Sex bias of females in survival from cancer and infections. Is X the answer?

Major differences in survival of men and women from infectious diseases and cancers have been highlighted by death rates from COVID-19 infections. In cancer, attention has been focussed on differences in gene expression from X chromosomes in men and women with a preponderance of genes involved in immune responses being expressed in women. Important findings have been that some of the genes are important epigenetic regulators that play fundamental roles in immune responses.Subject terms: Cancer epigenetics, Oncology

One of the striking features of the coronavirus disease 2019 (COVID-19) outbreak has been the higher death rates in men even though the infection rates seem similar between men and women.¹ Similar findings were reported from Wuhan where men had 2.4 times the death rate of women² and in New York where press releases stated twice the death rate of men compared with women.³ Although men had higher rates of comorbidities, these differences were not considered sufficient to explain the higher death rates and other explanations have been sort. Women are considered to have stronger immune responses against infective diseases and a higher rate of autoimmune diseases, so this has questioned whether the lower death rate may have an immune basis.A sex bias is not only seen in infections, but also in cancers where a strong sex bias in survival from cancer is well documented.^4,5 For example, women in Australia have approximately half the death rates from melanoma as males.⁶ A number of explanations have been proposed to account for these major differences in melanoma, such as higher sun exposure in males⁷ and higher mutation rates⁸ in melanoma from males. When stringent statistical analyses are carried out, however, female sex remains as the major contributor to longer survival.⁴Melanoma is not the only cancer to show improved survival in females and previous researchers have asked whether this may be due to differences in the sex chromosomes between male and females. In a mammoth study, Dunford and colleagues examined information in The Cancer Genome Atlas (TCGA) from 21 different tumour types from 4100 cancers.⁵ They found that 6 out of 783 X chromosome genes had loss-of-function mutations with tumour-suppressive function in males but not in females. There were no similar differences in 18,055 non-X autosomal genes. Importantly, four of the six genes were known epigenetic regulators, such as KDM6A (lysine-specific demethylase 6A), KDM5C (lysine-specific demethylase 5C), ATRX (Alpha thalassaemia/mental retardation syndrome X-linked) and DDX3X (DEAD-box helicase 3 X-linked).⁵These findings point to important differences in X chromosomes between the sexes. The Y chromosome codes mainly for genes that determine male sex, but X chromosomes are quite large and code for >800 genes many of which are involved in immune responses.⁹ To equalise the number of genes between the sexes, one of the X chromosomes in females undergoes inactivation (Xi) of its genes.¹⁰ The silencing process is, however, not perfect and between 10 and 20% of the genes on the X may be expressed in females depending on the tissue involved. It is probably of significance that failure to silence genes may be particularly high in activated lymphocytes.¹¹ As a result of this phenomenon, females have double expression of many genes involved in immune responses compared with males. Biologists have speculated that this is an evolutionary mechanism to protect the species by enhancing immune responses in females against harmful infections.Analysis of data in the TCGA on 458 melanoma patients revealed that KDM6A expression was strongly related to improved survival from melanoma in female patients. ATRX had prognostic significance in both sexes. Analysis of another series of 678 patients with earlier melanoma referred to as the Leeds Melanoma Cohort confirmed the association with KDM6A expression and also identified KDM5C and DDX3X as being related to improved survival.¹² Immune responses are known to be critical in survival from melanoma and the TCGA analysis allowed us to link high KDM6A to components of the immune system considered important in killing of melanoma. This was particularly so in the production of interferon γ in female patients which is a key cytokine needed by the immune system to kill cancer cells. Gene set analysis also showed downregulation of Myc and other oncogenic pathways that may have contributed to the improvement in survival.¹²These data add to a number of studies implicating KDM6A in immune responses against viral infections and in autoimmune diseases.¹³ At a molecular level, KDM6A is known to have an opposing role to EZH2 (enhancer of Zeste homologue 2) in the PRC2 complex in methylation of Lys 27 on H3 histone. This role may explain some of the effects of KDM6A on the immune system in that we previously reported that EZH2 was associated with the repression of several genes associated with antigen presentation and chemokines involved in T cell responses.¹⁴Although these studies are compelling in linking KDM6A to immune responses, it is still questionable whether it has a role in immune responses against COVID-19. If this was the case, we would expect that women being treated for severe COVID-19 infections in intensive care would have lower KDM6A expression than those with infections not requiring such care.¹⁵ We examined the RNA-seq data from blood samples of 102 COVID-19 patients. This included 38 women and 64 men, where 17 women and 34 men were admitted to intensive care unit. The analysis of KDM6A levels in the women showed that treatment in intensive care unit was associated with higher KDM6A expression ({"type":"entrez-geo","attrs":{"text":"GSE157103","term_id":"157103"}}GSE157103,¹⁵ data not shown). Although this was unexpected, it may indicate that KDM6A expression was linked to stronger responses causing higher inflammation in organs such as the lungs. No differences in KDM6A levels were detected in men irrespective of whether they were admitted to intensive care or not.Female patients with bi-allelic expression of KDM6A may induce the expression of interferon γ pathways which enhance anti-tumour immunity by recruiting immune modulatory cells (Fig. 1). These results point to the need for a better understanding of the role of X-linked genes in immune responses and whether EZH2-mediated suppression of immune modulatory genes have a role in infections as well as in cancer. In cancers and infections that have worst outcomes in males versus females, one approach might be to target (inhibit) the EZH2 epigenetic regulator that opposes KDM6A (Fig. 1). Another option may be to increase levels of KDM6A by administration of oestrogens. Oestrogen α receptors are expressed in practically all lymphocytes and were shown to physically interact with KDM6A to create a permissive chromatin state on endoplasmic reticulum (ER) targets such as C-X-C chemokine motif receptor 4.¹⁶ It was transactivated by ER to form a feed-forward loop. Administration of 17β-oestradiol has been suggested by others as treatment for COVID-19 infections.¹⁷Open in a separate windowFig. 1Proposed model of sex-biased role of the X-linked KDM6A gene in promoting immunity.Males harbour one X chromosome with no functional Y chromosome homologue. Hence, mutation in the X-linked epigenetic modifier KDM6A with tumour-suppressive or immunomodulatory role will probably lead to cancer or infection in males. Immune-related genes will be repressed by EZH2-mediated H3K27me3 deposition resulting in low KDM6A protein and immune evasion in male patients. In females, with two X chromosome, [one active (Xa) and one inactive (Xi)], a single mutation (m) in KDM6A is less likely to develop cancer or infections since another functional allele escapes X inactivation. Cells with high KDM6A level would be expected to demethylate H3K27me3 resulting in activation of the interferon γ pathway resulting in inactivation of natural killer (NK), dendritic or cytotoxic T cells to induce anti-tumour immunity and adaptive immunity against virus-infected cells.These studies have therefore raised many questions that require more detailed study to identify how the powerful survival benefits of the X-linked epigenetic regulators might be used to improve the therapeutic outcome in patients.     

