Crystallins Play a Crucial Role in Glaucoma and Promote Neuronal Cell Survival in an In Vitro Model Through Modulating Müller Cell Secretion.

Purpose: The aim of this study was to explore the roles of crystallins in the context of aging in glaucoma and potential mechanisms of neuroprotection in an experimental animal model of glaucoma. Methods: Intraocular pressure (IOP) was significantly elevated for 8 weeks in animals at different ages (10 days, 12 weeks, and 44 weeks) by episcleral vein cauterization. Retinal ganglion cells (RGCs) were quantified by anti-Brn3a immunohistochemical staining (IHC). Proteomics using ESI-LTQ Orbitrap XL-MS was used to analyze the presence and abundance of crystallin isoforms the retinal samples, respectively. Neuroprotective property and localization of three selected crystallins CRYAB, CRYBB2, and CRYGB as most significantly changed in retina and retinal layers were determined by IHC. Their expressions and endocytic uptakes into Müller cells were analyzed by IHC and Western blotting. Müller cell secretion of neurotrophic factors into the supernatant following CRYAB, CRYBB2, and CRYGB supplementation in vitro was measured via microarray. Results: IOP elevation resulted in significant RGC loss in all age groups (P < 0.001). The loss increased with aging. Proteomics analysis revealed in parallel a significant decrease of crystallin abundance - especially CRYAB, CRYBB2, and CRYGB. Significant neuroprotective effects of CRYAB, CRYBB2, and CRYGB after addition to retinal cultures were demonstrated (P < 0.001). Endocytic uptake of CRYAB, CRYBB2, and CRYGB was seen in Müller cells with subsequent increased secretion of various neurotrophic factors into the supernatant, including nerve growth factor, clusterin, and matrix metallopeptidase 9. Conclusions: An age-dependent decrease in CRYAB, CRYBB2, and CRYGB abundance is found going along with increased RGC loss. Addition of CRYAB, CRYBB2, and CRYGB to culture protected RGCs in vitro. CRYAB, CRYBB2, and CRYGB were uptaken into Müller cells. Secretion of neurotrophic factors was increased as a potential mode of action.

Delineating the Switch between Senescence and Apoptosis in Cervical Cancer Cells under Ciclopirox Treatment.

The iron-chelating drug ciclopirox (CPX) may possess therapeutic potential for cancer treatment, including cervical cancer. As is observed for other chemotherapeutic drugs, CPX can induce senescence or apoptosis in cervical cancer cells which could differently affect their therapy response. The present study aims to gain insights into the determinants which govern the switch between senescence and apoptosis in cervical cancer cells. We performed proteome analyses, proliferation studies by live-cell imaging and colony formation assays, senescence and apoptosis assays, and combination treatments of CPX with inhibitors of oxidative phosphorylation (OXPHOS) or glycolysis. We found that CPX downregulates OXPHOS factors and facilitates the induction of apoptosis under limited glucose availability, an effect which is shared by classical OXPHOS inhibitors. Under increased glucose availability, however, CPX-induced apoptosis is prevented and senescence is induced, an activity which is not exerted by classical OXPHOS inhibitors, but by other iron chelators. Moreover, we show that the combination of CPX with glycolysis inhibitors blocks cervical cancer proliferation in a synergistic manner. Collectively, our results reveal that the phenotypic response of cervical cancer cells towards CPX is strongly dependent on glucose availability, link the pro-apoptotic and pro-senescent activities of CPX to its bifunctionality as an OXPHOS inhibitor and iron chelator, respectively, and provide a rationale for combining CPX with glycolysis inhibitors.

Effects of Metformin on the virus/host cell crosstalk in human papillomavirus-positive cancer cells.

