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Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.

Summary: Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses. : Recent outbreaks and lack of effective treatments against dengue and Zika virus have caused public concerns. Estoppey et al. have identified cavinafungin as exerting potent and selective antiviral activity by targeting the signal-binding cleft of the catalytic subunit of the endoplasmic reticulum signal peptidase. Keywords: Zika virus, dengue virus, cavinafungin, signal peptidase, SEC11A, SEC11, CRISPR/Cas9, chemogenomic profiling

Selective and specific inhibitors of Plasmodium falciparum lysyl-tRNA synthetase represent promising therapeutic antimalarial avenues. Cladosporin was identified as a potent P. falciparum lysyl-tRNA synthetase inhibitor, with an activity against parasite lysyl-tRNA synthetase >100-fold more potent than that of the activity registered against the human enzyme. Despite its compelling activity, cladosporin exhibits poor oral bioavailability; a critical requirement for antimalarial drugs. Thus, the quest to develop metabolically stable cladosporin-derived analogues, while retaining similar selectivity and potency to that of the natural compound, has begun. Chemogenomic profiling of a designed library allowed an entirely innovative structure-activity relationship study to be initiated; this shed light on structural evidence of a privileged scaffold with a unique activity against tRNA synthetases.

Obesity is having an increasingly significant impact on health care services across the developed world. Although initially laparoscopic surgery was thought to be contraindicated in cases of obesity, surgeons, including gynaecologists, are now routinely performing laparoscopic surgery on obese patients. Limited research has been conducted into the effect of obesity on outcomes of major laparoscopic pelvic surgery. Some authors report worsening outcomes in obese women having laparoscopic hysterectomies; others suggest that complication rates do not increase, but operating times are longer. Here, we report our experience from 250 total laparoscopic hysterectomies performed for benign, malignant and pre-malignant conditions, and we compare outcomes among normal, overweight, obese and morbidly obese patients. We used a composite score index calculated on the basis of operating and theatre times, estimated blood loss, length of stay and number and severity of complications for our comparisons. Our conclusions suggest that meticulous, consistent surgical technique may produce similar outcomes in normal and obese patients having total laparoscopic hysterectomy, with longer operating/theatre times noted only at BMI levels >40.

Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections., Biosynthesis of glycosylphosphatidylinositol (GPI) is essential for the integrity of the fungal cell wall. Here, the authors show that the natural product jawsamycin inhibits GPI biosynthesis by targeting a subunit of the fungal UDP-glycosyltransferase, and displays pronounced activity against pathogenic fungi of the order Mucorales.

Invasive fungal infections are accompanied by high mortality rates that range up to 90%. At present, only three different compound classes are available for use in the clinic, and these often suffer from low bioavailability, toxicity, and drug resistance. These issues emphasize an urgent need for novel antifungal agents. Herein, we report the identification of chemically versatile benzamide and picolinamide scaffolds with antifungal properties. Chemogenomic profiling and biochemical assays with purified protein identified Sec14p, the major phosphatidylinositol/phosphatidylcholine transfer protein in Saccharomyces cerevisiae, as the sole essential target for these compounds. A functional variomics screen identified resistance-conferring residues that localized to the lipid-binding pocket of Sec14p. Determination of the X-ray co-crystal structure of a Sec14p-compound complex confirmed binding in this cavity and rationalized both the resistance-conferring residues and the observed structure-activity relationships. Taken together, these findings open new avenues for rational compound optimization and development of novel antifungal agents.

Pregnancy in a uterine rudimentary horn carries a high risk of uterine rupture with severe and potentially lethal intra-abdominal haemorrhage. There is now growing evidence that this condition can be safely managed by minimally invasive surgery. We report a case of an unruptured 11-week rudimentary horn pregnancy that was diagnosed and treated laparoscopically. We have performed a literature review using PubMed, Embase and Cochrane Database of Systematic Reviews to identify relevant cases and draw conclusions with regards to their management. We have collated 20 published cases of rudimentary horn pregnancies that were managed by laparoscopy. The surgical technique appears consistent among these cases with few variations. In advanced gestations, feticide may need to be performed. Morcellation has been shown to be possible without compromising patient safety from trophoblast spill. The possibility of uncommon presentations such as duplicated or absent ureter should be taken into account. Extracorporeal Roeder knot can be used safely to secure unusually dilated vascular pedicles. Overall, laparoscopy appears to be as safe as and potentially superior to laparotomy for the management of rudimentary horn pregnancies.

Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.

