The Spatial Patterns and Determinants of Cerebrospinal Fluid Circulation in the Human Brain.

The circulation of cerebrospinal fluid (CSF) is essential for maintaining brain homeostasis and clearance, and impairments in its flow can lead to various brain disorders. Recent studies have shown that CSF circulation can be interrogated using low b-value diffusion magnetic resonance imaging (low-b dMRI). Nevertheless, the spatial organization of intracranial CSF flow dynamics remains largely elusive. Here, we developed a whole-brain voxel-based analysis framework, termed CSF pseudo-diffusion spatial statistics (CΨSS), to examine CSF mean pseudo-diffusivity (MΨ), a measure of CSF flow magnitude derived from low-b dMRI. We showed that intracranial CSF MΨ demonstrates characteristic covariance patterns by employing seed-based correlation analysis. Importantly, we applied non-negative matrix factorization analysis to further elucidate the covariance patterns of CSF MΨ in a hypothesis-free, data-driven way. We identified distinct CSF spaces that consistently displayed unique pseudo-diffusion characteristics across multiple imaging datasets. Our study revealed that age, sex, brain atrophy, ventricular anatomy, and cerebral perfusion differentially influence MΨ across these CSF spaces. Notably, individuals with anomalous CSF flow patterns displayed incidental findings on multimodal neuroradiological examinations. Our work sets forth a new paradigm to study CSF flow, with potential applications in clinical settings.

Choice Architecture in Opioid Safety Alerting.

The need for effective and efficient clinical decision support (CDS) embedded in electronic health record (EHR) processes is growing. Using choice architecture design strategies may increase effectiveness of CDS solutions. The authors describe implementation of an opioid risk alert and subsequent revisions of that alert to increase effectiveness and reduce alert volumes. The first version of the alert used an opt-in choice architecture when recommending naloxone and the second version used an active choice design. The percentage of opioid prescriptions ordered with naloxone prescribed within the last 12 months increased significantly after implementation of the first version of the alert and then further increased significantly after implementation of the second version. Alert volumes decreased over the same timeframe. An education campaign was also implemented during the timeframe studied and likely also contributed to the naloxone outcomes seen.

Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

Can the Sperm Class Analyser (SCA) CASA-Mot system for human sperm motility analysis reduce imprecision and operator subjectivity and improve semen analysis?

Semen analysis (SA) is considered mandatory for suspected male infertility although its clinical value has recently become questionable. Sperm motility is an essential parameter for SA, but is limited by high measurement uncertainty, which includes operator subjectivity. Computer-assisted sperm analysis (CASA) can reduce measurement uncertainty compared with manual SA. The objective of this study was to determine whether the Sperm Class Analyser (SCA) CASA-Mot system could reduce specific components of sperm motility measurement uncertainty compared with the World Health Organization (WHO) manual method in a single laboratory undertaking routine diagnostic SA. The study examined: (i) operator subjectivity; (ii) precision, (iii) accuracy against internal and external quality standards; and (iv) a pilot sub-study examining the potential to predict an IVF fertilisation rate. Compared with the manual WHO method of SA on 4000 semen samples, SCA reduces but does not completely eliminate operator subjectivity. Study SCA and CASA-Mot are useful tools for well-trained staff that allow rapid, high-number sperm motility categorization with less analytical variance than the manual equivalent. Our initial data suggest that SCA motility may have superior predictive potential compared with the WHO manual method for predicating IVF fertilization.

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

Visualization of early events in mRNA vaccine delivery in non-human primates via PET-CT and near-infrared imaging.

Visualization of the spatio-temporal trafficking of vaccines after their delivery would help evaluate the efficacy of candidate formulations and aid their rational design for preclinical and translational studies. Here, we show that a dual radionuclide-near-infrared probe allows for quantitative, longitudinal and non-invasive monitoring, via positron emission tomography-computed tomography and near-infrared imaging of cynomolgus macaques, of the trafficking dynamics to draining lymph nodes of a model messenger RNA vaccine labelled with the probe. After intramuscular administration of the vaccine to the monkeys, we observed the dynamics of the mRNA vaccine at the injection site and in the draining lymph nodes, performed cellular analyses of the involved tissues using flow cytometry and identified through immunofluorescence that professional antigen-presenting cells are the primary cells containing the injected mRNA and encoding the antigen. This approach may reveal spatio-temporal determinants of vaccine efficacy in preclinical and translational studies employing large mammals.

Corrected cholesterol, a novel marker for predicting semen post-thaw quality: a pilot study.

Semen cryopreservation is the recommended method for protecting the fertility of men before they undergo iatrogenic treatments. However, post-thaw sperm quality is highly variable between and within individuals. Thus, prediction of post-thaw quality has not been achieved with great certainty. Men (n = 101) attending our andrology clinic each produced a single ejaculate for semen analysis and semen cryopreservation. Post-thaw semen quality was examined for associations with traditional and novel semen and seminal plasma parameters. This current research demonstrates that the cholesterol concentration of human sperm is associated with ejaculate tolerance to cryopreservation. Corrected cholesterol, calculated as the difference between the cholesterol of semen and the cholesterol of cell free seminal plasma, is predictive of post-thaw motility. This may have potential as a method to guide the number of ejaculates stored for individual patients before iatrogenic treatment and/or for the appropriate selection of ART with frozen/thawed semen.

Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection.

The lung is a critical prophylaxis target for clinically important infectious agents, including human respiratory syncytial virus (RSV) and influenza. Here, we develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via aerosol, to prevent RSV infections. First, we express palivizumab, which reduces RSV F copies by 90.8%. Second, we express engineered, membrane-anchored palivizumab, which prevents detectable infection in transfected cells, reducing in vitro titer and in vivo RSV F copies by 99.7% and 89.6%, respectively. Finally, we express an anchored or secreted high-affinity, anti-RSV F, camelid antibody (RSV aVHH and sVHH). We demonstrate that RSV aVHH, but not RSV sVHH, significantly inhibits RSV 7 days post transfection, and we show that RSV aVHH is present in the lung for at least 28 days. Overall, our data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could potentially be a promising pulmonary prophylaxis approach.

In Vitro Transcribed mRNA Vaccines with Programmable Stimulation of Innate Immunity.

In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manufactured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing translation of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immunostimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tethered IVT mRNA-TLR7 agonists leads to enhanced local immune responses, and to antigen-specific cell-mediated and humoral responses. We believe this system represents a potential platform compatible with any adjuvant of interest to enable specific programmable stimulation of immune responses.

Human sperm cryopreservation in cancer patients: Links with deprivation and mortality.

Evidence is mounting for a relationship between human semen quality and environmental/lifestyle/socioeconomic factors including long term health outcomes such as mortality. The relationship between pre-freeze and post-thaw semen quality in cancer patients and these factors are unknown. Frozen semen from 217 cancer patients was thawed and analysed using a validated CASA method. Post-thaw quality was matched and compared with WHO semen analysis performed prior to storage. The English Indices of Deprivation 2010 were matched with patients and then examined for relationships with pre-freeze and post-thaw semen quality. There is a relationship between semen quality and deprivation in cancer patients. Compared with pre-freeze semen quality, post-thaw semen quality has a stronger relationship with deprivation. Sperm cryopreservation may have potential as a systemic health diagnostic test and is predictive of cancer patient mortality.

Trends and usage in a London National Health Service Sperm Bank for cancer patients.

Sperm cryopreservation is the only method currently available that offers men with cancer insurance against sterilising iatrogenic treatments. We carried out two cohort and cross-sectional audits to identify trends with sperm cryopreservation referral rates and sample usage rates for men diagnosed with cancer and who banked sperm at The Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust. These retrospective audits revealed that a total of 4362 men with cancer successfully banked sperm between 1976 and 2013. Truncating the dataset to 2009 to allow for lag times between storage and use, the overall sample usage rate for cancer patients was 6.0% with 75 live births. Increased median age at referral influences the cancer profile of men seen at the bank, which is highlighted by a disproportionate rise in the number of men with prostate cancer. Among men who use banked sperm, a large rise in the use of intracytoplasmic sperm injection has occurred over time. The number of patients requiring the service is sharply increasing year on year as are the number of patients who go on to use their sample in assisted conception. The historical use rates of frozen sperm are likely to be significant underestimations of future use.

Validation of the sperm class analyser CASA system for sperm counting in a busy diagnostic semen analysis laboratory.

Sperm counts have been linked to several fertility outcomes making them an essential parameter of semen analysis. It has become increasingly recognised that Computer-Assisted Semen Analysis (CASA) provides improved precision over manual methods but that systems are seldom validated robustly for use. The objective of this study was to gather the evidence to validate or reject the Sperm Class Analyser (SCA) as a tool for routine sperm counting in a busy laboratory setting. The criteria examined were comparison with the Improved Neubauer and Leja 20-µm chambers, within and between field precision, sperm concentration linearity from a stock diluted in semen and media, accuracy against internal and external quality material, assessment of uneven flow effects and a receiver operating characteristic (ROC) analysis to predict fertility in comparison with the Neubauer method. This work demonstrates that SCA CASA technology is not a standalone 'black box', but rather a tool for well-trained staff that allows rapid, high-number sperm counting providing errors are identified and corrected. The system will produce accurate, linear, precise results, with less analytical variance than manual methods that correlate well against the Improved Neubauer chamber. The system provides superior predictive potential for diagnosing fertility problems.

Decoding the rat forelimb movement direction from epidural and intracortical field potentials.

Brain-machine interfaces (BMIs) use signals from the brain to control a device such as a computer cursor. Various types of signals have been used as BMI inputs, from single-unit action potentials to scalp potentials. Recently, intermediate-level signals such as subdural field potentials have also shown promise. These different signal types are likely to provide different amounts of information, but we do not yet know what signal types are necessary to enable a particular BMI function, such as identification of reach target location, control of a two-dimensional cursor or the dynamics of limb movement. Here we evaluated the performance of field potentials, measured either intracortically (local field potentials, LFPs) or epidurally (epidural field potential, EFPs), in terms of the ability to decode reach direction. We trained rats to move a joystick with their forepaw to control the motion of a sipper tube to one of the four targets in two dimensions. We decoded the forelimb reach direction from the field potentials using linear discriminant analysis. We achieved a mean accuracy of 69 ± 3% with EFPs and 57 ± 2% with LFPs, both much better than chance. Signal quality remained good up to 13 months after implantation. This suggests that using epidural signals could provide BMI inputs of high quality with less risk to the patient than using intracortical recordings.