Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

Ground-Based Mobile Measurements to Track Urban Methane Emissions from Natural Gas in 12 Cities across Eight Countries.

To mitigate methane emission from urban natural gas distribution systems, it is crucial to understand local leak rates and occurrence rates. To explore urban methane emissions in cities outside the U.S., where significant emissions were found previously, mobile measurements were performed in 12 cities across eight countries. The surveyed cities range from medium size, like Groningen, NL, to large size, like Toronto, CA, and London, UK. Furthermore, this survey spanned across European regions from Barcelona, ES, to Bucharest, RO. The joint analysis of all data allows us to focus on general emission behavior for cities with different infrastructure and environmental conditions. We find that all cities have a spectrum of small, medium, and large methane sources in their domain. The emission rates found follow a heavy-tailed distribution, and the top 10% of emitters account for 60-80% of total emissions, which implies that strategic repair planning could help reduce emissions quickly. Furthermore, we compare our findings with inventory estimates for urban natural gas-related methane emissions from this sector in Europe. While cities with larger reported emissions were found to generally also have larger observed emissions, we find clear discrepancies between observation-based and inventory-based emission estimates for our 12 cities.

Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.

Posttranscriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22 nt) noncoding RNAs capable of specifically targeting the miRNA-induced silencing complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct in situ interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering (Bassett G et al., Nat Commun 5, 4640, 2014). In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).

Ventricular-arterial coupling assessed by PWV/GLS ratio in hypertensive patients.

OBJECTIVE: The physiological interaction between the left ventricle (LV) and the arterial system, defined as ventricular-arterial coupling (VAC), facilitates the optimal volume of cardiac work and cardiovascular performance. The aim of this study was to evaluate the benefit of PWV/GLS ratio associated with other vascular and cardiac performance parameters in hypertensive patients compared to age-matched healthy controls. PATIENTS AND METHODS: We calculated the ratio of pulse wave velocity (PWV), as a marker of arterial stiffness, to global longitudinal strain (GLS), as a marker of left ventricular function in 135 patients divided in 3 groups, as follows: group 1 (HT + CAD) enrolled 54 hypertensive patients with coronary artery disease, group 2 (HT) enrolled 43 hypertensive patients and group 3 (CON) represented the control group consisting of 38 age-matched healthy subjects. RESULTS: GLS values were significantly reduced in HT+CAD (-17.50±7.2) vs. HT (-17.95±5.3) vs. control (-20.13±4.6) (p-value <0.001). PWV values were higher in HT+CAD (9.90±3.1) and HT (9.70±2.5) vs. control (7.85±3.2) (p-value <0.001). VA coupling measured by the PWV/GLS ratio showed significantly lower values in HT+CAD and HT vs. control (p-value <0.001). The ROC curve identified a threshold of -0.054 of the PWV/GLS ratio to detect altered ventricular-arterial coupling AUROC = 0.836, 95% CI [0.762; 0.909]. CONCLUSIONS: This study demonstrated that assessment of the PWV/GLS ratio represents a useful tool to detect altered ventricular-arterial coupling in hypertensive patients. The perspectives of future use could include monitoring of earlier development of multiple organ damage in hypertensive patients and the efficacy of the different hypertensive medications. Extensive prospective studies are needed to confirm this hypothesis.

RNA-Responsive gRNAs for Controlling CRISPR Activity: Current Advances, Future Directions, and Potential Applications.

CRISPR-Cas9 has emerged as a major genome manipulation tool. As Cas9 can cause off-target effects, several methods for controlling the expression of CRISPR systems were developed. Recent studies have shown that CRISPR activity could be controlled by sensing expression levels of endogenous transcripts. This is particularly interesting, as endogenous RNAs could harbor important information about the cell type, disease state, and environmental challenges cells are facing. Single-guide RNA (sgRNA) engineering played a major role in the development of RNA-responsive CRISPR systems. Following further optimizations, RNA-responsive sgRNAs could enable the development of novel therapeutic and research applications. This review introduces engineering strategies that could be employed to modify Streptococcus pyogenes sgRNAs with a focus on recent advances made toward the development of RNA-responsive sgRNAs. Future directions and potential applications of these technologies are also discussed.

