Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

Cognition and psychological wellbeing in hypopituitary patients.

Hypopituitarism (HP) frequently occurs in patients presenting with sellar masses and despite recent advances in therapeutic options, HP patients consistently suffer from impaired quality of life due to psychological distress and cognitive dysfunction. These neurocognitive complications tend to persist in spite of surgical or biochemical remission of the disease making it especially challenging to segregate the effect of HP per se from other comorbidities such as the effect of tumour, surgery, radiation therapy, or complications caused by excess hormone production. Regardless, there is ample evidence to suggest that receptors for various pituitary hormones are abundantly expressed in key areas of central nervous system that are associated with memory and behaviour function and HP is also associated with poor sleep which can further exacerbate neurocognitive dysfunction. There is also evidence that hormonal replacement in HP patients partially restores these neurocognitive functions and improves sleep disorders. However, there is a need for creating better awareness among healthcare providers interacting with HP patients to enhance an earlier recognition of these disorder and their impact on quality of life despite initial remission. Importantly, there is a need to not only develop better and more cost-effective replacement therapies that would closely mimic the physiological hormonal release patterns, but also develop coping strategies for HP patients suffering from these complications.

Neuropsychological complications of hypoprolactinemia.

Prolactin (PRL) is primarily produced by the pituitary lactotrophic cells and while initially named for its role in lactation, PRL has several other biological roles including immunomodulation, osmotic balance, angiogenesis, calcium metabolism, and appetite regulation. Most of the PRL-related literature has traditionally focused on hyperprolactinemia, whereas hypoprolactinemia has received little attention. There is evidence to suggest that PRL receptors are widely distributed within the central nervous system including the limbic system. Furthermore, PRL has been shown to play key role in the stress regulation pathway. Recent data also suggest that hypoprolactinemia may be associated with increased sexual dysfunction, anxiety, and depression. In this paper we discuss the current understanding regarding the neuropsychological impact of hypoprolactinemia and highlight the need for adequately defining hypoprolactinemia as an entity and consideration for future replacement therapies.

Brd4-bound enhancers drive cell-intrinsic sex differences in glioblastoma.

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.

Laparoscopic prepregnancy transabdominal cerclage: operative and pregnancy outcomes for a novel technique of suture placement using port closure device.

Transabdominal cerclage is an effective surgical intervention for preterm birth prevention. Placement of cervical sutures using a port closure device for prepregnancy laparoscopic abdominal cerclage has been used at our unit in recent years. We report the operative and pregnancy outcomes for prepregnancy laparoscopic abdominal cerclage using the port closure device and compare it with the outcomes of the traditional approach. For prepregnancy laparoscopic transabdominal cerclage (n=52), the port closure device approach was associated with less blood loss during surgery (0.95±4.4 mL vs 5.4±15.7 mL; P=.007) and a shorter hospital length of stay (0.0; 0.0-0.0 days vs 1.0; 0.0-1.0 days; P<.001). There were also trends toward shorter operating times (41.4±15.3 minutes vs 50.1±18.0 minutes; P=.167) and lower perioperative complication rates (0.0%; 0/21 vs 16.1%; 5/31; P=.065) when compared with the traditional technique. There was no significant difference between the port closure device technique and the traditional approach in the rate of preterm birth in a subsequent pregnancy (0.0%; 0/9 vs 22.6%; 7/39; P=.248). Use of the port closure device for suture placement during prepregnancy laparoscopic cerclage for preterm birth prevention was reported. This technique was associated with less blood loss and a shorter hospital length of stay, had trends toward shorter operating times and lower perioperative complication rates, and had similar rates of preterm birth.

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.

Is Lipoprotein(a) Clinically Actionable with Today's Evidence? The Answer is Yes.