Oncogenic types of human papillomaviruses (HPVs) are major human carcinogens. The viral E6/E7 oncogenes maintain the malignant growth of HPV-positive cancer cells. Targeted E6/E7 inhibition results in efficient induction of cellular senescence, which could be exploited for therapeutic purposes. Here we show that viral E6/E7 expression is strongly downregulated by Metformin in HPV-positive cervical cancer and head and neck cancer cells, both at the transcript and protein level. Metformin-induced E6/E7 repression is glucose and PI3K-dependent but-other than E6/E7 repression under hypoxia-AKT-independent. Proteome analyses reveal that Metformin-induced HPV oncogene repression is linked to the downregulation of cellular factors associated with E6/E7 expression in HPV-positive cancer biopsies. Notably, despite efficient E6/E7 repression, Metformin induces only a reversible proliferative stop in HPV-positive cancer cells and enables them to evade senescence. Metformin also efficiently blocks senescence induction in HPV-positive cancer cells in response to targeted E6/E7 inhibition by RNA interference. Moreover, Metformin treatment enables HPV-positive cancer cells to escape from chemotherapy-induced senescence. These findings uncover profound effects of Metformin on the virus/host cell interactions and the phenotype of HPV-positive cancer cells with implications for therapy-induced senescence, for attempts to repurpose Metformin as an anticancer agent and for the development of E6/E7-inhibitory therapeutic strategies.

Adhesions after Laparoscopic Myomectomy: Incidence, Risk Factors, Complications, and Prevention.

Uterine fibroids or uterine myomas are one of the most common benign diseases of the uterus. Symptoms associated with myomas can make surgical removal of myomas necessary. Besides the traditional abdominal route, laparoscopic myomectomy (LM) has gained more acceptances over the last few decades, and it is anticipated that laparoscopy is associated with lower adhesion development. Therefore, we conducted this review to analyze the evidence on adhesions after LM. The PubMed database was searched using the search terms "myomectomy" alone and in combination with "adhesions," "infertility OR fertility outcome," and "laparoscopy" among articles published in English and German. Although the well-known advantages of laparoscopy, for example, less pain, less blood loss, or shorter hospital stay, myomectomy belongs to high-risk operations concerning adhesion formation, with at least every fifth patient developing postsurgical adhesions. In laparoscopic surgery, surgeons´ experience as well tissue trauma, due to desiccation and hypoxia, are the underlying mechanisms leading to adhesion formation. Incisions of the posterior uterus may be associated with a higher rate of adhesions compared to anterior or fundal incisions. Adhesions can be associated with severe complications such as small bowel obstruction, chronic pelvic pain, complications in further operations, or impaired fertility. Tissue trauma and the experience of the surgeon in laparoscopic surgery are most of the influencing factors for adhesion formation after myomectomy. Therefore, every surgeon should adopt strategies to reduce adhesion development in daily routine, especially when it conducted to preserve or restore fertility.

Effects of the antifungal agent ciclopirox in HPV-positive cancer cells: Repression of viral E6/E7 oncogene expression and induction of senescence and apoptosis.

The malignant growth of human papillomavirus (HPV)-positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV-positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV-positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of prosenescent agents, CPX-induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53-independent Caspase-3/7 activation and induction of apoptosis. CPX also eliminates HPV-positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound antiviral and antiproliferative effects in HPV-positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV-inhibitory, prosenescent and proapoptotic agents in both normoxic and hypoxic environments.

The HPV E6/E7 Oncogenes: Key Factors for Viral Carcinogenesis and Therapeutic Targets.

Human papillomavirus (HPV)-induced cancers are expected to remain a major health problem worldwide for decades. The growth of HPV-positive cancer cells depends on the sustained expression of the viral E6 and E7 oncogenes which act in concert with still poorly defined cellular alterations. E6/E7 constitute attractive therapeutic targets since E6/E7 inhibition rapidly induces senescence in HPV-positive cancer cells. This cellular response is linked to the reconstitution of the antiproliferative p53 and pRb pathways, and to prosenescent mTOR signaling. Hypoxic HPV-positive cancer cells could be a major obstacle for treatment strategies targeting E6/E7 since they downregulate E6/E7 but evade senescence through hypoxia-induced mTOR impairment. Prospective E6/E7 inhibitors may therefore benefit from a combination with treatment strategies directed against hypoxic tumor cells.

