) and disease-free survival of patients with hypopharyngeal squamous cell carcinoma (T stage: HR=1.26, 95% CI: 1.06-1.51; N stage: HR=1.25, 95% CI: 1.13-1.37; retropharyngeal lymph node metastasis: HR=2.24, 95% CI: 1.56-3.21; radiotherapy: HR=0.55, 95% CI: 0.40-0.77). Conclusions: Metastasis of retropharyngeal lymph nodes in hypopharyngeal squamous cell carcinoma is not rare. Enhanced CT is of low accuracy and limited value in diagnosing retropharyngeal lymph node metastasis. Primary lesions located in the posterior wall of the hypopharyngx, N2 stage, and N3 stage are independent high-risk factors for retropharyngeal lymph node metastasis. The prognosis of hypopharyngeal cancer patients with retropharyngeal lymph node metastasis is worse, and active surgical exploration and clearance can effectively reduce the mortality caused by retropharyngeal lymph node metastasis.

Deciphering migraine pain mechanisms through electrophysiological insights of trigeminal ganglion neurons.

Migraine is a complex neurological disorder that affects millions of people worldwide. Despite extensive research, the underlying mechanisms that drive migraine pain and related abnormal sensation symptoms, such as hyperalgesia, allodynia, hyperesthesia, and paresthesia, remain poorly understood. One of the proposed mechanisms is cortical spreading depression (CSD), which is believed to be involved in the regulation of trigeminovascular pathways by sensitizing the pain pathway. Another mechanism is serotonin depletion, which is implicated in many neurological disorders and has been shown to exacerbate CSD-evoked pain at the cortical level. However, the effects of CSD and serotonin depletion on trigeminal ganglion neurons, which play a critical role in pain signal transmission, have not been thoroughly studied. In this study, we aimed to investigate the association between CSD and serotonin depletion with peripheral sensitization processes in nociceptive small-to-medium (SM) and large (L) -sized trigeminal ganglion neurons at the electrophysiological level using rat models. We divided the rats into four groups: the control group, the CSD group, the serotonin depletion group, and the CSD/serotonin depletion group. We induced CSD by placing KCl on a burr hole and serotonin depletion by intraperitoneal injection of PCPA (para-chlorophenoxyacetic acid). We then isolated trigeminal ganglion neurons from all groups and classified them according to size. Using patch-clamp recording, we recorded the excitability parameters and action potential (AP) properties of the collected neurons. Our results showed that in SM-sized trigeminal ganglion neurons, the CSD-SM and CSD/serotonin depletion groups had a higher positive resting membrane potential (RMP) than the control-SM group (p = 0.001 and p = 0.002, respectively, post-hoc Tukey's test). In addition, the gap between RMP and threshold in the CSD-SM group was significantly narrower than in the control-SM group (p = 0.043, post-hoc Tukey's test). For L-sized neurons, we observed prolongation of the AP rising time, AP falling time, and AP duration in neurons affected by CSD (p < 0.05, pairwise comparison test). In conclusion, our study provides new insights into the underlying mechanisms of migraine pain and abnormal somatosensation. CSD and serotonin depletion promote the transmission of pain signals through the peripheral sensitization process of nociceptive small-to-medium-sized trigeminal ganglion neurons, as well as nociceptive and non-nociceptive large-sized trigeminal ganglion neurons.

Definition of protocols for cryopreservation and three-dimensional in vitro culture of prepubertal goat testicular tissue after histomorphological, ultrastructural, and functional analysis.

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.

In vitro- and in vivo-derived early antral follicles have comparable in vitro follicular growth and oocyte maturation rates in goats.

Recent in vitro follicle culture (IVFC) studies in caprine have yielded lower maturation rates using late preantral follicles compared to early antral follicles. Thus, research focusing on developing stage-specific customized culture systems able to improve the efficiency of IVFC for late preantral follicles are warranted. This study aimed to compare the morphometric features, estradiol production, and gene expression between early antral caprine follicles produced in vitro and in vivo. In vitro-derived early antral follicles were produced after a 6-day in vitro culture of late preantral follicles, while in vivo-derived early antral follicles were yielded immediately after isolation from the ovaries; antral follicles were, thereafter, cultured for 18 days. In vitro-derived antral follicles were cultured either in a medium developed for preantral follicles (PF medium) or in a medium developed for antral follicles (AF medium). In vivo-derived early antral follicles, on the other hand, were cultured in AF medium (Control treatment). Results demonstrated that in vitro-derived antral follicles cultured in PF medium produced higher estradiol concentration, and m-RNA expression for matrix metalloproteinase-9 (MMP-9), and insulin receptor when compared to both in vitro- and in vivo-derived antral follicles cultured in AF medium. Remarkably, in vitro-derived antral follicles cultured in PF medium had similar MII and oocytes ≥110 µm rates compared with in vivo-derived antral follicles (Control treatment). In conclusion, when cultured in a single and appropriate medium (i.e., PF medium), in vitro-derived early antral follicles had comparable oocyte maturation rates to the in vivo-derived early antral follicles.

