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Symptoms in the urogenital organs are common in multiple system atrophy (MSA), also in the years preceding the MSA diagnosis. It is unknown how MSA is triggered and these observations in prodromal MSA led us to hypothesize that synucleinopathy could be triggered by infection of the genitourinary tract causing ɑ-synuclein (ɑSyn) to aggregate in peripheral nerves innervating these organs. As a first proof that peripheral infections could act as a trigger in MSA, this study focused on lower urinary tract infections (UTIs), given the relevance and high frequency of UTIs in prodromal MSA, although other types of infection might also be important triggers of MSA. We performed an epidemiological nested-case control study in the Danish population showing that UTIs are associated with future diagnosis of MSA several years after infection and that it impacts risk in both men and women. Bacterial infection of the urinary bladder triggers synucleinopathy in mice and we propose a novel role of ɑSyn in the innate immune system response to bacteria. Urinary tract infection with uropathogenic E. coli results in the de novo aggregation of ɑSyn during neutrophil infiltration. During the infection, ɑSyn is released extracellularly from neutrophils as part of their extracellular traps. Injection of MSA aggregates into the urinary bladder leads to motor deficits and propagation of ɑSyn pathology to the central nervous system in mice overexpressing oligodendroglial ɑSyn. Repeated UTIs lead to progressive development of synucleinopathy with oligodendroglial involvement in vivo. Our results link bacterial infections with synucleinopathy and show that a host response to environmental triggers can result in ɑSyn pathology that bears semblance to MSA. ispartof: ACTA NEUROPATHOLOGICA vol:145 issue:5 pages:541-559 ispartof: location:Germany status: published

To describe the first case of Vaginal natural orifice transluminal endoscopic surgery (vNOTES) approach to repair of an uterine isthmocele.Stepwise explanation of the surgical technique using original video footage. This study was exempted from requiring hospital IRB.Department of Obstetrics and Gynaecology, Imelda Hospital, Belgium.A 30-year-old (G2P2) patient presented with symptoms of intermenstrual bleeding and secondary subfertility following two uncomplicated cesarean section deliveries (elective for breech, elective repeat). The patient had a transvaginal US demonstrating an isthmocele with a defect of 8.1x 7.0mm with a myometrial thickness of 2mm over the defect and proceeded to undergo surgery due to persistent symptoms.

Vaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES) combines the advantages of vaginal surgery with those of laparoscopic surgery. Feasibility of vNOTES for other indications such as hysterectomy, ovarian cystectomy and myomectomy has been established. In this article, we describe a standardised step by step process to perform adnexal surgery by vNOTES.

Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). So far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that polyamine transport inhibitors are considered for cancer therapy. Here, we tested whether upregulation of candidate polyamine transporters of the P5B- transport ATPase family is responsible for the increased PTS in the well-studied breast cancer cell line MCF7 compared to the non-tumorigenic epithelial breast cell line MCF10A. We found that MCF7 cells present elevated expression of a previously uncharacterized P5B-ATPase ATP13A4, which is responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells are more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, overexpression of ATP13A4 WT in MCF10A cells induces a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labelled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we establish ATP13A4 as a new polyamine transporter in the human PTS and show that ATP13A4 may play a major role in the increased polyamine uptake of breast cancer cells. ATP13A4 therefore emerges as a candidate therapeutic target for anticancer drugs that block the PTS.

In Parkinson's disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear, but they are hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, and LRRK2 kinase activity has been shown to be involved in pS129-aSyn inclusion modulation. We observed selective downregulation of the novel PD risk factor RIT2 in vitro and in vivo. Rit2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rit2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, Rit2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. On the other hand, reduction of Rit2 levels leads to defects in the ALP, similar to those induced by the G2019S-LRRK2 mutation. Our data indicate that Rit2 is required for correct lysosome function, inhibits overactive LRRK2 to ameliorate ALP impairment, and counteracts aSyn aggregation and related deficits. Targeting Rit2 could represent an effective strategy to combat neuropathology in familial and idiopathic PD. ispartof: NPJ PARKINSONS DISEASE vol:9 issue:1 ispartof: location:United States status: published