PURPOSE OF REVIEW: Lipoprotein(a) is an independent risk factor for cardiovascular disease. We review the ongoing shifts in consensus guidelines for the testing and management of Lp(a) and provide insight into whether current evidence suggests that awareness and testing of Lp(a) is clinically actionable. RECENT FINDINGS: GWAS and Mendelian randomization studies have established causal links between elevated Lp(a) and forms of CVD, including CAD and calcific aortic valve disease. Testing of Lp(a) identifies patients with similar risk to that of heterozygous FH, enhances risk stratification in patients with borderline/intermediate risk as determined through traditional factors, and facilitates the assessment of inherited CVD risk through cascade screening in patients with known family history of elevated Lp(a). Reductions in Lp(a) through non-targeted therapies including PCSK9 inhibition and lipoprotein apheresis have demonstrated reductions in ASCVD risk that are likely attributable to lowering Lp(a). Targeted therapies to potently lower Lp(a) are in clinical development. Lp(a) is actionable, and can be used to identify high risk patients for primary prevention and their family members through cascade screening, and to guide intensification of therapy in primary and secondary prevention of ASCVD.

The use of ketamine infusion to dramatically reduce opioid requirements in a patient whose high-dose intrathecal opioid pump was inadvertently cut during surgery.

BACKGROUND: Chronic opioid therapy may lead to high level tolerance development, hyperalgesia, and central sensitization, which further complicates long-term therapeutic management of chronic pain patients. In this case, we encounter a patient who was receiving over 15,000 morphine milligram equivalents through their intrathecal pain pump. Unfortunately, the intrathecal pump was inadvertently cut during a spinal surgery. It was deemed unsafe to delivery IV equivalent opioid therapy in this case; instead, the patient was admitted to the ICU and given a four-day ketamine infusion. METHOD: The patient was started on a ketamine infusion at a rate of 0.5mg/kg/h, which was continued for three days. On the fourth day, the infusion rate was tapered over 12 h before being completely stopped. No coinciding opioid therapy was given during this time, which was only restarted in the outpatient setting. RESULTS: Despite chronic high levels of opioid therapy immediately prior to the ketamine infusion, the patient did not experience florid withdrawals during the infusion period. Additionally, the patient experienced remarkable improvement in their subjective pain rating, which decreased from 9 to 3-4 on an 11-point Number Rating Scale, while simultaneously being managed on an MME <100. These results were sustained through a 6-month follow-up period. CONCLUSION: Ketamine may play an important role in attenuating not only tolerance but also acute withdrawal in a setting where rapid or instant weaning from high dose chronic opioid therapy is needed.

Outcome predictors of immune-mediated necrotizing myopathy-a retrospective, multicentre study.

OBJECTIVES: Evidence-based treatment protocols are currently lacking for immune-mediated necrotizing myopathy (IMNM). In this multicentre retrospective study, we examined baseline clinical characteristics and treatment variables that may predict short-term outcomes of patients with IMNM. METHODS: Muscle biopsies from the John Hunter Hospital and the Royal Adelaide Hospital obtained between 2012 and 2019 were reviewed at a single laboratory at South Australia Pathology. All biopsies with histological features of IMNM were identified. Demographics of study subjects, clinical information and myositis-specific antibody status were recorded along with muscle strength, serum creatine kinase (CK) and treatment regimens at baseline and 3 and 6 months. Primary outcome measures were muscle strength and serum CK at 3 and 6 months. Mixed-effects regression models in a Bayesian framework were performed using the R statistical package. RESULTS: Female sex, older age, initial prednisone dose and i.v. methylprednisolone were associated with greater improvement in serum CK. In patients with moderate-severe disease at baseline, early IVIG was associated with greater improvement in hip flexor strength at 6 months. CONCLUSION: Early IVIG was associated with clinical improvement in the short-term follow-up in IMNM. Female sex, older age, initial oral prednisone dose and initial use of i.v. methylprednisolone were associated with better biochemical improvement.

Hyperbaric effects on cold stored whole blood following a military dive mission profile.