In vitro cytotoxic effects of 5-Fluorouracil on isolated murine ovarian preantral follicles.

5 fluorouracil (5FU), an antineoplastic drug, is often utilized in the therapeutic regimen for several types of cancer, including the hepatoblastoma in children. The effects of 5FU on the population of ovarian preantral follicles, which is the largest oocyte reservoir, is still poorly understood. The integrity of the ovarian preantral follicle pool is important for lifelong fertility. The better understanding of such effects may favor intervention strategies to protect fertility in 5FU-treated children and women coping with cancer. To analyze the effects of 5FU on isolated murine secondary follicles in vitro, ovaries were collected from young mice (28-30 days old), and secondary follicles were isolated and cultured for 12 days in basic culture medium, with or without 5FU at concentrations of 0.3 mM, 1 mM, 3 mM, 10 mM, and 30 mM. In the in vitro study, we analyzed the percentage of morphologically normal follicles, antrum formation, follicular diameter, and hormone production. On day 12, oocytes were recovered for in vitro maturation. 5FU treatment did not alter the percentage of morphologically normal follicles. On day 12, only 1, 10, and 30 mM 5FU significantly reduced the percentage of antrum. From day 4 onwards, 5FU treatments significantly reduced follicle diameter. The meiosis resumption rate was significantly lower in all 5FU treatments. 5FU concentrations ≥3 mM reduced estradiol levels. In conclusion, 5FU does not affect follicular morphology. However, 5FU deleteriously affects follicular growth, estradiol production, and oocyte maturation in isolated ovarian follicles.

Effects of in vitro exposure of sheep ovarian tissue to zearalenone and matairesinol on preantral follicles.

The aim of this study was to evaluate the effect of 1 µmol/l zearalenone (ZEN) and 1 µmol/l matairesinol (MAT), alone or in combination, on the morphology of in vitro-cultured ovarian preantral follicles. Ovaries from four adult sheep were collected at a local slaughterhouse and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN. The plant lignan MAT alone did not maintain the morphology of the ovarian follicles; its combination with ZEN counteracted the negative effects observed when follicles were cultured in the presence of the mycotoxin alone. However, MAT was not able to promote the in vitro development of the ovarian follicles.

In vitro embryo production from early antral follicles of goats fed with a whole full-fat linseed based diet.

The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300-400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus-oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

[EEFSEC knockdown inhibits proliferation, migration and invasion of prostate cancer cells in vitro].

OBJECTIVE: To investigate the role of selenocysteine-tRNA specific eukaryotic elongation factor (EEFSEC) in regulating the proliferation, migration, and invasion of human prostate cancer 22Rv1 cells. METHODS: We detected EEFSEC mRNA expression levels in human normal prostate cell line RWPE1 and human prostate cancer cell lines 22Rv1, LNCaP, Vcap and PC-3 using qRT-PCR and EEFSEC protein expression in surgical specimens of prostate cancer and adjacent tissues using Western blotting. 22Rv1 cells were infected with a lentiviral vector carrying EEFSEC shRNA or a control lentivirus and the interference efficiency was determined using Western blotting. XTT assay was used to assess the changes in the viability of the infected cells, and Transwell chamber assay was used to examine the changes in cell migration and invasion. The effect of EEFSEC knockdown on cell cycle progression was determined with flow cytometry and by detecting the expressions of cell cycle proteins using qRT-PCR. RESULTS: EEFSEC was significantly upregulated in prostate cancer cells (P < 0.05), and a high expression of EEFSEC was associated with a poor prognosis of the patients with prostate cancer. In 22Rv1 cells, EEFSEC knockdown significantly suppressed the proliferation (P < 0.001), migration (P < 0.001) and invasion (P < 0.001) of the cells, resulted in cell cycle arrest in G0/G1 phase, obviously inhibited the expression of C-myc and CCNB1, and significantly increased the expression of p15. CONCLUSION: EEFSEC knockdown can inhibit the proliferation, migration, and invasion of prostate cancer cells in vitro possibly by down-regulating the expression of C-myc.