Effects of cancer screening restart strategies after COVID-19 disruption

Background Many breast, cervical, and colorectal cancer screening programmes were disrupted due to the COVID-19 pandemic. This study aimed to estimate the effects of five restart strategies after the disruption on required screening capacity and cancer burden.Methods Microsimulation models simulated five restart strategies for breast, cervical, and colorectal cancer screening. The models estimated required screening capacity, cancer incidence, and cancer-specific mortality after a disruption of 6 months. The restart strategies varied in whether screens were caught up or not and, if so, immediately or delayed, and whether the upper age limit was increased.Results The disruption in screening programmes without catch-up of missed screens led to an increase of 2.0, 0.3, and 2.5 cancer deaths per 100 000 individuals in 10 years in breast, cervical, and colorectal cancer, respectively. Immediately catching-up missed screens minimised the impact of the disruption but required a surge in screening capacity. Delaying screening, but still offering all screening rounds gave the best balance between required capacity, incidence, and mortality.Conclusions Strategies with the smallest loss in health effects were also the most burdensome for the screening organisations. Which strategy is preferred depends on the organisation and available capacity in a country.Subject terms: Health policy, Population screening, Cancer screening, Cancer screening     

Tractography of the Cerebellar Peduncles in Second- and Third-Trimester Fetuses

BACKGROUND AND PURPOSE:Little is known about microstructural development of cerebellar white matter in vivo. This study aimed to investigate developmental changes of the cerebellar peduncles in second- and third-trimester healthy fetuses using motion-corrected DTI and tractography.MATERIALS AND METHODS:3T data of 81 healthy fetuses were reviewed. Structural imaging consisted of multiplanar T2-single-shot sequences; DTI consisted of a series of 12-direction diffusion. A robust motion-tracked section-to-volume registration algorithm reconstructed images. ROI-based deterministic tractography was performed using anatomic landmarks described in postnatal tractography. Asymmetry was evaluated qualitatively with a perceived difference of >25% between sides. Linear regression evaluated gestational age as a predictor of tract volume, ADC, and fractional anisotropy.RESULTS:Twenty-four cases were excluded due to low-quality reconstructions. Fifty-eight fetuses with a median gestational age of 30.6 weeks (interquartile range, 7 weeks) were analyzed. The superior cerebellar peduncle was identified in 39 subjects (69%), and it was symmetric in 15 (38%). The middle cerebellar peduncle was identified in all subjects and appeared symmetric; in 13 subjects (22%), two distinct subcomponents were identified. The inferior cerebellar peduncle was not found in any subject. There was a significant increase in volume for the superior cerebellar peduncle and middle cerebellar peduncle (both, P < .05), an increase in fractional anisotropy (both, P < .001), and a decrease in ADC (both, P < .001) with gestational age. The middle cerebellar peduncle had higher volume (P < .001) and fractional anisotropy (P = .002) and lower ADC (P < .001) than the superior cerebellar peduncle after controlling for gestational age.CONCLUSIONS:A robust motion-tracked section-to-volume registration algorithm enabled deterministic tractography of the superior cerebellar peduncle and middle cerebellar peduncle in vivo and allowed characterization of developmental changes.