BACKGROUND: Whole blood (WB) is carried by special operations forces as part of a remote damage control resuscitation strategy. The effects of an underwater mission on the quality and coagulation profile of WB were simulated by exposure to hyperbaric pressures in a chamber. METHODS: WB units collected in CPDA-1 were exposed to three different combinations of hyperbaric pressure and duration of exposure: Group A 153.52 kPa (15.24 msw; 1.52 atm) for 4 h; n = 9, Group B 506.63 kPa (50.29 msw; 5.00 atm) for 1 h; n = 9, Group C 153.52 kPa (15.24 msw; 1.52 atm) for 1 h; n = 7. The following parameters were measured on each unit: prothrombin time/international normalized ratio, activated partial thromboplastin time, thromboelastography and concentration determinations of platelets, lactate, fibrinogen, and lactate dehydrogenase. Each sample underwent baseline, prepressurization, immediate postpressurization, and 6 h postpressurization laboratory testing. RESULTS: Six hours following hyperbaric exposure, the lactate concentration in group C was higher than prepressurization measurement and the platelet concentration in Group A was lower than prepressurization measurement. There were no changes in any of the other analyzed biochemical, coagulation and thromboelastogram parameters following exposure to hyperbaric stress. DISCUSSION: These data suggest that pressurization of WB up to 5 atm did not impact parameters tested. Changes observed in lactate and platelet count need further study, as well as complementary testing of red blood cell integrity. Further investigation of the hyperbaric extremes is necessary to determine if there is a damage inducing pressure to which WB should not be exposed.

CRABP2 - A novel biomarker for high-risk endometrial cancer.

OBJECTIVE: Investigate the clinical and functional implications of elevated CRABP2 expression in endometrial cancer (EC) patients. METHODS: Patients were stratified into high and low CRABP2 expression groups using a decision tree classifier. Univariate and multivariate statistical analyses determined the prognostic and clinicopathological consequences of increased CRABP2 expression. A CRABP2 gene signature was generated using differential expression analysis, and analyzed using network-based approaches. The findings were validated in The Clinical Proteomic Tumor Analysis Consortium (CPTAC), a newly generated cohort of 120 endometrial tissues, and The Cancer Dependency Map (DepMap). RESULTS: 60 (11%) patients in TCGA had high CRABP2 expression, whilst 468 (89%) had low expression. High expression was associated with serous EC, reduced overall survival, advanced stage and grade. Downstream retinoic acid receptors (RARG and RARA) were correlated with CRABP2 expression and were associated with worse prognosis in serous EC. The CRABP2 gene signature was enriched for Polycomb target gene sets, and was regulated by ELP3 and BMP7. BMP7 expression was increased in the CRABP2-high group, was associated with worse prognosis, and CRISPR-Cas9 screens revealed correlations in its cell-fitness score with CRABP2 following gene knockout. The opposite was true for ELP3, suggesting opposing effects from both master regulators. CONCLUSIONS: CRABP2 expression is associated with poor prognosis and advanced EC. The expression of RARA and RARG correlates with CRABP2 and are associated with worse prognosis in advanced histological subtypes. Polycomb target gene sets and two master regulators, ELP3 and BMP7, were identified as functionally relevant mechanisms driving aberrant CRABP2 expression.

Botrytis cinerea Loss and Restoration of Virulence during In Vitro Culture Follows Flux in Global DNA Methylation.

Pathogenic fungi can lose virulence after protracted periods of culture, but little is known of the underlying mechanisms. Here, we present the first analysis of DNA methylation flux at a single-base resolution for the plant pathogen B. cinerea and identify differentially methylated genes/genomic regions associated with virulence erosion during in vitro culture. Cultures were maintained for eight months, with subcultures and virulence testing every month. Methylation-sensitive amplified polymorphisms were performed at monthly intervals to characterise global changes to the pathogen's genome during culture and also on DNA from mycelium inoculated onto Arabidopsis thaliana after eight months in culture. Characterisation of culture-induced epialleles was assessed by whole-genome re-sequencing and whole-genome bisulfite sequencing. Virulence declined with time in culture and recovered after inoculation on A. thaliana. Variation detected by methylation-sensitive amplified polymorphisms followed virulence changes during culture. Whole-genome (bisulfite) sequencing showed marked changes in global and local methylation during culture but no significant genetic changes. We imply that virulence is a non-essential plastic character that is at least partly modified by the changing levels of DNA methylation during culture. We hypothesise that changing DNA methylation during culture may be responsible for the high virulence/low virulence transition in B. cinerea and speculate that this may offer fresh opportunities to control pathogen virulence.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Polysomally protected viruses.