To describe a new technique for performing an isthmic retroperitoneal cerclage via vaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES).Stepwise explanation of the surgical technique using original video footage. This study was exempted from requiring hospital IRB.Department of Obstetrics and Gynaecology, Imelda Hospital, Belgium.A circumferential vaginal incision is made around the cervix after infiltration of the tissue with local anaesthetic and adrenaline. The bladder is deflected from the cervix up to the level of the isthmus but the peritoneum is not opened. The rectum is deflected from the cervix up to the level of the isthmus but the peritoneum is not opened. A Gelpoint vPath (9.5 cm) (Applied Medical, Rancho Santa Margarita) is used as a vNOTES port. The inner ring of the Alexis is inserted into the retroperitoneal dissection space around the cervix, covering the bladder anteriorly and the rectum posteriorly. An insufflation stabilization bag (ISB) is connected to the Gelseal cap to minimize pressure fluctuations within the confines of the retroperitoneal operative space. The paracervical space is opened just inferior to the uterine artery. A paracervical tunnel is made from the anterior to the posterior retroperitoneal space just lateral to the cervix and just inferior to the uterine artery. This procedure is performed bilaterally. A vessel loop is inserted from anterior to posterior through the paracervical tunnel on each side of the cervix. The Gelseal cap is removed and an Ethibond-2 suture is tied to the vessel loop. The Gelseal cap is placed again. By pulling on the vessel loop, it is replaced by the Ethibond suture. The Ethibond suture is now tied endoscopically to complete the cerclage; alternatively the suture could be tied vaginally without endoscopic instruments to the surgeon's preference. The vNOTES port is removed and the vaginal mucosa is sutured to the cervix again using a resorbable suture.Abdominal cerclage is the preferred approach to treat patients with refractory cervical insufficiency [1]. Over the last 20 years this technique increasingly gets performed laparoscopically with similar success rates [2]. The new transvaginal approach demonstrated in this video article may help reduce the invasivess even more by avoiding abdominal incisions and opening the peritoneum, while still benefiting from the vNOTES endoscopic visualization and minimally invasive instruments [3]. The cerclage is placed permanently at the level of the isthmus, similar to an abdominal cerclage. It is placed more cranially than a McDonald or Shirodkar cerclage and leaves no non-resorbable sutures in the vagina [4]. It can be an alternative to a conventional transvaginal cervicoisthmic cerclage [5,6] in patients with a narrow vagina and without uterine descensus. Following the guidelines of the IDEAL collaboration we present this technique to be peer reviewed in its early developmental phase before starting further studies [7].This is a first feasibility and technique description report on performing a retroperitoneal isthmic cervical via vNOTES. This technique should be considered new, not to be performed on pregnant patients or outside of clinical trials, and only in carefully counselled patients.

Study objective The first ever report of vaginal natural orifice transluminal endoscopic surgery (vNOTES) for benign gynaecological was reported in 2012. There has been an exponential uptake of the number of surgeons performing such procedures worldwide with no official guidance to ensure the safe implementation of this technique into gynaecological practice due its recency. The objective of this study is to report an international consensus-based statement to help guide a basis for adopting vNOTES into clinical practice. Study design The consensus-based statement was developed amongst 39 international experts using the Delphi methodology over three successive rounds. Consensus was pre-defined as an agreement of 80% or more by the experts. Consensus sought over eight key concepts pertaining to vNOTES including patient selection, perioperative management, surgical technique, instruments, anatomy, training, registries and trials and definition of the surgical technique. Recommendations from an expert anaesthetist and urogynaecologist were also sought to give a broader perspective with respect to the implementation of vNOTES. Results Fifty nine international surgeons were invited to participate and 39 (66%) agreed to participate based on being involved in a minimum of 20 vNOTES procedures. They were from 13 countries across 5 continents (Europe, North America, South America, Australia and Asia). Participation was 100% on all three rounds. Overall, consensus was reached in 50 of the 56 questions (89%) with the remaining 6 questions where consensus was not reached pertaining to the domain of patient selection. Conclusion An international expert based vNOTES statement is presented here to help guide adoption of vNOTES based on the experience of early adopters. Consensus was achieved on most components of this consensus statement. Given the recency of this technique, until high-level evidence becomes available, this statement provides an appropriate guidance to the safe implementation of vNOTES into gynaecological practice.

Objective To compare adnexectomy by vaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES) versus laparoscopy. Design Parallel group, 1:1 single-centre single-blinded randomised trial, designed as non-inferiority study with a margin of 15%. Setting Belgian teaching hospital POPULATION: Non-pregnant non-virgin women with an intact uterus and without obliteration of the pouch of Douglas scheduled to undergo removal of an adnexal mass assessed to be benign on ultrasound by IOTA criteria. Methods Randomisation to laparoscopy (control group) or vNOTES (experimental group). Stratification according to adnexal size. Blinding of participants and outcome assessors by sham incisions. Main outcome measures The primary outcome measure was adnexectomy by the allocated technique. Secondary outcomes included duration of surgery, pain scores and analgesics used, quality of life and adverse events. Results We randomly assigned 67 participants (34 to the vNOTES group and 33 to the laparoscopy group). The primary endpoint was always reached in both groups: there were no conversions. We performed a sensitivity analysis for the primary outcome, assuming one conversion in the vNOTES group and no conversions in the laparoscopy group: the one-sided 95% upper limit for the differences in proportions of conversion was estimated as 13%, which is below the predefined non-inferiority margin of 15%. The secondary outcomes demonstrated a shorter duration of surgery, lower pain scores, lower total dose of analgesics and a trend for more adverse events in the vNOTES group. Conclusions vNOTES is non-inferior to laparoscopy for a successful adnexectomy without conversion. vNOTES allowed shorter operating times and less postoperative pain but there was a trend for more adverse events.