[Analysis of drug resistance pattern and genes of Salmonella.spp isolated from human infections in Anhui Province].

Objective: To investigate the drug resistance pattern and drug resistance genotypes of Salmonella. spp isolated from fecal specimens and anal swabs of diarrhea cases in Anhui Province. Methods: The 149 strains of Salmonella.spp isolated from feces and anal swabs of diarrhea cases in Anhui Province from April to October 2017 were selected. The serotypes of Salmonella.spp were identified by slide agglutination. The susceptibility of all strains to 14 antibiotics were determined by micro-broth dilution method. Sixty of the cephalosporin-resistant antibiotics were selected. The ß-lactamase encoding genes bla(TEM), bla(SHV), bla(OXA-1), bla(OXA-2), bla(PER), bla(CMY), bla(CTX-M), and colistin resistance genes mcr-1 and mcr-2 were performed using the multi-PCR method. Results: Of the 149 diarrhea cases, the median (P(25), P(75)) of the age was 5.0 (1.1, 38.5). The 92 of them were male and 54.4% were children. Of the 149 strains of Salmonella.spp, 105 strains had different degrees of resistance to 13 antibiotics other than imipenem. The resistance rate of ampicillin was 55.0% (82/149), which was the highest. 53.0% strains (79 strains) were multidrug resistant, main of which were Salmonella typhimurium and Salmonella enteritidis. A total of 53 resistance patterns were detected, and 10 strains were resistant to ampicillin-ampicillin/sulbactam-tetracycline-chloramphenicol-cefazolin-trimethoprim/sulfamethoxazole, which was the most common resistance pattern. Among the 60 cephalosporin resistant strains, 45 strains carried bla(TEM-1), 6 of which also carried bla(CTX-M-14) and 3 of which also carried bla(CTX-M-65). All the 32 strains carried only bla(TEM-1) show resistance to ampicillin and 31 of them show resistance to cefazolin. There were 2 strains showing negative results of gene detection. mcr-1 was detected in a multidrug resistant strain. Conclusion: The resistance of Salmonella.spp to ampicillin shows a serious situation in this region, and there were a number of multidrug resistant strains. The bla(TEM-1) was the major drug resistance gene detected in this research. Detection of the mcr-1 suggests the emergence of surveillance to colistin resistance of Salmonella.spp in this area.

Anethole improves blastocysts rates together with antioxidant capacity when added during bovine embryo culture rather than in the in vitro maturation medium.

We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2-4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus-oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.

[The oncologic and functional outcomes of supracricoid partial laryngectomy for the treatment of laryngeal cancer].

Objective: To evaluate the oncologic and functional outcomes of laryngeal squamous cell carcinomas treated by supracricoid laryngectomy. Methods: The clinical data of 134 patients with laryngeal cancer who underwent supracricoid laryngectomy with cricohyoidoepiglottopexy (CHEP) or cricohyoidopexy (CHP) between July 2005 and April 2014 at Shandong Provincial ENT Hospital were retrospectively reviewed. Ninety-one patients including 31 cases of stage Ⅰ, 36 of stage Ⅱ, 18 of stage Ⅲ and 6 of stage Ⅳ underwent CHEP and 43 patients underwent CHP. Two patients received CHEP due to recurrence after open surgery and laser surgery. Three patients received CHP due to the recurrence of disease after open surgery and postradiotherapy persistence of disease. The Kaplan-Meier method was used to calculate the 3-year and 5-year survival rates. The Chi-square test was used to compare the survival rates between different surgical procedures. Results: All 91 patients who underwent CHEP had successful removals of PEG tubes, and 88 (96.7%) of them had tracheostomy tube decannulation. Among 43 patients with CHP, 42(97.6%) cases removal of PEG tubes(97.6%), including and 40(93.0%) cases with tracheostomy tube decannulation. There was one patient with local recurrence in all cases. In CHEP group, 3-year local control rate was 98.2%; 3-year and 5-year overall survival rate were 94.5% and 93.9%, respectively. In CHP group, 3-year local control rate was 97.6%; 3-year and 5-year overall survival rates were 86.0% and 83.3%, respectively. Pharyngeal fistula appeared in 2 cases of CHEP group and 4 cases of CHP group, and all of them were cured by conservative treatment. Conclusion: Supracricoid laryngectomy shows excellent oncologic and functional results for treatment of laryngeal cancer while maintaining laryngeal functions, especially in terms of local control rate and tracheostomy tube decannulation.