In the second half of pregnancy, the cerebellum is growing rapidly and is extremely vulnerable.¹ Despite the increasingly recognized association of antenatal and perinatal cerebellar injury with adverse motor and neurologic outcomes later in life,^2-5 little is known about normal cerebellar developmental in the later part of gestation, in particular with regard to changes in microstructure. In fact, most existing fetal MR imaging data addresses primarily changes in cerebellar volume with gestational age (GA) or changes in volume and their association with specific diseases such as congenital heart disease.^6-8In vivo evaluation of cerebellar microstructure using fetal MR imaging has been limited by the technical challenges related to imaging the gravid abdomen, particularly patient motion. However, data from ex vivo MR imaging studies are promising. For instance, Takahashi et al^9,10 performed high-resolution ex vivo DTI of fetal specimens and demonstrated the feasibility of using tractography to outline the cerebellar peduncles prenatally. Even though tractography of the cerebellar peduncles has been sporadically reported in vivo in technical articles or general review articles on fetal DTI,¹¹ the GA-related microstructural changes that occur in the cerebellar peduncles in the second half of pregnancy remain largely unexplored.Recent advances in hardware and software have improved fetal MR imaging substantially. The use of 3T magnets, which have been shown to be safe, results in improvement of the SNR and spatial resolution, which is advantageous to image the small structures of the fetal brain.^12,13 In addition, postprocessing algorithms that enable reconstruction of motion-corrected fetal DTI data are increasingly available and have been used by several groups to characterize the development of the supratentorial white matter tracts in vivo.^14-16 We hypothesize that fetal DTI performed at 3T and processed with a robust section-to-volume motion-correction and registration¹⁴ algorithm will enable tractography of the cerebellar peduncles in fetuses in the second and third trimesters of pregnancy. We aimed to characterize fetal cerebellar tract microstructure and to investigate tract-specific developmental changes.     

Rapid behavioral assessment of barriers and opportunities to improve vaccination coverage among displaced Rohingyas in Bangladesh,January 2018

Background

In November 2017, the World Health Organization received initial reports of suspected diphtheria cases in camps established for displaced Rohingyas in Cox’s Bazar district, Bangladesh. By January 11, 2018, over 4,000 suspected cases of diphtheria and 30 deaths were reported. The Bangladesh government and partners implemented a diphtheria vaccination campaign in December 2017. Outbreak response staff reported anecdotal evidence of vaccine hesitancy. Our assessment aimed to understand vaccination barriers and opportunities to enhance vaccine demand among displaced Rohingyas in Bangladesh.

Prolonged mechanical ventilation after cardiac surgery: substudy of the Transfusion Requirements in Cardiac Surgery III trial

Purpose

Prolonged mechanical ventilation (MV) is a major complication following cardiac surgery. We conducted a secondary analysis of the Transfusion Requirements in Cardiac Surgery (TRICTS) III trial to describe MV duration, identify factors associated with prolonged MV, and examine associations of prolonged MV with mortality and complications.

Methods

Four thousand, eight hundred and nine participants undergoing cardiac surgery at 71 hospitals worldwide were included. Prolonged MV was defined based on the Society of Thoracic Surgeons definition as MV lasting 24 hr or longer. Adjusted associations of patient and surgical factors with prolonged MV were examined using multivariable logistic regression. Associations of prolonged MV with complications were assessed using odds ratios, and adjusted associations between prolonged MV and mortality were evaluated using multinomial regression. Associations of shorter durations of MV with survival and complications were explored.

Results

Prolonged MV occurred in 15% (725/4,809) of participants. Prolonged MV was associated with surgical factors indicative of complexity, such as previous cardiac surgery, cardiopulmonary bypass duration, and separation attempts; and patient factors such as critical preoperative state, left ventricular impairment, renal failure, and pulmonary hypertension. Prolonged MV was associated with perioperative but not long-term complications. After risk adjustment, prolonged MV was associated with perioperative mortality; its association with long-term mortality among survivors was weaker. Shorter durations of MV were not associated with increased risk of mortality or complications.

Conclusion

In this substudy of the TRICS III trial, prolonged MV was common after cardiac surgery and was associated with patient and surgical risk factors. Although prolonged MV showed strong associations with perioperative complications and mortality, it was not associated with long-term complications and had weaker association with long-term mortality among survivors.

Study registration

www.ClinicalTrials.gov (NCT02042898); registered 23 January 2014. This is a substudy of the Transfusion Requirements in Cardiac Surgery (TRICS) III trial.

     

Opioid use disorder and the COVID 19 pandemic: A call to sustain regulatory easements and further expand access to treatment

Abstract

We highlight the critical roles that pharmacists have related to sustaining and advancing the changes being made in the face of the current COVID-19 pandemic to ensure that patients have more seamless and less complex access to treatment. Discussed herein is how the current COVID-19 pandemic is impacting persons with substance use disorders, barriers that persist, and the opportunities that arise as regulations around treatments for this population are eased.     