Study Objective The Hominis surgical system is a novel robot-assisted system, designed specifically for robotic vaginal natural orifice transluminal endoscopic surgery (RvNOTES). We presented our experience of the first 30 RvNOTES hysterectomies assessing the feasibility and safety of this technology. Design A two-center prospective study. Setting Academic tertiary referral centers. The ethics committees approved the study in both centers. Patients Thirty women with benign indication for hysterectomy. Intervention RvNOTES hysterectomy performed by the Hominis surgical system. Measurements and Main Results The primary outcome of the study was the rate of conversion to open or conventional laparoscopic approaches. Secondary outcomes included intra- and postoperative adverse events, operative time, estimated blood loss, length of hospital stay, and 6-week follow-up assessment. A total of 15 women were enrolled at each site. The median age was 59 years (range: 37–79) and the median body mass index was 25.4 kg/m2 (range: 17.6–40.0). Twenty-four women (80%) had comorbidities. All the procedures were completed successfully without conversion to open abdominal, traditional vaginal, or conventional laparoscopic surgery. No intraoperative complications were observed. Median blood loss and procedure duration were 50 mL (range: 20–400) and 57 minutes (range: 24–88), respectively. Postoperative pain was minimal, with a median visual analog scale of 3 (range: 1–5) for the first 24 hours following surgery. The median hospital stay was 3 days (range: 2–8). According to the treating physicians’ evaluations, the vaginal cuff was fully healed in all patients at the 6-week postoperative follow-up visit. Conclusions This is the first publication of robot-assisted vaginal hysterectomy using the Hominis surgical system. The positive results of this study show this new technology to be a safe and effective tool for vaginal natural orifice transluminal endoscopic surgery, enabling surgeons to operate vaginally with the known advantages of robotic modality.

The melatonin hormone, which is a multifunctional molecule in vertebrates, has been shown to exert complex actions on the immune system of mammals. In teleosts, the immunomodulatory capacity of this hormone has seldom been investigated. In the present experiment, we exposed ex vivo spleen and head kidney tissues of pike-perch to melatonin (Mel) and cortisol (Cort). We applied three concentrations of both hormones, alone and in combination, namely (1) Mel (10, 100 or 1000 pg mL−1) (2) Cort (50, 500 or 5000 ng mL−1) (3) Mel + Cort (10 + 50, 100 + 500 or 1000 pg mL−1+5000 ng mL−1). Pure medium without Mel or Cort served as control. After 15 h of incubation, we assessed the expression of a set of immunity-related genes, including genes encoding for pro-inflammatory proteins (il-1β, cxcl8 and tnf-α), acute-phase proteins (fgl2, fth1, hepc, hp and saa1) and key factors of the adaptive immune system (fκbp4 and tcrg). Both Mel and Cort, when used alone or combined at physiological concentrations, significantly influenced immune gene expressions that may lead to a global immune stimulation. Our results support both, an indirect action of the Mel hormone on the immune system through the regulation of intermediates such as Cort, as well as a direct action on immune targets through specific receptors.

The development of disease-modifying therapies for Parkinson’s disease is a major challenge which would be facilitated by a better understanding of the pathogenesis. Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are key players in Parkinson’s disease, but their relationship remains incompletely resolved. Previous studies investigating the effect of LRRK2 on α-synuclein–induced neurotoxicity and neuroinflammation in preclinical Parkinson’s disease models have reported conflicting results. Here, we aimed to further explore the functional interaction between α-synuclein and LRRK2 and to evaluate the therapeutic potential of targeting physiological LRRK2 levels. We studied the effects of total LRRK2 protein loss as well as pharmacological LRRK2 kinase inhibition in viral vector–mediated α-synuclein–based Parkinson’s disease models developing early- and late-stage neurodegeneration. Surprisingly, total LRRK2 ablation or in-diet treatment with the LRRK2 kinase inhibitor MLi-2 did not significantly modify α-synuclein–induced motor deficits, dopaminergic cell loss, or α-synuclein pathology. Interestingly, we found a significant effect on α-synuclein–induced neuroinflammatory changes in the absence of LRRK2, with a reduced microglial activation and CD4(+) and CD8(+) T cell infiltration. This observed lack of protection against α-synuclein–induced toxicity should be well considered in light of the ongoing therapeutic development of LRRK2 kinase inhibitors for idiopathic Parkinson’s disease. Future studies will be crucial to understand the link between these neuroinflammatory processes and disease progression as well as the role of α-synuclein and LRRK2 in these pathological events. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01007-8.