Social, environmental and microbiologic aspects of endemic mycoses in Brazil.

Many factors that lead to host immunosuppression are clearly known to predispose the host to fungal diseases, significantly influencing the occurrence of mycoses. However, little or nothing has been discussed regarding social or economic factors that can influence the occurrence of diseases caused by fungi. In this minireview, we discuss several factors that may affect the occurrence of mycoses in Brazil, a continentally extended country that is marked by large climatic variations and severe socioeconomic distortions that may limit access to health services for the population.

Effects of cytochalasin E on Paracoccidioides brasiliensis.

AIMS: To determine the effects of cytochalasin E, isolated from the extremophile fungus Aspergillus felis, on the cells of Paracoccidioides brasiliensis Pb18. METHODS AND RESULTS: Cytochalasin E showed a minimal inhibitory concentration of 3·6 µmol l-1 and minimum fungicidal concentration of 7·2 µmol l-1 on P. brasiliensis by in vitro microdilution and IC50 >964·0 µmol l-1 on murine macrophages. Its selectivity index (>263) indicated that this compound has selectivity for fungal cells. Morphological alterations were determined by optical and fluorescence microscopy, as well as scanning and transmission electron microscopy. Cytochalasin E affected P. brasiliensis bud-forming pseudohyphae, cell morphology, cell walls and cell membranes; caused the release of cellular material; and resulted in the production of reactive oxygen species. In murine macrophages, it affected cytoskeletal actin and inhibited phagocytosis. CONCLUSION: Cytochalasin E may be useful as an antifungal prototype against P. brasiliensis and in studies on phagocytosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM). Treatment is prolonged to control the clinical manifestations and prevent relapse. The study on the effects of cytochalasin E in P. brasiliensis is important because it can be used as a prototype for new antifungal drugs and consequently, broadens the treatment options for PCM.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles.

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 µg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 µg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

[Treatment and prognosis of 264 patients with hypopharyngeal carcinoma].

Objective: To investigate the key factors influencing the prognosis of hypopharyngeal carcinoma and the therapeutic methods improving the efficacy of treatments for hypopharyngeal carcinoma. Methods: Two hundred and sixty-four cases of hypopharyngeal squamous cell carcinoma treated from May 2010 to May 2015 were analyzed retrospectively. There were 211 cases of pyriform sinus carcinoma, 37 cases of posterior pharyngeal wall carcinoma, and 16 cases of postcricoid carcinoma. According to UICC 2002 criteria, 2 cases were for stage Ⅰ, 14 for stage Ⅱ, 32 for stage Ⅲ and 216 for stage Ⅳ. Postoperative circumferential defects existed in 112 (42.4%) cases, and 86 of them were reconstructed with free jejunum transplantation. Among all cases, 54 patients (20.5%) had the preservation of laryngeal functions after surgery and 210 patients (79.5%) with total laryngectomy; 238 cases (90.2%) underwent bilateral cervical lymph node dissection and 203 patients received posterior pharyngeal lymph node exploration and dissection, with positive metastases for posterior pharyngeal lymph nodes in 36 cases (17.7%). Eight cases with cervical lymph node metastasis extensively involving the soft tissue, prevertebral fascia or encases carotid artery received preoperative radiotherapy of 50 Gy. After surgery 13 patients received concurrent radiotherapy and chemotherapy, 337 underwent adjuvant radiotherapy with a dose of 50-60 Gy each, and 14 patients did not receive radiotherapy or did not completed their radiotherapy programs. SPSS 13.0 saftware was used to analyze the data. Results: All patients were followed up for more than 2 years. With Kaplan-Meier method, the 2-, 3- and 5-years survival rates were 69.6%, 62.8% and 51.3%, respectively. There were significant differences in 3-year survival rates between T1-2 group (75.5%) and T3-4 group (59.2%) (χ(2)=4.282 P=0.039), N0 group (81.6%) and N+ group (58.2%) (χ(2)=6.802 P=0.009), laryngeal functions preserved (81.8%) and unpreserved group (58.9%) (χ(2)=5.314 P=0.021). Multivariate Logistic regression analysis showed that cervical lymph node metastasis was an independent prognostic factor (P=0.027). The success rate of free jejunum transplantation was 98.8%. Local recurrence, cervical lymph node recurrence, second primary cancer, and distant metastasis accounted respectively for 11.2%, 18.8%, 12.5% and 45.0% of death cases. Conclusions: The prognosis-associated factors for hypopharyngeal carcinoma should be taken into account, including the evaluation of the carcinogenesis of the mucosal area, early screening of premalignant lesion or second primary cancer in the esophagus and dissection of the posterior pharyngeal lymph nodes, which will help to improve the local control rate and recent survival rate in patients with hypopharyngeal cancer.