Patterns of Use of Induction Therapy for T2N0 Esophageal Cancer

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A Diagnostic Algorithm for Posterior Fossa Tumors in Children: A Validation Study

BACKGROUND AND PURPOSE:Primary posterior fossa tumors comprise a large group of neoplasias with variable aggressiveness and short and long-term outcomes. This study aimed to validate the clinical usefulness of a radiologic decision flow chart based on previously published neuroradiologic knowledge for the diagnosis of posterior fossa tumors in children.MATERIALS AND METHODS:A retrospective study was conducted (from January 2013 to October 2019) at 2 pediatric referral centers, Children''s Hospital of Philadelphia, United States, and Great Ormond Street Hospital, United Kingdom. Inclusion criteria were younger than 18 years of age and histologically and molecularly confirmed posterior fossa tumors. Subjects with no available preoperative MR imaging and tumors located primarily in the brain stem were excluded. Imaging characteristics of the tumors were evaluated following a predesigned, step-by-step flow chart. Agreement between readers was tested with the Cohen κ, and each diagnosis was analyzed for accuracy.RESULTS:A total of 148 cases were included, with a median age of 3.4 years (interquartile range, 2.1–6.1 years), and a male/female ratio of 1.24. The predesigned flow chart facilitated identification of pilocytic astrocytoma, ependymoma, and medulloblastoma sonic hedgehog tumors with high sensitivity and specificity. On the basis of the results, the flow chart was adjusted so that it would also be able to better discriminate atypical teratoid/rhabdoid tumors and medulloblastoma groups 3 or 4 (sensitivity = 75%–79%; specificity = 92%–99%). Moreover, our adjusted flow chart was useful in ruling out ependymoma, pilocytic astrocytomas, and medulloblastoma sonic hedgehog tumors.CONCLUSIONS:The modified flow chart offers a structured tool to aid in the adjunct diagnosis of pediatric posterior fossa tumors. Our results also establish a useful starting point for prospective clinical studies and for the development of automated algorithms, which may provide precise and adequate diagnostic tools for these tumors in clinical practice.

In the past 10 years, there has been an exponential increase in knowledge of the molecular characteristics of pediatric brain tumors, which was only partially incorporated in the 2016 World Health Organization Classification of Tumors of the Central Nervous System.¹ The main update in the 2016 Classification was the introduction of the molecular profile of a tumor as an important factor for predicting different biologic behaviors of entities which, on histology, look very similar or even indistinguishable.² A typical example is the 4 main groups of medulloblastoma: wingless (WNT), sonic hedgehog (SHH) with or without the p53 mutation, group 3, and group 4. Although they may appear similar on microscopy, these categories have distinct molecular profiles, epidemiology, prognosis, and embryologic origin.³Subsequent to the publication of the 2016 World Health Organization Classification, further studies have identified even more molecular subgroups of medulloblastoma with possible prognostic implications⁴ and also at least 3 new molecular subgroups of atypical teratoid/rhabdoid tumor (AT/RT)⁵ and several subgroups of ependymoma.⁶ MR imaging shows promise as a technique for differentiating histologic tumors and their molecular subgroups. This capability relies on not only various imaging characteristics but also the location and spatial extension of the tumor, evident on MR imaging, which can be traced to the embryologic origin of the neoplastic cells.^5,7-10One approach to the challenge of identifying imaging characteristics of different tumors in children is to use artificial intelligence. Yet despite this exciting innovation, correctly identifying the location of the mass and its possible use as an element for differential diagnosis still requires the expertise of an experienced radiologist. Previously, D''Arco et al¹¹ proposed a flow chart (Fig 1) for the differential diagnosis of posterior fossa tumors in children based on epidemiologic, imaging signal, and location characteristics of the neoplasm. The aims of the current study were the following: 1) to validate, in a retrospective, large cohort of posterior fossa tumors from 2 separate pediatric tertiary centers, the diagnostic accuracy of that flow chart, which visually represents the neuroadiologist''s mental process in making a diagnosis of posterior fossa tumors in children, 2) to describe particular types of posterior fossa lesions that are not correctly diagnosed by the initial flow chart, and 3) to provide an improved, clinically accessible flow chart based on the results.Open in a separate windowFIG 1.Predesigned radiologic flow chart created according to the literature before diagnostic accuracy analysis. The asterisk indicates brain stem tumors excluded from the analysis. Double asterisks indicate relative to gray matter. Modified with permission from D''Arco et al.¹¹     

How Postdoctoral Research in Paul Greengard's Laboratory Shaped My Scientific Career,Although I Never Did Another Phosphorylation Assay

In this short review, I describe from personal experience how every step in the career of any scientist, no matter how disjointed and pragmatic each might seem at the time, will almost inevitably meld together, to help us all tackle novel projects. My postdoctoral research in Paul Greengard''s laboratory, where I investigated neurotransmitter-mediated phosphorylation of Synapsin I, was instrumental in my career progression, and Paul''s support was instrumental in my ability to make a leap into independent research.