Trans-vaginal natural orifice transluminal endoscopic surgery (vNOTES) is a recently popularised minimally invasive surgical procedure, aimed at minimising abdominal wall scars and improving pain and patient recovery times. Although vNOTES has been studied in the context of post-operative pain and cosmesis, women’s acceptance of the technique has only been cursorily examined. In this survey-based observational study, we assessed the acceptability of this technique among a cohort of Middle Eastern women. A cohort of 175 Middle Eastern women were surveyed using a 13-item questionnaire at a single gynaecology centre. The survey used was a translated version of a questionnaire from a previous study (1) and comprised open-response, five-point Likert Scale and agree-disagree items. Among 175 Middle Eastern women participated in this study most of them holding neutral view on abdominal and gynaecological procedures via vagina. 47% of participants were unsure regarding the effect of surgery via vagina on their sexual function. Although 61% of the participants showed no preference towards vNOTES over laparoscopic cholecystectomy, more than half of them indicated preference if vNOTES shown to be as effective and safe as laparoscopic cholecystectomy. The gender of the surgeon was shown to have no influence on the perspectives of the majority of participants to undergo vNOTES. vNOTES may hold value for women who have conservative upbringing and/or value cosmesis. This study provides information regarding Middle Eastern women’s perspectives on vNOTES, which may be of considerable clinical use as the popularity of this surgical technique continues to increase.

STUDY OBJECTIVE To evaluate the safety and feasibility of vaginal natural orifice transluminal endoscopic surgery (vNOTES) for benign gynaecological indications. STUDY DESIGN Prospective observational study (Canadian Task Force classification II-3) in a non-university teaching hospital. One thousand consecutive patients (2013-2018) who were eligible for vNOTES approach to benign gynaecological surgery were included. RESULTS Patient data and perioperative outcomes were included for analysis after the vNOTES surgical approach was standardized and made the default route of surgery. Of the 1000 vNOTES cases performed during this study period, hysterectomy (73%) was the most common followed by adnexal surgery (18%) and salpingectomy (4%). The mean age of the patient cohort was 46 yrs of age (22-83) and mean BMI, 26 kg/m2(16.5-52). There was a conversion rate of 0.4% (4 cases), 3 to conventional laparoscopy and 1 to laparotomy. The mean operating time was 42 minutes (14-250 minutes). The total complication rate was 3.9% (39) of which 1% (10) was intraoperative complications and 2.9% (29) were post-operative complications. When the hysterectomy cohort was sub analyzed, the total complication rate was 5.2% in this group (intraoperative 1.4% and post-operative 3.8%) with only a 0.4% complication rate in the non-hysterectomy sub-group. Of the complications in the hysterectomy group there were 9 cystotomies (1.2%). The mean specimen weight for all hysterectomies was 172 g (20-3361 g) with an average operating time of 46 minutes (20-250). CONCLUSION The perioperative outcomes of this large vNOTES case series for benign gynaecological indications is comparable to other vNOTES procedures reported in the literature apart from the risk of cystotomy which is a hysterectomy specific risk. The outcomes are that of a single high-volume surgeon incorporating cases within his respective learning curve of the surgical technique and as such should be interpreted accordingly. Since 2015 the International NOTES Society has initiated a prospective complication database where all vNOTES surgeons are invited to register their cases.

OBJECTIVE The aim of this study was to describe our experience with transvaginal natural orifice transluminal endoscopic surgery (vNOTES) uterosacral ligament suspension (USLS) and to evaluate the learning curve of mastering the skills to operate this procedure. METHODS This is a prospective study of the first 35 vNOTES hysterectomy and USLS surgeries performed between October 2018 and April 2019 at Rambam Health Care Campus (Israel) (n = 26) and Imelda Hospital (Belgium) (n = 9). The primary outcome was surgical time. Secondary outcomes included intraoperative bleeding, length of hospitalization, pain during the first 24 hours postoperative pain, and the need for analgesia, intraoperative, and postoperative adverse events. RESULTS The median age was 55 years (range, 40-81). The median operative time was 113 minutes (range, 89-233). For the procedures performed at Rambam, total operative time decreased from the first 13 to the following 13: from 149 minutes (range, 89-233 minutes) to 103 (89-170) (

In many experiments with simultaneous interpreting (SI), researchers use source speeches that are not only written and read aloud, but also pre-recorded, reducing their ecological validity. This st...