[Significance of retropharyngeal node dissection in treatment of hypopharyngeal carcinoma].

Objective: investigate the incidence of retropharyngeal lymph node (RPLN) metastasis and the risk factors for RPLN metastasis in hypopharyngeal cancer, and the relationship of planned dissection of the RPLN with the survival and tumor control rates in patients with hypopharyngeal cancer. Methods: A total of 203 patients with hypopharyngeal squamous cell carcinoma who underwent radical surgery as initial treatment from February 2011 to July 2015 were analyzed retrospectively. There were 167 cases of pyriform sinus carcinoma, 23 cases of posterior pharyngeal wall carcinoma, and 13 cases of postcricoid carcinoma. Results: The incidence of RPLN metastasis in HPC was 17.7%, with a highest rate of 43.5% in pharyngeal wall carcinoma. The incidence of RPLN metastasis in T3-4 pyriform sinus carcinoma was 18.3%, which significantly higher than 2.8% in T1-2 cases(χ(2)=5.360, P=0.020). The rate of RPLN metastasis was 23.8% in N2b-3 and 8.6% in N0-2a, with a statistically significant difference(χ(2)=7.637, P=0.006). There was no statistically significant difference in overall survival rates between patients with and without RPLN metastasis(P>0.05). Data were analyzed by SPSS 13.0 software. Conclusions: RPLN metastasis is not rare in hypopharyngeal carcinoma. Planned dissection of the RPLN should be performed with the initial surgery in patients with advanced hypopharyngeal cancer, especially posterior pharyngeal wall carcinoma, T3-4 pyriform sinus carcinoma and staged N2b-3 disease, which can reduce the regional recurrence rate and provided with a better prognosis.

[Endoscopic screening for upper gastrointestinal second primary malignancies in patients with hypopharyngeal squamous cell carcinoma].

Objective: To evaluate the usefullness of flexible esophagoscopy and chromoendoscopy with Lugol's solution in the detection of synchronous esophageal neoplasm in patients with hypopharyngeal squamous cell carcinoma (HSCC). Methods: A retrospective review of 96 cases with HSCC that received surgical treatment from March 2016 to March 2017 was accomplished. In these patients, the site of origin were pyriform sinus (n=75), posterior pharyngeal wall (n=11) and postcricoid (n=10). Esophagoscopy was prospectively performed on all patients before treatment for HSCC. All patients underwent conventional white-light endoscopic examination with Lugol chromoendoscopy and narrow band image. Suspicious areas of narrow band image or Lugol-voiding lesions were observed and biopsied. The treatment strategy of primary HSCC was modified according to the presence of synchronous esophageal squamous cell neoplasms by a multidisciplinary approach. Results: Ninety-six patients were enrolled (age ranging from 37-80 years). All patients did not have previous treatment.Histopathological analysis revealed middle to high-grade dysplasia in 5 cases, Tis cancer in 5 cases, cancer in 16 cases and inflammation or normal findings in the others. Four cases were treated with endoscopic submucosal dissection before hypopharygeal surgery, 3 cases with lower esophageal cancers were treated with gastric pull-up combined with free jejunal flap after total circumferential pharyngolaryngectomy (TCPL) and certical esophagectomy, and 14 cases were treated with TCPL, total esophagectomy and gastric pull-up. Conclusions: Esophagoscopy is a feasible and justified procedure in HSCC cases as it enhances the detection of premalignant lesion or second primary cancer. Routine esophagoscopy for detecting synchronous second primary tumor should be recommended for patients with HSCC. The treatment strategy for primary HSCC is modified according to the presence of synchronous second primary tumor.