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SummaryThe membrane-localized phosphatidylinositol (PI) 3-kinase (PI3K)/Akt pathway regulates cell growth and is aberrantly activated in cancer. Recent studies reveal a distinct nuclear PI3K/Akt pathway involving PI phosphate (PIP) kinases that bind the tumor suppressor protein p53 (wild-type and mutant) to generate nuclear p53-polyphosphoinositide (PIPn) complexes that activate Akt. In the membrane pathway, PI transfer proteins (PITPs) transport PI, the precursor of PIPns, to endomembranes to enable PIPnsynthesis. In contrast, nuclear PIPnsignaling relies on poorly characterized non-membranous PIPnpools. Here we show that PITPs accumulate in the non-membranous nucleoplasm in response to stress and are necessary to generate nuclear PIPnpools. Class I PITPα/β bind p53 to form p53-PIPncomplexes that activate nuclear Akt in response to stress, which inhibits apoptosis. These findings demonstrate an unexpected function for PITPα/β in nuclear PIPnsignaling by generating membrane-free, protein-linked PIPnpools that are modified by PIP kinases/phosphatases to regulate protein function.In briefPhosphatidylinositol transfer proteins initiate the nuclear protein-associated PIPnnetwork in membrane-free regions.

BackgroundPolycystic ovary syndrome (PCOS) is a heterogeneous disorder, affecting around 10% of women of reproductive age, with infertility, depression or anxiety, obesity, insulin resistance and type 2 diabetes as risk factors. The cause of PCOS is not known but there is a predisposition to developing PCOS in adult life that arises during fetal or perinatal life. PCOS also has a genetic predisposition and a number of genetic loci associated with PCOS have been identified. These loci contain 25 candidate genes which are currently being studied to define the syndrome. Although the name PCOS suggests a syndrome of the ovary, PCOS has also been associated with the central nervous system and other organ systems in the body due to the wide variety of symptoms it presents.MethodsHere, we examined the expression patterns of PCOS candidate genes in gonadal (ovary and testis), metabolic (heart, liver and kidney) and brain (brain and cerebellum) tissues during the first half of human fetal development and postnatally until adulthood using public RNA sequencing data. This study is an initial step for more comprehensive and translational studies to define PCOS.ResultsWe found that the genes were dynamically expressed in the fetal tissues studied. Some genes were significantly expressed in gonadal tissues, whilst others were expressed in metabolic or brain tissues at different time points prenatally and/or postnatally. HMGA2, FBN3 and TOX3 were highly expressed during the early stages of fetal development in all tissues but least during adulthood. Interestingly, correlation between expression of HMGA2/YAP1 and RAD50/YAP1 were significant in at least 5 of the 7 fetal tissues studied. Notably, DENND1A, THADA, MAPRE1, RAB5B, ARL14EP, KRR1, NEIL2 and RAD50 were dynamically expressed in all postnatal tissues studied.ConclusionsThese findings suggest that these genes have tissue- or development-specific roles in multiple organs, possibly resulting in the various symptoms associated with PCOS. Thus the fetal origin of a predisposition to PCOS in adulthood could arise via the effects of PCOS candidate genes in the development of multiple organs.

The tumor suppressor p53 and phosphoinositide 3-kinase (PI3K)-Akt pathway have fundamental roles in regulating cell growth and apoptosis, and they are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear Akt activation by a p53-dependent mechanism that is distinct from the canonical membrane-localized PI3K-Akt pathway. Upon genotoxic stress, a nuclear PI3K binds to p53 in the non-membranous nucleoplasm to generate a p53-PI3,4,5P(3) complex, which recruits Akt and PDK1 and mTORC2 that are required to activate Akt, and phosphorylates FOXOs, thereby inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear Akt in an on/off fashion upon stress, whereas mutant p53 dose dependently stimulates high basal Akt activity. The p53-PI3,4,5P(3) complex is dephosphorylated to p53-PI4,5P(2) by the phosphatase and tensin homolog (PTEN) to inhibit Akt activation. The nuclear p53-phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, underscoring its therapeutic relevance.