Key content Injury of the urinary tract is the most common major complication of gynaecological laparoscopic surgery. Injury to either bladder or ureter results in significant morbidity for the patient and may lead to litigation. Knowledge of pelvic anatomy, training and meticulous technique are of paramount importance in reducing the incidence of urinary tract injury. Ideally an injury should be identified and repaired during the primary operation, but vigilance in the immediate postoperative period may result in early recognition and intervention. Learning objectives To understand the common risk factors of urinary tract injury at laparoscopy. To learn strategies to prevent injury where possible. To learn strategies for intraoperative and postoperative recognition and repair of such injuries. To understand the significance of multi-disciplinary management of such injuries. Ethical issues Limited evidence shows that laparoscopic hysterectomy may carry a higher risk of urinary tract injury compared with abdominal hysterectomy. Should patients be counselled accordingly?

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway, however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context, and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides, and defined a discrete set of residues near the RNA binding motif which confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.

SummaryWith renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.

Objective To design a new method for oral preparation of urine for sperm retrieval after retrograde ejaculation (RE) and to test the motility of sperm exposed to prepared and unprepared urine. Design In vitro testing of urine conditions and sperm motility. Setting Assisted conception unit at a teaching hospital in the United Kingdom. Patient(s) Ten healthy volunteers to provide urine and sperm specimens from men attending the unit for semen analysis. Intervention(s) Various solutions of sodium bicarbonate and sodium chloride were drunk by a single subject until a suitable regimen was achieved. This regimen (called the Liverpool solution) was then tested on 10 volunteers. Samples of sperm were then added to prepared urine, unprepared urine, and culture medium, and the motility was analyzed. Main Outcome Measure(s) Urinary pH and osmolarity, sperm motility. Result(s) Urine produced by the 10 volunteers had a mean pH of 7.47 (range, 7.23–7.79) and a mean osmolarity of 289 mOsmol/L (range, 225–412 mOsmol/L), similar to that of medium. The progressive motility of sperm exposed to the unprepared urine was reduced (42.4% of sperm in medium), whereas that in the prepared urine was similar to that in the control medium. Conclusion(s) Liverpool solution can be used in any unit treating couples with RE, and it is a noninvasive and inexpensive regimen that may optimize urine pH and osmolarity for sperm survival after RE.

Radical trachelectomy is an operation developed as an alternative to radical hysterectomy for patients with small-volume, early stage cervical cancer, who wish to retain their fertility. The body of the uterus is left in place, so that future pregnancies can occur. Patients who have undergone radical trachelectomy may face problems conceiving naturally and may request assisted conception. This article explains the operation and the difficulties that those working in reproductive medicine may face.

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae—Erg26p, Homo sapiens—NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound., FR171456 is a bioactive chemical produced by some microorganisms. Here, the authors identify the enzyme NSDHL of the sterol synthesis pathway as the molecular target of FR171456, rendering it the first compound to specifically target this class of enzyme in yeast and mammalian cells.

Phenotypic screens are effective starting points to identify compounds with desirable activities. To find novel antifungals, we conducted a phenotypic screen in Saccharomyces cerevisiae and identified two discrete scaffolds with good growth inhibitory characteristics. Lack of broad-spectrum activity against pathogenic fungi called for directed chemical compound optimization requiring knowledge of the molecular target. Chemogenomic profiling identified effects on geranylgeranyltransferase I (GGTase I), an essential enzyme that prenylates proteins involved in cell signaling, such as Cdc42p and Rho1p. Selection of resistant mutants against both compounds confirmed the target hypothesis and enabled mapping of the compound binding site to the substrate binding pocket. Differential resistance-conferring mutations and selective substrate competition demonstrate distinct binding modes for the two chemotypes. Exchange of the S. cerevisiae GGTase I subunits with those of Candida albicans resulted in an absence of growth inhibition for both compounds, thus confirming the identified target as well as the narrow antifungal spectrum of activity. This prenylation pathway is reported to be nonessential in pathogenic species and challenges the therapeutic value of these leads while demonstrating the importance of an integrated target identification platform following a phenotypic screen.

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.

A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest ‘decatransin’ as the name for this new decadepsipeptide translocation inhibitor.

Objective To describe the laparoscopic management of an interstitial gestation of a heterotopic pregnancy. Design Case report and technique description. Setting Tertiary-level private practice. Patient(s) Woman with a 6-week gestation spontaneous heterotopic twin pregnancy: one twin intrauterine, one interstitial. Intervention(s) A purse-string suture was applied to the proximal portion of the interstitial heterotopic pregnancy. Main Outcome Measure(s) To enable a cornual resection to be performed with minimal bleeding and without recourse to laparotomy. Result(s) At 8 weeks gestation an ultrasound scan confirmed a viable singleton intrauterine pregnancy, but a scan at 12 weeks showed a missed miscarriage. Conclusion(s) The embedding of the suture into the uterine serosa prevents slipping of the ligature that could occur with a pretied loop.