Atrial Functional Tricuspid Regurgitation as a Distinct Pathophysiological and Clinical Entity: No Idiopathic Tricuspid Regurgitation Anymore.

Functional tricuspid regurgitation (FTR) is a strong and independent predictor of patient morbidity and mortality if left untreated. The development of transcatheter procedures to either repair or replace the tricuspid valve (TV) has fueled the interest in the pathophysiology, severity assessment, and clinical consequences of FTR. FTR has been considered to be secondary to tricuspid annulus (TA) dilation and leaflet tethering, associated to right ventricular (RV) dilation and/or dysfunction (the "classical", ventricular form of FTR, V-FTR) for a long time. Atrial FTR (A-FTR) has recently emerged as a distinct pathophysiological entity. A-FTR typically occurs in patients with persistent/permanent atrial fibrillation, in whom an imbalance between the TA and leaflet areas results in leaflets malcoaptation, associated with the dilation and loss of the sphincter-like function of the TA, due to right atrium enlargement and dysfunction. According to its distinct pathophysiology, A-FTR poses different needs of clinical management, and the various interventional treatment options will likely have different outcomes than in V-FTR patients. This review aims to provide an insight into the anatomy of the TV, and the distinct pathophysiology of A-FTR, which are key concepts to understanding the objectives of therapy, the choice of transcatheter TV interventions, and to properly use pre-, intra-, and post-procedural imaging.

Right heart chambers geometry and function in patients with the atrial and the ventricular phenotypes of functional tricuspid regurgitation.

AIMS: Atrial functional tricuspid regurgitation (A-FTR) is a recently defined phenotype of functional tricuspid regurgitation (FTR) associated with persistent/permanent atrial fibrillation. Differently from the classical ventricular form of FTR (V-FTR), patients with A-FTR might present with severely dilated right atrium and tricuspid annulus (TA), and with preserved right ventricular (RV) size and systolic function. However, the geometry and function of the right ventricle, right atrium, and TA in patients with A-FTR and V-FTR remain to be systematically evaluated. Accordingly, we sought to: (i) study the geometry and function of the right ventricle, right atrium, and TA in A-FTR by two- and three-dimensional transthoracic echocardiography; and (ii) compare them with those found in V-FTR. METHODS AND RESULTS: We prospectively analysed 113 (44 men, age 68 ± 18 years) FTR patients (A-FTR = 55 and V-FTR = 58) that were compared to two groups of age- and sex-matched controls to develop the respective Z-scores. Severity of FTR was similar in A-FTR and V-FTR patients. Z-scores of RV size were significantly larger, and those of RV function were significantly lower in V-FTR than in A-FTR (P < 0.001 for all). The right atrium was significantly enlarged in both A-FTR and V-FTR compared to controls (P < 0.001, Z-scores > 2), with similar right atrial (RA) maximum volume (RAVmax) between A-FTR and V-FTR (P = 0.2). Whereas, the RA minimum volumes (RAVmin) were significantly larger in A-FTR than in V-FTR (P = 0.001). CONCLUSION: Despite similar degrees of FTR and RAVmax size, A-FTR patients show larger RAVmin and smaller TA areas than V-FTR patients. Conversely, V-FTR patients show dilated, more elliptic and dysfunctional right ventricle than A-FTR patients.

Optimising the quarantining and response sequence towards SARS-CoV-2 outbreaks on board cargo vessels