Laparoscopy or laparotomy as the way of entrance in myoma enucleation.

PURPOSE: Uterine myomas are the main cause of benign uterine diseases in premenopausal women. Objective of the present paper is to investigate the current best treatment modalities of myomas, depending on age of the patient and her desire to preserve fertility, as well as on clinical presentation of this pathology, such as size, number, and location of fibroids and, furthermore, on surgical experience of the gynecologist. METHODS: The design of our work is a systematic literature review of existing studies, reviews, and meta-analysis conducted in PubMed and Cochrane Library to identify relevant literature. Commonly, the myomectomy is to be recommended in women desiring to preserve fertility when myomas are associated with symptoms such as excessive bleeding, pelvic discomfort, or palpable abdominally fibroids. The decision of surgical approach for myomectomy should be individualized, depending on size and location, as well as on surgeon's experience. The different modalities of myomectomy, laparoscopic, hysteroscopic, robotic-assisted, or laparotomic are in detail presented in the paper, according to Society of Obstetricians and Gynecologists of Canada (SOGC) clinical practice guidelines and taking into consideration possible concerns of myomectomy such as uterine rupture, development of adhesions, and myomas' recurrence.

Uterine perforation following a fractional curettage successfully treated with the modified polysaccharide 4DryField® PH: a case report.

BACKGROUND: Uterine perforation is the most common complication of curettage and may result in bleeding. Therefore, urgent control of bleeding from the uterine wall perforation is necessary to avoid an emergency hysterectomy or blood transfusion, to prevent peritoneal adhesion formation, possible chronic pelvic pain, and infertility. In the present case, an active bleeding secondary to a perforation of the uterus during curettage, for diagnosis of endometrial carcinoma, was instantaneously and successfully treated with only the application of a novel modified polysaccharide powder. This is, to the best of our knowledge, the first time that the agent 4DryField® has been used for this purpose. CASE PRESENTATION: A 71-year-old German woman with serometra and endometrial hyperplasia suffered a perforation of the anterior wall of the uterus during the hysteroscopic resection of submucosal polyps and a fractional curettage. Subsequently, an immediate laparoscopy showed an active bleeding from the wound, which was promptly stopped with only the application of the hemostatic and anti-adhesion polysaccharide powder, 4DryField®. There were no postoperative complications. Nine weeks later, a laparoscopic hysterectomy with bilateral salpingoophorectomy for endometrial carcinoma (histology: stage IA, pT1a, cN0, L0 V0 M0/G2) was performed. The former injured area looked slightly prominent, was completely healed, and showed a shiny serosa. All her pelvic organs were free of adhesions, and there was one 0.5-mm calcified granuloma in the Douglas pouch. CONCLUSIONS: The efficient hemostasis combined with the adhesion prevention effect of 4DryField®, allowed a fast control of the uterine wall bleeding, saved operation time, avoided the risks of other procedures for bleeding control and contributed to the normal healing of the uterine wall without any adhesion formation.

Gynaecological endoscopic surgical education and assessment. A diploma programme in gynaecological endoscopic surgery.

In recent years, training and education in endoscopic surgery has been critically reviewed. Clinicians, both surgeons as gynaecologist who perform endoscopic surgery without proper training of the specific psychomotor skills, are at higher risk to increased patient morbidity and mortality. Although the apprentice-tutor model has long been a successful approach for training of surgeons, recently, clinicians have recognised that endoscopic surgery requires an important training phase outside the operating theatre. The Gynaecological Endoscopic Surgical Education and Assessment programme (GESEA) recognises the necessity of this structured approach and implements two separated stages in its learning strategy. In the first stage, a skill certificate on theoretical knowledge and specific practical psychomotor skills is acquired through a high-stake exam; in the second stage, a clinical programme is completed to achieve surgical competence and receive the corresponding diploma. Three diplomas can be awarded: (a) the Bachelor in Endoscopy, (b) the Minimally Invasive Gynaecological Surgeon (MIGS) and (c) the Master level. The Master level is sub-divided into two separate diplomas: the Master in Laparoscopic Pelvic Surgery and the Master in Hysteroscopy. The complexity of modern surgery has increased the demands and challenges to surgical education and the quality control. This programme is based on the best available scientific evidence, and it counteracts the problem of the traditional surgical apprentice-tutor model. It is seen as a major step toward standardisation of endoscopic surgical training in general.