It is conceivable that an RNA virus could use a polysome, that is, a string of ribosomes covering the RNA strand, to protect the genetic material from degradation inside a host cell. This paper discusses how such a virus might operate, and how its presence might be detected by ribosome profiling. There are two possible forms for such apolysomally protected virus, depending upon whether just the forward strand or both the forward and complementary strands can be encased by ribosomes (these will be termed type 1 and type 2, respectively). It is argued that in the type 2 case the viral RNA would evolve anambigrammaticproperty, whereby the viral genes are free of stop codons in a reverse reading frame (with forward and reverse codons aligned). Recent observations of ribosome profiles of ambigrammatic narnavirus sequences are consistent with our predictions for the type 2 case.

The SALT-Readout ASIC for Silicon Strip Sensors of Upstream Tracker in the Upgraded LHCb Experiment.

SALT, a new dedicated readout Application Specific Integrated Circuit (ASIC) for the Upstream Tracker, a new silicon detector in the Large Hadron Collider beauty (LHCb) experiment, has been designed and developed. It is a 128-channel chip using an innovative architecture comprising a low-power analogue front-end with fast pulse shaping and a 40 MSps 6-bit Analog-to-Digital Converter (ADC) in each channel, followed by a Digital Signal Processing (DSP) block performing pedestal and Mean Common Mode (MCM) subtraction and zero suppression. The prototypes of SALT were fabricated and tested, confirming the full chip functionality and fulfilling the specifications. A signal-to-noise ratio of about 20 is achieved for a silicon sensor with a 12 pF input capacitance. In this paper, the SALT architecture and measurements of the chip performance are presented.

Are trainees working in obstetrics and gynecology confident and competent in the care of frail gynecological oncology patients?

INTRODUCTION: Older patients undergoing cancer surgery are at increased risk of post-operative complications, prolonged hospital stay, and mortality. Identification of frailty can help predict patients at high risk of peri-operative complications and allow a collaborative, multidisciplinary team approach to their care. A survey was conducted to assess the confidence and knowledge of trainees in obstetrics and gynecology regarding identification and management of peri-operative issues encountered in frail gynecological oncology patients. METHODS: A web-based survey was distributed via the Audit and Research in Gynaecological Oncology (ARGO) collaborative and UK Audit and Research Collaborative in Obstetrics and Gynaecology (UKARCOG) . The survey on the management of frail peri-operative patients was disseminated to doctors-in-training (trainees) working in obstetrics and gynecology in the United Kingdom (UK) and Ireland. Specialty (ST1-7), subspecialty, and general practice trainees, non-training grade doctors, and foundation year doctors currently working in obstetrics and gynecology were eligible. Consultants were excluded. Study data were collected using REDCAP software hosted at the University of Manchester. Responses were collected over a 6-week period between January and February 2020. RESULTS: Of the 666 trainees who participated, 67% (425/666) reported inadequate training in peri-operative management of frail patients. Validated frailty assessment tools were used by only 9% (59/638) of trainees and less than 1% (4/613) were able to correctly identify all the diagnostic features of frailty. Common misconceptions included the use of chronological age and gender in frailty assessments. The majority of trainees (76.5%, 448/586) correctly answered a series of questions relating to mental capacity; however, only 6% (36/606) were able to correctly identify all three diagnostic features of delirium. A total of 87% (495/571) of trainees supported closer collaboration with geriatricians and a multidisciplinary approach. CONCLUSIONS: Obstetrics and gynecology trainees reported inadequate training in the peri-operative care of frail gynecological oncology patients, and overwhelmingly favored input from geriatricians. Routine use of validated frailty assessment tools may aid diagnosis of frailty in the peri-operative setting. There is an unmet need for formal education in the management of frail surgical patients within the UK and Irish obstetrics and gynecology curriculum.