Significance Mutations in ATP13A2 cause a spectrum of related neurodegenerative disorders. ATP13A2 is a lysosomal exporter of polyamines that contributes to lysosomal health and controls cellular polyamine content. Conversely, loss of ATP13A2 leads to lysosomal dysfunction, a hallmark of neurodegeneration. Here, we show that polyamines transported by ATP13A2 provide cellular protection by lowering reactive oxygen species (ROS), which may relate to the antioxidant properties of polyamines. Consequently, dysfunctional ATP13A2 sensitizes cells to oxidative stress, which impairs mitochondria, and induces toxicity and cell death. ATP13A2-mediated polyamine transport represents a conserved pathway that protects against mitochondrial oxidative stress. The combined protective impact of ATP13A2 on lysosomal health and mitochondrial oxidative stress may explain why ATP13A2 exerts potent neuroprotective effects., Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Background and objective Transvaginal natural orifice transluminal endoscopic surgery (vNOTES) has been applied massively in the gynecological field in recent years. The aim of the current study is to present the surgical technique of vNOTES omentectomy and to evaluate the feasibility of this procedure. Methods A case series study of the first 5 vNOTES omentectomy procedures performed for surgical staging of suspicious early stage ovarian cancer, at Rambam Health Care Campus (Israel) and Imelda Hospital (Belgium) between November 2018 and August 2019. Sociodemographic and clinical data were retrieved from patients’ electronic charts. Primary points of interest included intra-operative bleeding, length of surgery, length of hospitalization, and surgical complications. Results The median age was 61 years (range 50–72), and the median BMI was 27 kg/m2 (range 23–33). All the operations were carried out to completion through the vaginal GelPOINT, without insertion of an assistant abdominal trocar or conversion to another surgical approach. The median omentectomy time was 45 min (range: 39–52). The median estimated intraoperative blood loss was 150 ml (range: 20–200). The median hospital stay was 2 days (range: 1–3). Conclusions vNOTES is a feasible technique for omentectomy in early stage ovarian cancer, with low rates of complications and improved cosmetic results.

Hormonal induction of spermiation, previously reported to be immunogenic in fishes, is a common hatchery practice in pikeperch, Sander lucioperca. The aim of the present study was to investigate the effects of repeated induction of spermiation in pikeperch, following application of either human chorionic gonadotropin (hCG) or salmon gonadoliberine analogue (sGnRHa) on sperm quality indices as well as on immune and stress response. Mature males of pikeperch (n = 7 per group) were stimulated twice with five days between injections of either hCG (hCG; 500 IU kg−1), sGnRHa (sGnRHa; 50 μg kg−1) or NaCl (control group; 1 ml kg−1) to assess spermatozoa motility with a computer-assisted sperm analysis (CASA) system. During second sampling, blood plasma was sampled for humoral innate immune (peroxidase and lysozyme activities, ACH50), stress (cortisol, glucose) and endocrine (testosterone) markers. In addition, the head kidney was dissected to assay the expression of several immune genes (such as il1, c3, hamp, tnf-α and lys genes). The results indicate that hormonal treatment significantly increased sperm production. Sperm sampled after the hormonal treatment maintained its quality throughout the study, regardless of the sampling time. However, it appears that the application of hCG induced elevated cortisol and glucose plasma levels compared to the control group. Almost all immune markers, except the relative expression of hepcidin (hamp gene), were unaffected by the two hormones applied. The results showed that the induction treatment of spermiation processes in pikeperch resulted in an important physiological stress response for which the intensity varied according to the hormonal agent used. However, this stress response (more profound following application of hCG) was weakly associated with innate immune functions. On the other hand, a significant negative correlation between the expression of several important immune markers (peroxidase activity, relative expression of c3 and il1 genes) and sperm quality indices indicates significant involvement of immune status on sperm quality. The results obtained shed light on immune-system-induced modifications to sperm quality. The data presented here highlight the need for careful revision of broodstock management and selection practices where welfare status as well as individual predispositions of fish to cope with the stress should be taken under the consideration.