Cryolipolysis uses tissue cooling to solidify lipids, preferentially damaging lipid-rich cells. Topical cooling is popular for the reduction of local subcutaneous fat. Injection of biocompatible ice-slurry is a recently introduced alternative. We developed and verified a quantitative model that simulates the heat exchange and phase changes involved, offering insights into ice-slurry injection for treating subcutaneous fat.Finite element method was used to model the spatial and temporal progression of heat transfer between adipose tissue and injected ice-slurry, estimating dose-response relationships between properties of the slurry and size of tissue affected by cryolipolysis. Phase changes of both slurry and adipose tissue lipids were considered. An in vivo swine model was used to validate the numerical solutions. Oils with different lipid compositions were exposed to ice-slurry in vitro to evaluate the effects of lipid freezing temperature. Microscopy and nuclear magnetic resonance (NMR) were performed to detect lipid phase changes.A ball of granular ice was deposited at the injection site in subcutaneous fat. Total injected ice content determines both the effective cooling region of tissue, and the duration of tissue cooling. Water's high latent heat of fusion enables tissue cooling long after slurry injection. Slurry temperature affects the rate of tissue cooling. In swine, when 30 ml slurry injection at -3.5°C was compared to 15 ml slurry injection at -4.8°C (both with the same total ice content), the latter led to almost twice faster tissue cooling. NMR showed a large decrease in diffusion upon lipid crystallization; saturated lipids with higher freezing temperatures were more susceptible to solidification after ice-slurry injection.Total injected ice content determines both the volume of tissue treated by cryolipolysis and the cooling duration after slurry injection, while slurry temperature affects the cooling rate. Lipid saturation, which varies with diet and anatomic location, also has an important influence.

In 2004, the identification of female germline or oogonial stem cells (OSCs) that can support post–natal oogenesis in ovaries of adult mice sparked a major paradigm shift in reproductive biology. Although these findings have been independently verified, and further extended to include identification of OSCs in adult ovaries of many species ranging from pigs and cows to non–human primates and humans, a recent study rooted in single–cell RNA sequence analysis (scRNA-seq) of adult human ovarian cortical tissue claimed that OSCs do not exist, and that other groups working with OSCs following isolation by magnetic-assisted or fluorescence-activated cell sorting have mistaken perivascular cells (PVCs) for germ cells. Here we report that rare germ lineage cells with a gene expression profile matched to OSCs but distinct from that of other cells, including oocytes and PVCs, can be identified in adult human ovarian cortical tissue by scRNA-seq after optimization of analytical workflow parameters. Deeper cell-by-cell expression profiling also uncovered evidence of germ cells undergoing meiosis-I in adult human ovaries. Lastly, we show that, if not properly controlled for, PVCs can be inadvertently isolated during flow cytometry protocols designed to sort OSCs because of inherently high cellular autofluorescence. However, human PVCs and human germ cells segregate into distinct clusters following scRNA-seq due to non–overlapping gene expression profiles, which would preclude the mistaken identification and use of PVCs as OSCs during functional characterization studies.

Background Toxicity data are routinely collected in phase 3 (neo)adjuvant breast cancer (BC) clinical trials, but ovarian toxicity is infrequently assessed, despite its impact on fertility and long-term health. Thus, little is known about the ovarian effects of newer BC therapies. This results in an information gap for women when making treatment decisions. We explored the barriers to collecting ovarian toxicity data and/or including ovarian function as an endpoint in BC trials of anti-cancer drugs. Methods Semi structured interviews were conducted with key stakeholders involved in BC clinical trials (clinicians, consumers, pharmaceutical companies, drug regulatory advisors). Participants were asked detailed questions about how trial endpoints are selected, whether effects on the ovary are assessed, and barriers to and benefits of collecting ovarian function data to assess ovarian toxicity. Interviews were transcribed verbatim, coded in NVivo software and analysed using inductive thematic analysis. Results Saturation was reached after 25 interviews (9 clinicians, 7 consumers, 5 drug regulatory advisors, 4 pharmaceutical company advisors); half were female. Participants were from North America (20%), Europe (52%), Australia (24%), Asia (4%). Median age was 50 years and median time in breast cancer research or drug regulation was 16 years. The main reported barrier to the collection of ovarian toxicity data in clinical trials was that this issue was rarely considered. Reasons included that these data are considered less important than survival data and are not required for regulatory approval. Other barriers included limited resources, lack of knowledge regarding how to assess ovarian side effects and lack of relevance in certain settings (further detail in Table 1). Most participants believed assessing ovarian toxicity in trials would be beneficial to clinicians and to patients (eg. assisting treatment and family planning decisions). Strategies to increase ovarian toxicity assessment included its inclusion in clinical trial design guidelines, improving familiarity with ovarian function markers among trial design decision makers, and increased stakeholder interest. A stronger consumer voice and regulatory and clinician advocacy were regarded as important. Regarding trial endpoint selection, pharmaceutical companies were almost always identified as the main decision maker, but clinicians including cooperative trial group researchers, consumers, regulators, and statisticians were also important contributors. While most consumers and pharmaceutical company advisors felt clinicians and consumers influenced trial design, in contrast, some clinicians and regulators reported consumers and clinicians had little influence. Factors identified as important considerations in determining trial endpoints included the main goal of the trial (eg. regulatory approval), established standardised endpoints, resources, and the investigational agent studied. All pharmaceutical advisors reported that meeting the requirements for regulatory approval was the major factor considered during endpoint selection. Conclusion This qualitative analysis evaluates the barriers to including measures of ovarian function in BC clinical trials. Increased awareness, stronger advocacy and guidelines might lead to more frequent inclusion of this important endpoint in future trials that include premenopausal women with early BC. Table 1.Emergent themes regarding ovarian toxicity assessment in BC trialsThemeDomainCategory 1: Barriers to assessment of ovarian function Not prioritisedNot discussed or thought about;Not the primary question studied by clinical trial;Less important than other endpoints/data;Not required for regulatory approval;Data not related to survival is infrequently published;More appropriate for a follow up/registry studyToo resource intensive A burden on investigators and patients;Difficult to collect good quality data;Assessment not considered feasible;Time to obtaining results too long;Too costlyLack of knowledgeLack of clinician knowledge regarding ovarian function side effects;Lack of consensus regarding which markers to assess;Lack of preclinical data suggestive of ovarian toxicityData not relevant in certain settingsConcurrent use of gonadotoxic chemotherapy;Trials mandate contraception use;Want to suppress ovarian function in some breast cancer phenotypes;Low numbers of premenopausal women enrolled;Investigational agent already known to cause ovarian toxicity/suppressionCategory 2: Perceived benefits of assessing ovarian function Data important to patientsImproved ability to make informed cancer treatment decisions;Improved ability to make fertility and ovarian preservation decisions;Infertility and early menopause are relevant and important to patients;Preservation of ovarian function is important for quality of life;To avoid potential harm to patientsData important to cliniciansImproved ability to counsel patients;Improved understanding of the investigational agent;Improved understanding of the impact of ovarian function on disease outcomes;Holistic understanding of a patient’s experience on treatment;Prospective information is more robust than retrospective dataCategory 3: Strategies to improve inclusionIncreased stakeholder interestIncreased clinician advocacy;Increased consumer voice;Increased regulatory buy in;Increased cooperative trial group interest;Increased reproductive specialist involvement;Increased pharmaceutical company interestIncreased awareness regarding ovarian function and onco-fertilityTrial design guidelines and recommendations;Increased discussion and education;Improved familiarity with ovarian markers and ease of incorporation/collection;Use of social media and internet resources Citation Format: Wanyuan Cui, Kelly-Anne Phillips, Prudence A Francis, Sherene Loi, Richard A Anderson, Ann H Partridge, Louise A Keogh. What are the barriers to assessment of ovarian toxicity in breast cancer clinical trials? [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-19-03.