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico . However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus . Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo .

We report the case of a 37-year-old woman who developed idiopathic brachial plexus neuritis, also referred to as Parsonage-Turner syndrome, after laparoscopic excision of endometriosis. The differential diagnosis between this non-position-related neuritis and brachial plexus injury is discussed. The aim of this report was to raise awareness on this distressing postoperative complication.

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

Objective To describe a case in which a midline laparotomy for two presumed malignant masses instead revealed parasitic fibroids. Design Case report. Setting Tertiary-level private hospital. Patient(s) Woman with 7- and 13-cm abdominopelvic masses 3 years after a total laparoscopic hysterectomy for fibroids. Intervention(s) Midline laparotomy, bilateral salpingooophorectomy, infracolic omentectomy, appendicectomy, para-aortic lymph node dissection, and high rectosigmoid resection with primary anastomosis. Main Outcome Measure(s) Histology of masses showed adenomyoma. Result(s) There was no evidence of malignancy. Conclusion(s) Morcellation of the fibroids during a total laparoscopic hysterectomy likely left fragments that formed iatrogenic parasitic fibroids, which led to the subsequent laparotomy and bowel resection for potential malignancy.

Herein is described an anterior approach to uterine artery ligation during laparoscopic myomectomy and total laparoscopic hysterectomy. The anterior leaf of the broad ligament is opened and the uterine artery is clipped lateral to its crossing over the ureter. Outcome measures were completion of the procedure laparoscopically and the need for transfusion postoperatively. Thirty-eight myomectomies and 28 difficult total laparoscopic hysterectomies (primarily uteri with large myomas) were performed, with 1 conversion to laparotomy during myomectomy and 1 during hysterectomy, and 1 transfusion after total laparoscopic hysterectomy. The anterior approach to uterine artery ligation is an alternative method for treatment of uterine artery occlusion during laparoscopic myomectomy or hysterectomy performed to treat large myomas.

BACKGROUND: We wanted to test the hypothesis that using abdominal ultrasound at the time of embryo transfer to guide replacement, improved pregnancy rates by at least 5%. METHODS: An RCT in a large assisted conception unit. A pilot study and power calculation suggested that at least 2000 embryo transfers were required to demonstrate a difference of 5%, for a test with 80% power and Type 1 error 0.05. Randomization, data entry and analysis were arranged independently. Randomization was stratified for age and fresh/frozen embryo transfer. Analysis was by intention to treat. RESULTS: There was no difference in clinical pregnancy or live birth rates between the two groups. The clinical pregnancy rate for ultrasound-guided embryo transfer was 22% and for non-ultrasoundguided embryo transfer was 23% (odds ratio: 0.96; 95% confidence interval: 0.79‐1.18). CONCLUSIONS: We set out to determine whether ultrasound-guided embryo transfer improved clinical pregnancy rates and live birth rates in assisted conception. We used an appropriately powered RCT design. We did not demonstrate a difference. This outcome is at odds with the UKs National Institute of Clinical Excellence recommendations for fertility treatment (Fertility Assessment and Treatment for People with Fertility Problems. London, UK: RCOG Press, 2004, 112.) which used a meta-analysis of four smaller trials (range 362‐800 patients, totalling 2051 embryo transfers) to conclude that ultrasound should be offered. We suggest that the current Cochrane review should be updated with data from our trial and recommend that consideration is given to accounting for heterogeneity between the included trials.

Incompetence of the internal urethral sphincter causes semen to pass into the bladder. Infertility can be successfully treated by restoring normal ejaculation or by sperm retrieval techniques.

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.

Objective To describe a potential new use of gonadotropin-releasing hormone (GnRH) antagonists. Design Case report. Setting Assisted conception unit at a teaching hospital in the United Kingdom. Patient(s) A 37-year-old woman undergoing in vitro fertilization (IVF) who accidentally stopped using GnRH agonists after starting ovarian stimulation. Intervention(s) A GnRH antagonist was used to avoid a luteinizing hormone (LH) surge and hence "rescue" the cycle. Result(s) Successful oocyte retrieval was carried out, two embryos transferred, and a viable twin pregnancy ensued. Conclusion(s) This may be a new indication for the use of GnRH antagonists.

end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.