When the Society for Neuroscience was soliciting articles for its new journal, I was a postdoctoral fellow in Paul Greengard''s laboratory, completing studies of neurotransmitter regulation of synapsin I phosphorylation. When Paul informed me that he been invited to contribute a paper, we knew it would be risky thing to volunteer one''s precious data because this was an untested journal. But it came at a good time because I was about to leave the laboratory and had data that complemented my recently accepted paper demonstrating phosphorylation of the presynaptic protein Synapsin I (which we called Protein I at the time) by a neurotransmitter (Dolphin and Greengard, 1981b). So I was very happy to publish in the initial volume of the Journal of Neuroscience, which was to be the first of my 17 papers so far in the journal. The data published in 1981 extended my work on serotonin-mediated phosphorylation and also demonstrated phosphorylation by another class of neuromodulator, acting at adenosine receptors (Dolphin and Greengard, 1981a). It was such a pleasure for me to reread that paper, as it brought back many memories of life in the Greengard laboratory, together with colleagues and friends, including Pietro De Camilli, Angus Nairn, Wieland Huttner, Clive Palfrey, Eric Nestler, Suzanne Lohmann, Ulrich Walter, Howard Schulman, Mary Kennedy, and many others.My interest in intracellular signaling pathways activated by neurotransmitters began when I was an undergraduate student in Biochemistry; I wrote a dissertation on cyclic AMP in prokaryotes and eukaryotes, and then conducted an undergraduate research project examining glycine as a neurotransmitter, using spinal cord synaptosomes as an experimental preparation. This interest propelled me toward Parkinson''s disease research as a graduate student in the laboratory of the neurologist David Marsden at the Institute of Psychiatry in London, where I examined the importance of dopaminergic and noradrenergic pathways in animal models of Parkinson''s disease. I then wrote to Paul Greengard, asking to do postdoctoral research under his mentorship, because I knew I needed more basic training in cell signaling pathways, since my PhD environment had been very focused on translational and clinical research. I could not think of anywhere better than Paul''s laboratory because of his seminal work on cyclic nucleotide signaling pathways (Beam and Greengard, 1976). He said his laboratory was full, but agreed to take me in a year''s time, subject to interview. Fortunately, I had also obtained a UK Medical Research Council French exchange fellowship to work in the laboratory of Joel Bockaert, at College de France in Paris, where I studied the coupling between β-adrenergic receptors and adenylate cyclase. This valuable experience cemented my desire to continue research in this area and work with Paul Greengard. Thus it was that Paul, on a visit to Paris, invited me to be interviewed in his hotel room. I was on my guard when I knocked on the door, and a voice called out to come in. As I entered, I could see nothing but a large bed, but I soon located Paul lying flat on the floor beyond; he explained it was because of his chronic back problem. I perched on the bed, and tried to answer his searching scientific questions. I later realized that Paul lay on the floor at every opportunity, often in our laboratory meetings and indeed during other interviews (Nestler, 2019).When I arrived in Paul''s laboratory then at Yale, in the summer of 1978, the protein Synapsin I had recently been identified as a major presynaptic phosphoprotein (Ueda and Greengard, 1977). It was then called “Protein 1” because it stood out as the main polypeptide in synaptosomes that was phosphorylated in both a cAMP- and a Ca²⁺-dependent manner (Sieghart et al., 1979). However, no neurotransmitters had yet been found to influence the phosphorylation of Synapsin I, and to identify such a pathway seemed to be the holy grail at the time. Paul''s world view of multiple signaling cascades terminating in the phosphorylation of many different target proteins, that would underlie long-term synaptic events, was often at odds with the then prevailing view of ion channel biologists, that neurotransmission was mediated by fast neurotransmitters acting on ion channels, a process in which there was no need to consider neurochemical mechanisms. Eric Kandel (from an electrophysiological standpoint), and Paul, from the basis of biochemical pathways, were both key to the acceptance of the view that a multitude of biochemical pathways must play a role in longer term events. Indeed, Paul was collaborating with Eric Kandel''s laboratory at that time to elucidate whether a phosphorylation cascade mediated the effects of serotonin (5-HT) in Aplysia, and many of us in the laboratory were very influenced by these elegant studies, combining, as they did, electrophysiology and biochemical approaches (Castellucci et al., 1980, 1982).Paul suggested that my project should be to examine whether neurotransmitters could change the state of phosphorylation of Synapsin I in the brain, but he left it entirely to me what tissue I should use for my study. There were ∼25 postdocs and students in his laboratory at the time, and so I had a lot of help and advice, although Paul was the opposite of a micro-manager. Influenced both by the work of Eric Kandel and that of my previous mentor Joel Bockaert (Enjalbert et al., 1978), I decided that serotonin would be a good neurotransmitter to study. Taking inspiration from the work of George Aghajanian, also a professor in the Pharmacology Department at Yale, whose studies had revealed the effects of serotonin on many types of neuron (Aghajanian et al., 1990), I chose to examine the tiny facial nucleus in the brainstem, since Aghajanian had extensively examined the electrophysiological properties of this nucleus, as