Long term follow-up concerning safety and efficacy of novel adhesion prophylactic agent for laparoscopic myomectomy in the prospective randomized ADBEE study.

We conducted a prospective randomized single blind - subject study in the University Clinic of Gynecology of Pius-Hospital Oldenburg. The primary objective of the ADBEE study was to assess the safety and manageability of ADBLOCK when used as an adjunct to laparoscopic surgery for the primary of myomas in women wishing to improve pregnancy outcomes. The study population included 32 women aged between 18-45 years, in good general health condition, who have not completed their family planning and who are undergoing primary ('virgin') laparoscopic myomectomy with an aim to improve pregnancy outcomes. The patients were randomized in 2 groups, ADBLOCK arm with 21 patients and surgery only arm with 11 patients. The study was single blind - subject and the investigators were blinded to treatment group assignment until completion of uterine suturing and prior to removal of the endoscope. A vigorous follow-up of subjects was organized, focusing on its two critical characteristics: completeness and duration. Completeness represented the percentage of subjects who returned to every planed follow - up appointments. The patients were evaluated in a specific period of time, which defined the duration of follow-up. Safety of the ADBLOCK was estimated after analyzing and documentation of any adverse events occurred, clinical and physical examination of patients as well as evaluation of laboratory measures. There were 25 adverse events reported in ADBLOCK treatment group and 12 events in NO-ADBLOCK group over the 24-months treatment. All adverse events in both treatment arms were not anticipated, with all events in the ADBLOCK group being resolved. At 28 days, there was no significant difference in proportion of events between the two treatments (p = 0.440). Overall, the number of events reported was low and the severity of events was generally mild with an unlikely or no relationship to treatment. There were no unanticipated device related adverse events seen in both treatment groups over the immediate post-operative period or during the 24 months follow up period. By 12 weeks all patients reported their wound as healing well or healed and at 6 months all wounds were reported as healed. There were no differences between both treatment groups regarding the use of painkillers over 24 months follow up period. This clinical first - in - human study, sustained by a rigorous follow-up of the subjects has demonstrated that ADBLOCK is a safe product, presenting no additional safety risk or burden to the patients over surgery alone. The device was relatively easy to use, with a low device failure rate that had no impact on the surgical procedures.

Gynaecological Endoscopic Surgical Education and Assessment. A diploma programme in gynaecological endoscopic surgery.

In recent years, training and education in endoscopic surgery has been critically reviewed. Clinicians, both surgeons as gynaecologist who perform endoscopic surgery without proper training of the specific psychomotor skills are at higher risk to increased patient morbidity and mortality. Although the apprentice-tutor model has long been a successful approach for training of surgeons, recently, clinicians have recognised that endoscopic surgery requires an important training phase outside the operating theatre. The Gynaecological Endoscopic Surgical Education and Assessment programme (GESEA), recognises the necessity of this structured approach and implements two separated stages in its learning strategy. In the first stage, a skill certificate on theoretical knowledge and specific practical psychomotor skills is acquired through a high stake exam; in the second stage, a clinical programme is completed to achieve surgical competence and receive the corresponding diploma. Three diplomas can be awarded: (a) the Bachelor in Endoscopy; (b) the Minimally Invasive Gynaecological Surgeon (MIGS); and (c) the Master level. The Master level is sub-divided into two separate diplomas: the Master in Laparoscopic Pelvic Surgery and the Master in Hysteroscopy. The complexity of modern surgery has increased the demands and challenges to surgical education and the quality control. This programme is based on the best available scientific evidence and it counteracts the problem of the traditional surgical apprentice tutor model. It is seen as a major step toward standardization of endoscopic surgical training in general.