Additional file 1: Figure S1. Phenotypic effect of 3xHS treatment. (A) Phenotype of soil-grown segregating PcUBI::LbCas12a::G7T and PcUBI::Cas9::G7T lines under control conditions or 3xHS regime. Plants displaying a pds3 phenotype are indicated with a white circle. (B) T2 plants containing AsCas12a, FnCas12a, LbCas12a or Cas9 and targeting PDS3. DNA was extracted for three to twelve individuals for each line and treatment, PCR products amplified from targeted loci were sequenced and analysed using ICE (https://ice.synthego.com). The KO-score is given for each sample, indicating indels that result in a frameshift or are 21+bp in length. Lines indicate mean KO-score per sample per treatment. n=3-12 per sample per treatment. (C) Phenotype of in vitro grown segregating PcUBI::Cas9::G7T and PcUBI::LbCas12a::G7T lines under control conditions or 3xHS regime. Picture taken 14 days after the last heat shock. White arrowheads indicate PcUBI::LbCas12a::G7T plants displaying pds3 phenotype after 3xHS. Figure S2. Critical temperature to induce genome editing. (A) Phenotypic effect of number of consecutive heat shocks on segregating Arabidopsis T2 lines expressing PcUBI::LbCas12a::G7T (two independent lines, Cas12a #3-#4) and a gRNA targeting PDS3. Transgenic lines and wild-type control (WT) were grown under control conditions (21 Phenotypic effect of number of consecutive heat shocks on segregating Arabidopsis T2 lines expressing PcUBI::LbCas12a::G7T (two independent lines, Cas12a #3-#4) and a gRNA targeting PDS3. Transgenic lines and wild-type control (WT) were grown under control conditions (21��C) or subjected to one, two, three or four heat shocks (1-4xHS; 37��C). Pictures taken 14 days after recovery. (C) Phenotypic distribution of wild-type (WT) and two independent transgenic lines containing PcUBI::LbCas12a::G7T subjected to control (C) conditions or 3xHS treatments at 30��C, 37��C or 42��C. N.G.: Not Germinated. n=100 per line per treatment. Figure S3. Effect of other stress conditions on genome editing efficiency. Segregating Arabidopsis T2 lines expressing PcUBI::LbCas12a::G7T (two independent lines) and a gRNA targeting PDS3 were grown under control conditions (21��C) on 1/2MS medium supplemented with Bleomycin (0,3mg/ml), Hydroxyurea (HU; 0,75mM), Mannitol (25mM or 50mM) or NaCl (50mM or 100mM). Additionally, transgenic lines were exposed to one, two or three 24h cycles of 40 W/m-2 light supplemented with 0,42 W/m-2 UV alternated with 24h of control light conditions. n=50 for each line and treatment. Figure S4. Heat-induced genome editing is independent of the transcriptional regulator. T2 segregating lines of four constructs (pB-ZmUBI-Cas9PTA-G7T, pB-RPS5PA-Cas9PTA-G7T, pB-35SP-Cas9PTA-G7T and pB-PcUBI-Cas9PTA-G7T) targeting PDS3 with one of four possible gRNAs) were grown under control conditions (C) or subjected to 3xHS (HS). pds3 phenotypes were scored according to the severity (albino, mosaic or spots) 14 days after final HS for each line and condition. Lines that show an increase in number and/or severity of pds3 phenotypes after 3xHS are indicated with *. n=25 for each line and condition. Figure S5. Effect of 3xHS on inducing large deletions. (A) Experimental set-up. Four type-II MC genes (MC4-7) are positioned in a tandem repeat on chromosome 1. We designed five gRNAs (A3, A9, A11, B3 and B7) to delete the 10,4kB region from the genome. (B) Six independent lines with different combinations of gRNAs were submitted to 3xHS (HS) or control treatment (C). 14 days after the last HS, plants were genotyped. The number of plants where the intended deletion was observed is indicated as a ratio (relative to the number of plants genotyped). (C) Molecular confirmation of deletion. For each independent line, the PCR product corresponding to a big deletion was sequenced for 4-6 individuals. Sequence reads were mapped to the expected deletions using the Geneious software. Alignments are shown for those lines where a single repair product was observed in the PCR product. (D) Example of the genotyping strategy. A11-B7 T2 lines grown under control conditions (in this example), an untransformed control (WT) and a no-template control (-) were genotyped with two pairs of primers: one control reaction amplifying a part of AtMC7 (ATMC7-FW + ATMC7-RV) and one reaction amplifying the intended deletion (ATMC7-FW + ATMC4-RV; see also panel A). Figure S6. Effect of 3xHS on Cas9-VQR activity. Two independent transgenic single-locus T2 lines containing PcUBI::Cas9VQR::Pea3AT were imposed to a 3xHS treatment or control conditions. Two weeks after the final heat shock, the two oldest leaves were genotyped by Sanger sequencing. n=121 (T2 Control), 119 (T2 Heat) or 30 (T3). Figure S7. Effect of 3xHS on HDR efficiency. (A) Experimental set-up to insert mRuby3 at the C-terminus of OLE1(AT4G25140). Schematic representation of the T-DNA constructs (pK LacI-AtCas9; top), the endogenous OLE1 locus (middle) and OLE1-mRuby3 HR product (bottom). Abbreviations: right border (RB), left border (LB), G7 terminator (G7T), kanamycin resistance cassette (nptII), nopaline synthase terminator (NosT). (B) Results of genotyping PCR two weeks after the last heat shock of T2 lines expressing Cas9 fused to LacI, a gRNA targeting OLE1 and a template containing mRuby and overhang sequences at both sides of the target region. For each line and condition (control or 3xHS), the number of plants where the right border of the insertion was amplified relative to the total number of plants genotyped is shown. A representative example is shown above the table; lines where the right border was amplified are indicated with a black arrow. (C) Left: Visual confirmation of putative HDR events in T3 lines. A functional OLE1-mRuby construct results in red fluorescent seeds, indicated with white arrows. Right: Quantification of HR efficiency in three independent T3 lines that were subjected to control conditions or 3xHS in the previous generation. Each dot indicated the HR efficiency per line per treatment, the number of lines with visual observation of HR relative to total number of screened lines is indicated below the graphs. (D) Molecular confirmation of HR events in 13 independent T3 individuals. The right border sequence was amplified, sequenced and mapped to the expected HR outcome using the Geneious software. Black bars indicate disagreements to the reference sequence.

Background Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. Results Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. Conclusions Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny., Research Foundation - Flanders (FWO) [12T3418N, BOF20/PDO/016, 3G038719]; Ghent University [12T3418N, BOF20/PDO/016, BOFMET2015000201]; EU through Erasmus+ grants, J.B. thanks the Research Foundation -Flanders (FWO) and Ghent University for a postdoctoral fellowship (12T3418N and BOF20/PDO/016). A.B. is supported by Research Foundation Flanders (FWO research project 3G038719). D.I. is supported by Ghent University (BOF-Methusalem project BOFMET2015000201). A.K., J.A.B. and J.J. were supported by the EU through Erasmus+ grants.