well as its ultrastructure (Aghajanian and McCall, 1980). His work indicated the facial nucleus had no interneurons, and therefore I thought it might lead to a more homogeneous phosphorylation response than many other brain areas. Indeed, serotonin did result in an increase in phosphorylation of Synapsin I in this preparation, but I initially found that the effects were small and very variable (Fig. 1, left). This was disappointing, and I remember blaming myself for being a cack-handed experimenter.Open in a separate windowFigure 1.Phosphorylation of Synapsin I by actions at serotonin and adenosine receptors. Stimulation of Synapsin I (then called Protein I) phosphorylation with serotonin in the presence and absence of IBMX (left) (from Dolphin and Greengard, 1981b, our Fig. 2) or 2-choloroadenosine (right) (from Dolphin and Greengard, 1981a, our Fig. 4). The “back phosphorylation” method was used, in which ³²P-ATP together with catalytic subunit of protein kinase A were used to phosphorylate residual nonphosphorylated sites on Synapsin I.A breakthrough came indirectly through family tragedy; my father died in 1979, and I returned to England for the funeral. Fortuitously, I also decided to investigate potential future positions in the UK, and visited Leslie Iversen''s Medical Research Council unit in Cambridge. While there, I showed my underwhelming phosphorylation data to John Dowling, who was on sabbatical in the unit at the time, working on dopamine-sensitive adenylate cyclase activity in the retina (Watling et al., 1979). I asked his advice and remember clearly that he suggested adding a low concentration of a phosphodiesterase inhibitor to the slices to potentiate the cAMP response to serotonin. On my return, I duly added isobutyl-methyl-xanthine (IBMX) to the facial nucleus slices, and the responses became much more robust (Dolphin and Greengard, 1981b) (Fig. 1, left). Similarly, Eric Nestler also found that dopamine and depolarization increased the phosphorylation of Synapsin I in superior cervical ganglion neurons (Nestler and Greengard, 1980).Something I could not understand in my data was that the serotonin-mediated phosphorylation of Synapsin I was Ca²⁺-dependent, whereas the phosphorylation mediated by stimulation of an adenosine receptor was not. This was the main topic of my follow-up paper in the first volume of Journal of Neuroscience (Dolphin and Greengard, 1981a) (Fig. 1, right). Everyone in the laboratory remembers that writing papers with Paul was exhausting, as we considered multiple versions of every sentence, and these were written and rewritten in pencil with many erasures. But I can still hear his voice in every phrase of this Journal of Neuroscience paper, and it remains a pleasure to reread each well-considered argument, for which I take absolutely no credit.In the Discussion, many possibilities are suggested to explain the surprising Ca²⁺ dependence of the serotonin-stimulated phosphorylation, and these points remain relevant, even now. For example, we point out that, in Aplysia neurons, serotonin stimulated a presynaptic adenylate cyclase, and this increased the influx of Ca²⁺ (Shimahara and Tauc, 1977; Klein and Kandel, 1978). We suggested that both processes might contribute to presynaptic Synapsin I phosphorylation. I also wonder now if we were seeing a combined effect of 5HT3 receptor activation, allowing Ca²⁺ entry, and subsequent Ca²⁺-calmodulin (CaM) kinase-dependent phosphorylation of Synapsin I, as well as activation of presynaptic serotonergic GPCRs that directly stimulated adenylate cyclase. Of course, the existence of 5HT3 receptors, which are Ca²⁺-permeable ion channels, and the many subtypes of G-protein-coupled 5-HT receptors was unknown at the time (Raymond et al., 2001).My study of neurotransmitter-dependent phosphorylation of Synapsin I was cut short because of family problems that called me back to the UK. But work on Synapsin I simultaneously involved many others in Paul''s laboratory, so research on its subcellular localization and function continued apace. For example, Wieland Huttner and Mary Kennedy both contributed to unraveling the process of Ca²⁺-dependent phosphorylation of Synapsin I (Huttner et al., 1981; Kennedy and Greengard, 1981). Indeed, cAMP-dependent protein kinase and CaM kinase both phosphorylated the same site on Synapsin I, and CaM kinase also phosphorylated additional sites (Huttner et al., 1981). Pietro De Camilli showed in elegant immuno-electron microscopic studies that Synapsin I was a cytoplasmic protein that was associated with synaptic vesicles (De Camilli et al., 1983). To date, there are over 2000 studies involving Synapsin I listed in PubMed, identifying its roles in synaptic function and pathology. These include many from Paul''s own laboratory, continuing throughout his career (Hilfiker et al., 2005; Venton et al., 2006).When I returned to UK, I had the good fortune to obtain a staff position at the Medical Research Council''s National Institute of Medical Research (NIMR) at Mill Hill in London. This was an institution that Paul knew well, since he had spent a postdoctoral period in Wilhelm Feldberg''s department at NIMR in the 1950s, recording from sympathetic nerve fibers. I find it fascinating that Paul was already interested in phosphorylation even then (Greengard and Straub, 1959a, 1959b). My post came about entirely through Paul''s suggestion and generosity: he wrote to Arnold Burgen, then the Director of the Institute, whom he must have known from his work on cholinergic transmission. Suddenly and miraculously, I was offered a job, during a period when Margaret Thatcher was Prime Minister, the UK was in a deep recession, and academic positions were few and far between.Before arriving, I knew very little about NIMR, and was thrilled to be appointed into the same Division of Neurophysiology and Neuropharmacology, which had