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Serine biosynthesis target in breast cancer An in vivo RNAi screen of metabolic enzymes and transporters is used to identify, among other genes, phosphoglycerate dehydrogenase (PHGDH) as a gene required for breast tumour growth. PHGDH resides in a region of chromosome 1p that is often amplified in breast cancers, leading to PHGDH overexpression. Elevated levels of PHGDH cause increased metabolic flux through the serine synthesis pathway, which in turn contributes significantly to the flux of glutamine to α-ketoglutarate through the tricarboxylic acid cycle. These observations suggest that targeting PHGDH or the serine biosynthesis pathway in general might be of therapeutic value in the subset of breast cancers with high PHGDH expression. Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation 1 , 2 . RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes 3 . Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase ( PHGDH ) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.

PurposeOur aim was to evaluate if maternal age at transfer following autologous oocyte cryopreservation is associated with live birth rate (LBR).MethodsWe performed a retrospective cohort study of all patients who thawed autologous oocytes and then underwent a single frozen euploid embryo transfer between 2011 and 2021 at a large urban university-affiliated fertility center. Each oocyte thaw patient was matched 2:1 to in vitro fertilization (IVF) patients who underwent single embryo transfer < 1 year after retrieval. Primary outcome was LBR. Secondary outcomes included implantation rates (IR) and spontaneous abortion rates (SABR).ResultsA total of 169 oocyte thaw patients were matched to 338 IVF patients. As expected, oocyte thaw patients were older (median age 42.5 vs. 37.6 years, p < 0.001) and waited longer between retrieval and transfer than in vitro fertilization patients (median time 59 vs. 1 month, p < 0.001). In univariate analysis, implantation and LBR differed among oocyte thaw and IVF patients (p < 0.05), but SABR did not (p = 0.57). Transfer outcomes in oocyte thaw patients did not differ based on transfer age group (IR: p = 0.18; SABR: p = 0.12; LBR: p = 0.24). In a multiple logistic regression model, age at transfer was not predictive of live birth when controlling for age at retrieval, embryo morphology, and day of blastulation.ConclusionsMaternal age at transfer after oocyte cryopreservation is not predictive of LBR; this suggests that “an aging womb” does not impair LBR after oocyte thaw and empowers patients to return for transfer when ready for childbearing.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

An in vivo imaging-based competitive transplant screen in zebrafish identifies epoxyeicosatrienoic acids as enhancers of haematopoietic stem and progenitor cell (HSPC) engraftment; these derivatives of arachidonic acid also promote zebrafish developmental HSPC specification through a PI(3)K-dependent AP-1 and runx1 transcriptional program and their pro-engraftment effect is conserved in mammals (indicating clinical potential). Enhancement of stem cell engraftment in a zebrafish model Haematopoietic stem and progenitor cell (HSPC) transplantation — with infusions of either bone marrow or peripheral blood progenitor cells — is used clinically to treat certain cancers and diseases of the blood and immune system, but we still understand very little about how HSPCs engraft to the host. Leonard Zon and colleagues have developed a competitive marrow transplantation system in adult zebrafish in which engraftment is measured by in vivo fluorescence imaging of the kidney — the adult haematopoietic site. Using this model to screen for engraftment enhancing activity, the authors identify epoxyeicosatrienoic acids, including 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, as agents able to enhance engraftment and HSPC specification through the activation of a transcription factor Runx1-mediated expression program. This activity of EET is conserved in mice, indicating possible clinical potential for EET to promote bone marrow transplants. Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids 1 , 2 that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation (1,2). RNAi-based loss of function screening has proven powerful for the identification of novel and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumor suppressor genes (3). Here, we developed a method for identifying novel cancer targets via negative selection RNAi screening in solid tumours. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumourigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of ER-negative breast cancers. PHGDH catalyzes the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have elevations in serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a TCA cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH over-expression and demonstrate the utility of in vivo negative selection RNAi screens for finding potential anticancer targets.

Little is known about ageing with autism spectrum disorder (ASD). We examined the characteristics of adults referred to a specialist diagnostic centre for assessment of possible ASD, 100 of whom received an ASD diagnosis and 46 did not. Few demographic differences were noted between the groups. Comorbid psychiatric disorders were high in individuals with ASD (58 %) and non-ASD (59 %). Individuals who received an ASD diagnosis had higher self-rated severity of ASD traits than non-ASD individuals. Within the ASD group, older age was associated with higher ratings of ASD traits and better cognitive performance. One interpretation is that general cognitive ability and the development of coping strategies across the lifespan, do not necessarily reduce ASD traits but may mitigate their effects.