Plastic neo-vaginal construction in Mayer-Rokitansky-Küster-Hauser syndrome: an expert opinion paper on the decision-making treatment process.

Vaginal agenesis is a congenital anomaly that affects the life of one of each four thousand women around the world. There is a trend that patients request immediate surgical correction, instead of passive vaginal dilatation. Therefore a differentiated counselling should be provided. We present a comparative chart, based on published evidence, with aspect to the available techniques, which will facilitate the decision-making process in the clinical practice. From our point of view, the best results are achieved with techniques that combine the advantages of the minimal-invasive surgery with those derived of the use of peritoneum as covering tissue of the neovagina. Nevertheless there is a lack on interdisciplinary consensus about the best option to restore the physical and sexual quality of life.

Adhesions are the major cause of complications in operative gynecology.

Adhesion formation has been found to be highly prevalent in patients with a history of operations or inflammatory peritoneal processes. These patients are at a high risk of serious intraoperative complications during a subsequent operation if adhesiolysis is performed. These complications include bowel perforation, ureteral or bladder injury, and vascular injury. In order to minimize the risk of these complications, adhesiolysis should only be performed by experienced surgeons, and intraoperative strategies must be adopted. The reduction of the overall incidence of adhesions is essential for subsequent surgical treatments. Anti-adhesion strategies must be adopted for preventing the reoccurrence of adhesions after abdominopelvic operations. The strategies employed to reduce the risk and the overall incidence of adhesions have been elucidated in this article.

How to avoid risks for patients in minimal-access trials: Avoiding complications in clinical first-in-human studies by example of the ADBEE study.

A clinical trial is a prospective study designed to establish the safety and efficacy of investigational devices in humans, in accordance with the strict guidelines of the Food and Drug Administration (FDA; USA) or European Medicines Agency (EMA; Europe). Before a clinical first-in-human study is initiated, preclinical studies of the investigational product are mandatory, and the results should be sufficient to indicate that the investigational device is acceptably safe for the proposed evaluation in human subjects. The present paper describes an experience of clinical trials, highlighting ways of avoiding possible complications in clinical first-in-human studies. For a better approach to our aim, we exemplified a prospective, randomized, single-blind study, ADBEE. The primary objective was to assess the safety of the ADBLOCK system when used as an adjunct to laparoscopic primary removal of myomas in women wishing to improve pregnancy outcomes.

Insufflation with humidified and heated carbon dioxide in short-term laparoscopy: a double-blinded randomized controlled trial.

BACKGROUND: We tested the hypothesis that warm-humidified carbon dioxide (CO2) insufflation would reduce postoperative pain and morphine requirement compared to cold-dry CO2 insufflation. METHODS: A double-blinded, randomized, controlled trial was conducted to compare warm, humidified CO2 and cold-dry CO2. Patients with benign uterine diseases were randomized to either treatment (n = 48) or control (n = 49) group during laparoscopically assisted vaginal hysterectomy. Primary endpoints of the study were rest pain, movement pain, shoulder-tip pain, and cough pain at 2, 4, 6, 24, and 48 hours postoperatively, measured by visual analogue scale. Secondary outcomes were morphine consumption, rejected boli, temperature change, recovery room stay, and length of hospital stay. RESULTS: There were no significant differences in all baseline characteristics. Shoulder-tip pain at 6 h postoperatively was significantly reduced in the intervention group. Pain at rest, movement pain, and cough pain did not differ. Total morphine consumption and rejected boli at 24 h postoperatively were significantly higher in the control group. Temperature change, recovery room stay, and length of hospital were similar. CONCLUSIONS: Warm, humidified insufflation gas significantly reduces postoperative shoulder-tip pain as well as morphine demand. This trial is registered with Clinical Trial Registration Number DRKS00003853 (German Clinical Trials Register (DRKS)).

Dysfunctional action control as a specific feature of Parkinson's disease.