INTRODUCTION Abiraterone acetate + prednisone (AAP) and docetaxel have proven their efficacy in the treatment of patients with newly diagnosed metastatic hormone-sensitive prostate cancer (mHSPC) in clinical trials. However, real-world data are scarce. The goal of this study is to evaluate real-world data on the efficacy and safety of these therapies in mHSPC patients. PATIENTS AND METHODS Records of 93 patients from 21 different centres were retrospectively reviewed. Primary and secondary endpoints were radiographic and PSA progression-free survival (RPFS - PSA-PFS) and cancer specific and overall survival (CSS - OS), respectively. Adverse events (AEs) were evaluated according to the Common Terminology Criteria for Adverse Events version 5.0. Differences in oncological outcome and AEs were evaluated between three treatment groups: ADT only (N=26) - ADT + AAP (N=48) - ADT + docetaxel (N=19). Survival analysis was performed using Kaplan-Meier statistics. RESULTS Median RPFS was 13 months (95% confidence interval [CI]: 9-17) for ADT only, 21 months (95% CI: 19-23) for ADT + AAP and 12 months (95% CI: 11-14) for ADT + docetaxel (p = 0.004). The 1-year PSA-PFS, CSS and OS were 73.5%, 90.7% and 88.7%, respectively, with no significant differences between the three groups. Adverse events of grade 3 or higher were not observed more frequently. CONCLUSION Retrospective real-world data show a significantly longer RPFS for mHSPC patients treated with ADT + AAP compared to ADT only or ADT + docetaxel at short-term follow-up. This can aid in counselling of mHSPC patients in daily clinical practice.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although being a powerful tool, off-target effects of these inhibitors are easily overlooked in a complex biological setting. Here we illustrate the implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is often used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the auxin catabolite indole-3-acetic acid (IAA)-Asp. By competing for the same enzyme, PA interferes with auxin conjugation, resulting in an increase in cellular auxin concentrations. These increased auxin levels likely further contribute to an increase in adventitious rooting previously observed upon PA-treatment. Despite the focus on GH3.6 in this report, PA is conjugated by an array of enzymes and their subsequent reduced activity on native substrates could potentially affect a whole set of physiological processes in the plant. We conclude that surrogate occupation of the endogenous conjugation machinery in the plant by exogenous compounds is likely a more general phenomenon that is rarely considered in pharmacological studies. Our results hereby provide an important basis for future reference in studies using chemical inhibitors.

We describe the first case of a vaginal NOTES total hysterectomy under spinal anesthesia in the head- down position without sedation or additive analgesia in a patient with end-stage COPD. This report adds to the growing evidence that neuraxial techniques are a good alternative for general anesthesia in select cases for laparoscopic surgery.

Oxidative Stress Cell Model preparation for intact cell cross-linking

Lysosomal membrane integrity

SPM Addition in cells for intact cell cross-linking

Intact cell cross-linking

Genes associated with endolysosomal function have been recently associated with familial Parkinson's disease and described as risk factors for sporadic cases. This indicates that deficits in this pathway predispose to parkinsonism. To better understand the role of these genes in disease development, rodent models have been created by targeting genes playing a role in endolysosomal function, such as LRRK2, DNAJC6, SYNJ1, VPS35, GBA1, ATP13A2 and TMEM175. Here, we review the latest findings describing parkinsonian features in these animal models secondary to endolysosomal dysfunction. Also, we provide suggestions for further development and application of these animal models to better understand the contribution of endolysosomal dysfunction in Parkinson's disease and provide novel models for testing therapeutic approaches.

Several neurodegenerative disorders are characterized by oligodendroglial pathology and myelin loss. Oligodendrogliopathies are a group of rare diseases for which there currently is no therapy. Gene delivery through viral vectors to oligodendrocytes is a potential strategy to deliver therapeutic molecules to oligodendrocytes for disease modification. However, targeting oligodendroglial cells in vivo is challenging due to their widespread distribution in white and gray matter. In this study, we aimed to address several of these difficulties by designing and testing different oligodendroglial targeting vectors in rat and mouse brain, utilizing different promoters, serotypes, and delivery routes. We found that different oligodendroglial promoters (myelin basic protein [MBP], cytomegalovirus-enhanced MBP, and myelin-associated glycoprotein [MAG]) vary considerably in their ability to drive oligodendroglial transgene expression and different viral vector serotypes (rAAV2/7, rAAV2/8, and rAAV2/9) exhibit varying efficacies in transducing oligodendrocytes. Different administration routes through intracerebral or intraventricular injection allow widespread targeting of mature oligodendrocytes. Delivery of rAAV2/9-MAG-GFP into the cerebrospinal fluid results in GFP expression along the entire rostrocaudal axis of the spinal cord. Collectively, these results show that oligodendrocytes can be targeted with high specificity and widespread expression, which will be useful for gene therapeutic interventions or disease modeling purposes.

Vaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES) is a minimally invasive, scar-free technique that uses a vaginal colpotomy to access the peritoneal cavity. Hysterectomy via vNOTES has shown to be technically feasible and safe, with shorter hospital stay and lower postoperative pain scores. Moreover, vNOTES adds the advantages of endoscopy to those of vaginal surgery and thereby broadens the indications of a conventional vaginal hysterectomy. Virginity has been contraindicated for vNOTES so far, as vaginal accessibility can be severely reduced in virgin women. Therefore, cases of vNOTES hysterectomy in virgin patients have never been reported in the literature. The objective of this study is to assess the technical feasibility and safety of hysterectomy in virgin women. We performed a retrospective analysis of patient files of all vNOTES hysterectomies performed on virgin women in our centre (Imelda Hospital, Bonheiden, Belgium) from July 2016 until June 2020 (N = 9). Despite limited vaginal accessibility, vNOTES hysterectomy was successfully performed in all nine patients, without conversion to laparoscopy or laparotomy and without clinically relevant complications. In this first IDEAL stage 1 study, vaginal NOTES hysterectomy appears to be technically feasible and safe in virgin women.Impact statementWhat is already known on this subject? Vaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES) is an emerging, minimally invasive technique that offers a safe alternative to laparoscopy and open surgery. Besides better cosmetics (scar-free technique), advantages of vNOTES hysterectomy include shorter hospital stay and less postoperative pain compared to laparoscopic hysterectomy. Moreover, vNOTES adds the benefits of endoscopy (visualisation of anatomy) to conventional vaginal hysterectomy. Virginity has been contraindicated for vNOTES so far, because the absence of uterine descent and narrow vagina in virgins is thought to complicate the vaginal approach. Therefore, vNOTES hysterectomy has never been reported in virgin patients.What do the results of this study add? In this study, we are the first to report the feasibility of performing a vNOTES hysterectomy in virgin patients. The results show that, in experienced hands, indications for vNOTES can be broadened and include virgin women.What are the implications of these findings for clinical practice and/or further research? This first report of vNOTES hysterectomy in virgin patients is only a small but important step in the evaluation of safety and efficacy of this emerging technique. Further research is needed to assess reproducibility of the findings and to carefully determine indications and contraindications of vNOTES.

Study Objective: Vaginal natural orifice transluminal endoscopic surgery (vNOTES) is a novel minimal invasive surgical technique allowing a variety of gynecological procedures. The current literature describes improved patient comfort, improved better cosmetic results, and reduced operation time. This is a first study to assess pregnancy outcome after fertility-preserving vNOTES procedures. Design/Participants/Materials/Setting/Methods: We performed a retrospective observational cohort study including 125 patients under 43 years that underwent fertility-preserving vNOTES over a 5-year period (2014–2019). The gynecological surgical procedures included were vNOTES myomectomy, vNOTES salpingectomy for ectopic pregnancy, vNOTES unilateral adnexectomy, and vNOTES cystectomy. A total of 26 pregnancies in 21 cases were observed, with deliveries between 2015 and 2020. Results: Retrospective analysis in this patient group showed that 18 pregnancies were diagnosed within 1 year after vNOTES (85.7%). Mean interval between surgery and pregnancy was 6 months. Fertility treatment was performed in 28.6%. In the 26 observed pregnancies, no vNOTES-related complications were observed and delivery was at term in all cases. Mode of delivery was a vaginal delivery in twenty cases (76.9% of total) of which 2 cases vacuum assisted (7.7%) and a Caesarean section in 6 cases (23.1% of total). Two cases of trial of labor after Caesarean are described after vNOTES surgery, both ended in an uncomplicated vaginal delivery. In case of vaginal delivery, the perineum was intact in 15%, a mediolateral episiotomy was performed in 50 and 35% a grade 1–2 perineal rupture was described. No grade 3–4 perineal ruptures are described. Limitations: A limitation of this study is the retrospective design which does not correct for confounding factors. Further larger multicenter studies are needed to validate these data. Conclusions: This is the first study describing pregnancy outcome after fertility-preserving vNOTES procedures. vNOTES did not affect the mode of delivery or cause pregnancy-related complications. vNOTES did not increase the risk of extensive perineal tears during vaginal delivery. These preliminary data show no adverse events when vNOTES is performed in women of reproductive age. Posterior colpotomy as performed in all vNOTES procedures is by itself not an indication for an elective Caesarean section.

imonoblotting protocol JMS

BackgroundDuring the pathogenesis of Parkinson’s disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo.MethodsThe disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or its dominant-negative T22N mutant as a control. The cellular integrity was quantified by morphological and biochemical analyses.ResultsIn primary neurons, WT Rab7 rescued the αSyn -induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn -induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain.ConclusionRab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.