Background Currently there are no established fertility preservation options for pre-pubertal boys facing cancer treatment. Granulocyte-colony stimulating factor (G-CSF) treatment has been proposed to be chemoprotective against spermatogonial cell loss in an alkylating chemotherapy model of busulfan treated adult mice. Having previously shown that exposure to the alkylating-like chemotherapy cisplatin resulted in a reduction in germ cell numbers in immature human testicular tissues, we here investigate whether G-CSF would prevent cisplatin-induced germ cell loss in immature human and mouse (fetal and pre-pubertal) testicular tissues. Methods Organotypic in vitro culture systems were utilised to determine the effects of clinically-relevant concentrations of G-CSF in cisplatin-exposed immature testicular tissues. Human fetal (n = 14 fetuses) and mouse pre-pubertal (n = 4 litters) testicular tissue pieces were cultured and exposed to cisplatin or vehicle control for 24 hrs and analysed at 72 and 240 hrs post-exposure. Combined G-CSF and cisplatin exposure groups explored varying concentrations and duration of G-CSF supplementation to the culture medium (including groups receiving G-CSF before, during and after cisplatin exposure). In addition, effects of G-CSF supplementation alone were investigated. Survival of total germ cell and sub-populations were identified by expression of AP2γ and MAGE-A4 for human gonocytes and (pre)spermatogonia, respectively, and MVH and PLZF, for mouse germ cells and putative spermatogonial stem cells (SSCs) respectively, were quantified. Results Exposure to cisplatin resulted in a reduced germ cell number in human fetal and mouse pre-pubertal testicular tissues at 240 hrs post-exposure. Germ cell number was not preserved by combined exposure with G-CSF using any of the exposure regimens (prior to, during or after cisplatin exposure). Continuous supplementation with G-CSF alone for 14 days did not change the germ cell composition in either human or mouse immature testicular tissues. Conclusions This study demonstrates that exposure to G-CSF does not prevent cisplatin-induced germ cell loss in immature human and mouse testicular tissues in an in vitro system.

Esophageal intramural pseudodiverticulosis (EIP) is a rare condition characterized by outpouchings from the esophageal lumen into the outer esophageal wall. It can lead to complications such as esophageal strictures presenting as dysphagia and esophagobronchial fistula (EBF) presenting as recurrent aspiration pneumonia. Only 1 other report of EIP with EBF has been reported in the literature. EIP itself is typically managed medically while a complication such as EBF, on the other hand, elicits far more invasive approaches in the form of open surgical intervention. We present the case of a 50 year-old male newly diagnosed with EIP complicated by EBF who was palliated with esophageal stenting. Upper endoscopy with esophageal stent placement offers a safe, feasible, and non-operative therapy for EIP and EBF.