Parkinson's disease (PD) is characterised by motor deficits as well as cognitive alterations, particularly concerning frontal lobe control. Here, we were interested in whether executive function is abnormal already early in PD, as well as whether this dysfunction worsens as a part of the dementia in PD. The following groups engaged in tasks addressing action control: PD patients with mild and advanced motor symptoms (aPD) without dementia, PD patients with dementia (PDD), patients with Alzheimer's disease (AD) and healthy subjects (CON). Subjects either had to perform or inhibit button presses upon go and no-go cues, respectively. These cues were preceded by pre-cues, either randomly instructive of right or left hand preparation (switch condition), or repetitively instructive for one side only (non-switch condition). PDD and aPD omitted more go responses than CON. Furthermore, PDD disproportionally committed failures upon no-go cues compared to CON. In the non-switch condition, PDD performed worse than AD, whose deficits increased to the level of PDD in the switch condition. Over all PD patients, task performance correlated with disease severity. Under the switch condition, task performance was low in both PDD and AD. In the non-switch condition, this also held true for advanced PD patients (with and without dementia), but not for AD. Thus, the deficits evident in PDD appear to develop from imbalanced inhibitory-to-excitatory action control generally inherent to PD. These results specify the concept of dysexecution in PD and differentiate the cognitive profile of PDD from that of AD patients.

The C2-domain protein QUIRKY and the receptor-like kinase STRUBBELIG localize to plasmodesmata and mediate tissue morphogenesis in Arabidopsis thaliana.

Tissue morphogenesis in plants requires communication between cells, a process involving the trafficking of molecules through plasmodesmata (PD). PD conductivity is regulated by endogenous and exogenous signals. However, the underlying signaling mechanisms remain enigmatic. In Arabidopsis, signal transduction mediated by the receptor-like kinase STRUBBELIG (SUB) contributes to inter-cell layer signaling during tissue morphogenesis. Previous analysis has revealed that SUB acts non-cell-autonomously suggesting that SUB controls tissue morphogenesis by participating in the formation or propagation of a downstream mobile signal. A genetic screen identified QUIRKY (QKY), encoding a predicted membrane-anchored C2-domain protein, as a component of SUB signaling. Here, we provide further insight into the role of QKY in this process. We show that like SUB, QKY exhibits non-cell-autonomy when expressed in a tissue-specific manner and that non-autonomy of QKY extends across several cells. In addition, we report on localization studies indicating that QKY and SUB localize to PD but independently of each other. FRET-FLIM analysis suggests that SUB and QKY are in close contact at PD in vivo. We propose a model where SUB and QKY interact at PD to promote tissue morphogenesis, thereby linking RLK-dependent signal transduction and intercellular communication mediated by PD.

Evaluation of SprayShield™ Adhesion Barrier in a single center: randomized controlled study in 15 women undergoing reconstructive surgery after laparoscopic myomectomy.

OBJECTIVE: The objective of this study was to evaluate the safety and efficacy of SprayShield™ Adhesion Barrier in preventing and/or reducing postoperative adhesion during gynecological surgery. DESIGN: This was a prospective, controlled, blinded, and randomized study. Patient blinding was performed intraoperatively. Subjects were randomly assigned to the SprayShield™ or the control group in a 2:1 ratio. SETTING: The study was conducted at the Clinic of Gynaecology and Obstetrics, at the University Hospital for Gynecology in Germany. PATIENTS: Fifteen patients participated in this study; nine patients were assigned to the SprayShield™ and six patients to the control group. INTERVENTIONS: During first operation (FLL) in the SprayShield™ group, the agent was applied to all myomectomy suture lines. Patients in the control group did not receive any anti-adhesion treatment, only good surgical practice. A second-look laparoscopy (SLL) was performed 8-12 weeks after myomectomy to evaluate adhesion formation. MAIN OUTCOME MEASURES: Main outcome measures were incidence, severity, and extent of uterine adhesions. RESULTS: No significant differences were found between the two study groups. CONCLUSIONS: SprayShield™ is easy to use. No serious adverse event related to SprayShield™ was observed. Efficacy data are inconclusive regarding the performance of SprayShield™. Further studies are needed to better understand this performance.