Resolution acuity across the visual field for mesopic and scotopic illumination.

We investigated the classical question of why visual acuity decreases with decreasing retinal illuminance by holding retinal eccentricity fixed while illumination varied. Our results indicate that acuity is largely independent of illuminance at any given retinal location, which suggests that under classical free-viewing conditions acuity improves as illumination increases from rod threshold to rod saturation because the retinal location of the stimulus is permitted to migrate from a peripheral location of maximum sensitivity but poor acuity to the foveal location of maximum acuity but poor sensitivity. Comparison with anatomical sampling density of retinal neurons suggests that mesopic acuity at all eccentricities and scotopic acuity for eccentricities beyond about 20° is limited by the spacing of midget ganglion cells. In central retina, however, scotopic acuity is further limited by spatial filtering due to spatial summation within the large, overlapping receptive fields of the A-II class of amacrine cells interposed in the rod pathway between rod bipolars and midget ganglion cells. Our results offer a mechanistic interpretation of the clinical metrics for low-luminance visual dysfunction used to monitor progression of retinal disease.

Mitogen-Activated Protein Kinase (MAPK) and Obesity-Related Cancer.

Obesity is a major public health concern worldwide. The increased risk of certain types of cancer is now an established deleterious consequence of obesity, although the molecular mechanisms of this are not completely understood. In this review, we aim to explore the links between MAPK signalling and obesity-related cancer. We focus mostly on p38 and JNK MAPK, as the role of ERK remains unclear. These links are seen through the implication of MAPK in obesity-related immune paralysis as well as through effects on the endoplasmic reticulum stress response and activation of aromatase. By way of example, we highlight areas of interest and possibilities for future research in endometrioid endometrial cancer and hepatocellular carcinoma associated with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and MAPK.

Terasaki spiral ramps and intracellular diffusion.

The sheet-like endoplasmic reticulum (ER) of eukaryotic cells has been found to be riddled with spiral dislocations, known as 'Terasaki ramps', in the vicinity of which the doubled bilayer membranes which make up ER sheets can be approximately modeled by helicoids. Here we analyze diffusion on a surface with locally helicoidal topological dislocations, and use the results to argue that the Terasaki ramps facilitate a highly efficient transport of water-soluble molecules both within the lumen of the endoplasmic reticulum, and in the adjacent cytoplasmic space.

An optimized, broadly applicable piggyBac transposon induction system.

The piggyBac (PB) transposon has been used in a number of biological applications. The insertion of PB transposons into the genome can disrupt genes or regulatory regions, impacting cellular function, so for many experiments it is important that PB transposition is tightly controlled. Here, we systematically characterize three methods for the post-translational control of the PB transposon in four cell lines. We investigated fusions of the PB transposase with ERT2 and two degradation domains (FKBP-DD, DHFR-DD), in multiple orientations, and determined (i) the fold-induction achieved, (ii) the absolute transposition efficiency of the activated construct and (iii) the effects of two inducer molecules on cellular transcription and function. We found that the FKBP-DD confers the PB transposase with a higher transposition activity and better dynamic range than can be achieved with the other systems. In addition, we found that the FKBP-DD regulates transposon activity in a reversible and dose-dependent manner. Finally, we showed that Shld1, the chemical inducer of FKBP-DD, does not interfere with stem cell differentiation, whereas tamoxifen has significant effects. We believe the FKBP-based PB transposon induction will be useful for transposon-mediated genome engineering, insertional mutagenesis and the genome-wide mapping of transcription factor binding.