previously housed the Yale triumvirate of Paul Greengard, Murdoch Ritchie, and Bill Douglas, and which currently contained an eclectic and exciting mix of groups studying pain, hearing, epilepsy, and hippocampal LTP. Wilhelm Feldberg also still had a laboratory there, and used to call me Delphine. I was, however, supernumerary, and given a small cupboard to work in, and an even smaller research consumables budget. In that environment, since I had become intrigued by presynaptic events, I decided to examine neurotransmitter release, and its modulation by activation of presynaptic receptors, including those activated by adenosine agonists, building on my knowledge of the very robust response of Synapsin I phosphorylation (Dolphin, 1983; Dolphin and Archer, 1983). I also rapidly developed an enjoyable collaboration with Tim Bliss and members of his group, including Mick Errington, working with them to measure field potentials and examine glutamate release from the dentate gyrus during the induction of LTP (Dolphin et al., 1982).Studying the regulation of neurotransmitter release naturally led me to further questions about regulation of the Ca²⁺ influx that triggers this release, but this was not something I could directly address at NIMR. Although it did not feel like it at the time, it was fortuitous that the rules for obtaining tenure changed while I was there, meaning that to apply for tenure I would have to reapply for my position and then remain untenured for another 5 years. Many of the affected staff started to look for university posts, and I was offered a lectureship at St. George''s Hospital Medical School, in the Department of Pharmacology. The interview went well, apart from being asked if I intended to have children, and I moved there in 1983, hoping again to pivot my research in a new direction. At that time, the Department was almost entirely populated by electrophysiologists, including the Head of Department, John Kelly, together with Vincenzo Crunelli, Mark Meyer, and Tom Bolton.In this environment, it would be feasible for me to study voltage-gated calcium channels and their modulation by neurotransmitters, and how this related to neurotransmitter release. Ironically, while I was at Yale, Richard Tsien was on the faculty in the Physiology department, doing beautiful work on cardiac electrophysiology, leading up to an understanding of how cAMP modulates cardiac calcium channels, and the existence of additional calcium channels in neurons (Bean et al., 1984; Nowycky et al., 1985). However, my main memory of Dick Tsien at the time was that he adjudicated on my Yale postdoctoral fellowship application. When I started investigating calcium channels, I very belatedly wished I had concentrated better during Physiology seminars at Yale, to take advantage of their wealth of knowledge. Nevertheless, through the enormous generosity particularly of John Kelly at St. George''s, I was able to learn how to record calcium currents in DRG neurons, which had already been established as a model to study presynaptic events (Dunlap and Fischbach, 1978). Together with my first postdoctoral research assistant and great friend, Rod Scott, we started to apply the same techniques to examine the mechanism of modulation of action potentials and calcium currents in these neurons by neurotransmitters, where we showed inhibition, particularly by activation of GABA-B and adenosine receptors (Dolphin et al., 1986).Tools were initially lacking to study the role of G proteins in the modulation of calcium channels and neurotransmitter release, but this changed for me when I visited Pietro De Camilli in Milan and attended the Fifth International Conference on Cyclic Nucleotides and Protein Phosphorylation, in July 1983. There I heard about the use of pertussis toxin to distinguish between different GTP binding protein mechanisms. Although it was not yet available commercially, I managed to locate a local source of pertussis toxin (entailing rather scary visits to the UK government Center for Applied Microbiology & Research at Porton Down, where research on dangerous toxins and pathogens was conducted). Using both pertussis toxin and nonhydrolyzable guanine nucleotide analogs (including photoactivatable forms, made by John Wootton and David Trentham at NIMR), we then investigated the involvement of pertussis toxin-sensitive G proteins in the modulation of both glutamate release (Dolphin and Prestwich, 1985) and calcium currents (Dolphin et al., 1986; Scott and Dolphin, 1986; Dolphin et al., 1988).One of the next big scientific questions was to examine the same modulatory processes using the calcium channel subunits that were being cloned at that time by several groups (Tanabe et al., 1987; Ellis et al., 1988; De Jongh et al., 1990; Mori et al., 1991; Starr et al., 1991; Williams et al., 1992). In 1989, I was asked to apply for the Chair of Pharmacology at the Royal Free Hospital School of Medicine in London, and there I reoriented our efforts to examine the role of specific calcium channel subunits in the modulation of native and cloned calcium currents by G proteins. In our work, we concentrated initially on the role of the calcium channel β subunits (Berrow et al., 1995). We then uncovered an essential role for the N-terminus of Ca_V2 calcium channels in their G-protein modulation (Page et al., 1998; Canti et al., 1999), and we demonstrated the importance of calcium channel β subunits in the G-protein modulation of Ca_V2.2 channels (Meir et al., 2000; Leroy et al., 2005), and also in PI3 kinase-mediated calcium channel modulation (Viard et al., 2004).At that time, very little work had been done on the role of the intriguing α₂δ subunits of calcium channels, whose topology and function were initially unclear (Brickley et al., 1995; Gurnett et al., 1996). We started working on these proteins through another fortunate encounter. When the Royal