A search for variants in coding exons of 25 genome-wide association study risk genes in a large cohort of autoimmune patients finds that rare coding-region variants at known loci have a negligible role in common autoimmune disease susceptibility, arguing against the previously proposed rare-variant synthetic genome-wide association hypothesis. In search of autoimmune disease heritability Although many common variants of modest-effect size have been identified in genome-wide association studies (GWAS), much of the heritability of complex traits remains unexplained. These authors looked for variants in coding exons of 25 GWAS risk genes in a large cohort of subjects with six autoimmune disease phenotypes and controls, and show that rare coding-region variants at known loci have at most a minor role in common autoimmune disease susceptibility. These results do not support the theory that the missing heritability for common autoimmune diseases is attributable to rare coding mutations at known loci, but are consistent with disease caused by many common-variant loci of weak effect. Genome-wide association studies (GWAS) have identified common variants of modest-effect size at hundreds of loci for common autoimmune diseases; however, a substantial fraction of heritability remains unexplained, to which rare variants may contribute 1 , 2 . To discover rare variants and test them for association with a phenotype, most studies re-sequence a small initial sample size and then genotype the discovered variants in a larger sample set 3 , 4 , 5 . This approach fails to analyse a large fraction of the rare variants present in the entire sample set. Here we perform simultaneous amplicon-sequencing-based variant discovery and genotyping for coding exons of 25 GWAS risk genes in 41,911 UK residents of white European origin, comprising 24,892 subjects with six autoimmune disease phenotypes and 17,019 controls, and show that rare coding-region variants at known loci have a negligible role in common autoimmune disease susceptibility. These results do not support the rare-variant synthetic genome-wide-association hypothesis 6 (in which unobserved rare causal variants lead to association detected at common tag variants). Many known autoimmune disease risk loci contain multiple, independently associated, common and low-frequency variants, and so genes at these loci are a priori stronger candidates for harbouring rare coding-region variants than other genes. Our data indicate that the missing heritability for common autoimmune diseases may not be attributable to the rare coding-region variant portion of the allelic spectrum, but perhaps, as others have proposed, may be a result of many common-variant loci of weak effect 7 , 8 , 9 , 10 .

PurposeOur aim was to describe the reproductive decisions and outcomes of BRCA-positive patients who used preimplantation genetic testing for monogenic disorders (PGT-M).MethodsWe performed a retrospective case series of all PGT-M cycles for BRCA variants between 2010–2021 at a large urban academic fertility center. All patients who underwent ≥ 1 cycle of IVF with PGT-M for BRCA1 or BRCA2 were included. The primary outcome was total number of BRCA-negative euploid embryos per patient.ResultsSixty four patients underwent PGT-M for BRCA variants. Forty-five percent (29/64) were BRCA1-positive females, 27% (17/64) were BRCA2-positive females, 16% (10/64) were BRCA1-positive males, 11% (7/64) were BRCA2-positive males, and one was a BRCA1 and BRCA2-positive male. There were 125 retrieval cycles with PGT-M, and all cycles included PGT for aneuploidy (PGT-A). Eighty-six percent (55/64) of patients obtained at least one BRCA- negative euploid embryo, with median of 1 (range 0–10) BRCA-negative euploid embryo resulted per cycle and median 3 (range 0–10) BRCA-negative euploid embryos accumulated per patient after a median of 2 (range 1–7) oocyte retrievals. Sixty-four percent (41/64) of patients attempted at least one frozen embryo transfer (FET) with a total of 68 FET cycles. Fifty-nine percent (40/68) of embryos transferred resulted in live births. Subgroup analysis revealed different reproductive pathways for BRCA1-positive females, BRCA2-positive females, and BRCA1/2-positive males (p < 0.05).ConclusionPGT-M is a viable option for BRCA-positive patients to avoid transmission while building their families. Most patients in our cohort achieved pregnancy with BRCA-negative euploid embryos.

BackgroundTraining procedural skills using proficiency-based progression (PBP) methodology has consistently resulted in error reduction. We hypothesised that implementation of metric-based PBP training and a valid assessment tool would decrease the failure rate of epidural analgesia during labour when compared to standard simulation-based training.MethodsDetailed, procedure-specific metrics for labour epidural catheter placement were developed based on carefully elicited expert input. Proficiency was defined using criteria derived from clinical performance of experienced practitioners. A PBP curriculum was developed to train medical personnel on these specific metrics and to eliminate errors in a simulation environment.Seventeen novice anaesthetic trainees were randomly allocated to undergo PBP training (Group P) or simulation only training (Group S). Following training, data from the first 10 labour epidurals performed by each participant were recorded. The primary outcome measure was epidural failure rate.ResultsA total of 74 metrics were developed and validated. The inter-rater reliability (IRR) of the derived assessment tool was 0.88. Of 17 trainees recruited, eight were randomly allocated to group S and six to group P (three trainees did not complete the study). Data from 140 clinical procedures were collected. The incidence of epidural failure was reduced by 54% with PBP training (28.7% in Group S vs 13.3% in Group P, absolute risk reduction 15.4% with 95% CI 2% to 28.8%, p=0.04).ConclusionProcedure-specific metrics developed for labour epidural catheter placement discriminated the performance of experts and novices with an IRR of 0.88. Proficiency-based progression training resulted in a lower incidence of epidural failure compared to simulation only training.Trial registration number NCT02179879. NCT02185079; Post-results.