Free School of Medicine was merged with University College London (UCL), I was asked to move to the UCL Gower Street campus in 1997, primarily to free up my space for Professor Geoff Burnstock, who had relinquished his role as Head of Department of Anatomy at UCL. There, I had the good fortune to collaborate with Michele Rees in the Pediatrics Department, on the molecular basis for the Ducky mouse mutation, that causes absence epilepsy and ataxia. The mutation turned out to be in a calcium channel auxiliary subunit gene, Cacna2d2, encoding α₂δ−2 (Barclay et al., 2001). This work fortuitously led us on to an extended series of studies on the biochemistry, physiology, and pharmacology of the α₂δ subunit family, about which little was then known. These investigations allowed me to combine my interests in biochemistry, from my undergraduate years, with all the many other techniques we have embraced, including electrophysiology. Among our findings were that the von Willebrand factor domain of α₂δ subunits is essential for their ability to augment calcium currents (Canti et al., 2005; Hoppa et al., 2012; Dahimene et al., 2018), that α₂δ proteins are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol linkage rather by a transmembrane domain (Davies et al., 2010), that the α₂δ subunits increase trafficking of calcium channels to the cell surface (Cassidy et al., 2014), and that proteolytic cleavage of α₂δ into α₂ and δ is an essential molecular switch for the augmentation of calcium currents and transmitter release (Kadurin et al., 2016; Ferron et al., 2018).Other studies, including work from our own laboratory, found that α₂δ−1 mRNA and protein are strongly upregulated in DRG neurons in rodent models of neuropathic pain (Luo et al., 2001; Newton et al., 2001; Bauer et al., 2009). Furthermore, α₂δ−1 is the main receptor for the antiepileptic drug gabapentin, which is also of therapeutic benefit in neuropathic pain (Gee et al., 1996). We contributed to showing that their binding to α₂δ−1 is essential for the action of gabapentinoids to alleviate neuropathic pain (Field et al., 2006). Since α₂δ proteins are auxiliary subunits, the molecular mechanism of action of these drugs in neuropathic pain was not immediately obvious, until we showed that gabapentinoids reduced calcium currents chronically, but not acutely, through an inhibitory effect on α₂δ trafficking, both in vitro (Hendrich et al., 2008; Tran-Van-Minh and Dolphin, 2010) and in vivo (Bauer et al., 2009). We then found, by making a functional extracellularly tagged version of Ca_V2.2, that as a consequence, gabapentin also reduced the trafficking and cell surface expression of the channel itself (Cassidy et al., 2014). A knock-in mouse containing this tagged Ca_V2.2 is now allowing us to identify that α₂δ−1 KO also reduces the cell surface localization of Ca_V2.2 in DRGs and their presynaptic terminals in vivo (Nieto-Rostro et al., 2018).The molecular mechanisms involved in the development and maintenance of neuropathic pain are the subject of research of many groups (for review, see Sexton et al., 2018; Alsaloum et al., 2020; Halievski et al., 2020). As a result of orienting our research toward the involvement of calcium channels in neuropathic pain, I encountered another unexpected advantage of moving to UCL. We have been able to benefit enormously from the knowledge and advice of the many experts in pain who work there, including John Wood, Tony Dickenson, and Maria Fitzgerald. It is remarkable that, during my time at UCL, threads from my past have been picked up and helped to shape my work. Indeed, we have collaborated extensively with Tony Dickenson, who was one of the other nontenured staff scientists who left NIMR at the same time as me, and who then obtained a lectureship directly at UCL (Bauer et al., 2009; Patel et al., 2013). Similarly, in an extension of the Greengard family, we had a valuable collaboration with Tim Ryan, who has also worked extensively with Paul Greengard''s laboratory, particularly on the role of Synapsins in vesicular release (Ryan et al., 1996; Chi et al., 2003). With Tim, we studied the role of α₂δ proteins in presynaptic Ca²⁺ entry and vesicular release (Hoppa et al., 2012), and this led us on to further examining the role of proteolytic cleavage of α₂δ in these processes (Kadurin et al., 2016; Ferron et al., 2018).I was honored to speak at Paul''s 80th birthday celebration, and then also to attend his 90th birthday symposium, both held at Rockefeller University, where I was very happy to meet up with so many previous colleagues (Fig. 2). At the latter meeting, I also had the privilege of being able to tell Paul of my election to the Royal Society, and to thank him for his support; and I could see his real pleasure in this news. Although I have never worked directly on Synapsins since leaving Paul''s laboratory, from these and other meetings, I have of course retained a keen interest in Paul''s wide-ranging research, maintained until his death at age 93 in 2019. His work unraveling the enormous complexity of the many pathways involving neuronal protein phosphorylation, and their physiological and pathologic roles (Hilfiker et al., 1999; Svenningsson et al., 2004; Brichta and Greengard, 2014), contributed to his being awarded the Nobel Prize in 2000, together with Arvid Carlsson and Eric Kandel.Open in a separate windowFigure 2.Eric Nestler, Paul Greengard, Annette Dolphin; December 11, 2015 at Paul''s 90th birthday celebration.For younger scientists, I have only a few messages. Like me, I hope you will find that all your scientific studies and experience will be of benefit in the long run, even if you change fields, and all your scientific mentors, colleagues, and friends will remain important throughout your career; but the value of chance, casual conversations, and fortuitous meetings cannot be overstated. Treasure them all.