The Coronavirus Disease (COVID-19) pandemic has brought significant impact onto the maritime activities worldwide, including disruption to global trade and supply chains. The ability to predict the evolution and duration of a COVID-19 outbreak on cargo vessels would inform a more nuanced response to the event and provide a more precise return-to-trade date. A SEIQ(H)R (Susceptibility--Exposed-Infected--Quarantine--(Hospitalisation)--Removed/Recovered) model is developed and fit-tested to simulate the transmission dynamics of COVID-19 on board cargo vessels of up to 60 crew. Due to specific living and working circumstances on board cargo vessels, instead of utilising the reproduction number, we consider the highest fraction of crew members who share the same nationality to quantify the transmissibility of the disease. The performance of the model is verified using case studies based on data collected during COVID-19 outbreaks on three cargo vessels in Western Australia during 2020. The simulations show that the model can forecast the time taken for the transmission dynamics on each vessel to reach their equilibriums, providing informed predictions on the evolution of the outbreak, including hospitalisation rates and duration. The model demonstrates that (a) all crew members are susceptible to infection; (b) their roles on board is a determining factor in the evolution of the outbreak; (c) an unmitigated outbreak could affect the entire crew and continue on for many weeks. The ability to model the evolution of an outbreak, both in duration and severity, is essential to predict outcomes and to plan for the best response strategy. At the same time, it offers a higher degree of certainty regarding the return to trade, which is of significant importance to multiple stakeholders.

Automated left atrial volume measurement by two-dimensional speckle-tracking echocardiography: feasibility, accuracy, and reproducibility.

AIMS: A byproduct of left atrial (LA) strain analysis is the automated measurement of LA maximal volume (LAVmax), which may decrease the time of echocardiography reporting, and increase the reproducibility of the LAVmax measurement. However, the automated measurement of LAVmax by two-dimensional speckle-tracking analysis (2DSTE) has never been validated. Accordingly, we sought to (i) assess the feasibility of automated LAVmax measurement by 2DSTE; (ii) compare the automated LAVmax by 2DSTE with conventional two-dimensional (2DE) biplane and three-dimensional echocardiography (3DE) measurements; and (iii) evaluate the accuracy and reproducibility of the three echocardiography techniques. METHODS AND RESULTS: LAVmax (34-197 mL) were obtained from 198/210 (feasibility 94%) consecutive patients (median age 67 years, 126 men) by 2DSTE, 2DE, and 3DE. 2DE and 2DSTE measurements resulted in similar LAVmax values [bias = 1.5 mL, limits of agreement (LOA) ± 7.5 mL], and slightly underestimated 3DE LAVmax (biases = -5 mL, LOA ± 17 mL and -6 mL, LOA ± 16 mL, respectively). LAVmax by 2DSTE and 2DE were strongly correlated to those obtained by cardiac magnetic resonance (CMR) (r = 0.946 and r = 0.935, respectively; P < 0.001). However, LAVmax obtained by 2DSTE (bias = -9.5 mL, LOA ± 16 mL) and 2DE (bias = -8 mL, LOA ± 17 mL) were significantly smaller than those measured by CMR. Conversely, 3DE LAVmax were similar to CMR (bias = -2 mL, LOA ± 10 mL). Excellent intra- and inter-observer intraclass correlations were found for 3DE (0.995 and 0.995), 2DE (0.990 and 0.988), and 2DSTE (0.990 and 0.989). CONCLUSION: Automated LAVmax measurement by 2DSTE is highly feasible, highly reproducible, and provided similar values to conventional 2DE calculations in consecutive patients with a wide range of LAVmax.

SARS-CoV-2 infections among Australian passengers on the Diamond Princess cruise ship: A retrospective cohort study.

BACKGROUND: Prolonged periods of confined living on a cruise ship increase the risk for respiratory disease transmission. We describe the epidemiology and clinical characteristics of a SARS-CoV-2 outbreak in Australian passengers on the Diamond Princess cruise ship and provide recommendations to mitigate future cruise ship outbreaks. METHODS: We conducted a retrospective cohort study of Australian passengers who travelled on the Diamond Princess from 20 January until 4 February 2020 and were either hospitalised, remained in Japan or repatriated. The main outcome measures included an epidemic curve, demographics, symptoms, clinical and radiological signs, risk factors and length of time to clear infection. RESULTS: Among 223 Australian passengers, 56 were confirmed SARS-CoV-2 positive. Forty-nine cases had data available and of these over 70% had symptoms consistent with COVID-19. Of symptomatic cases, 17% showed signs and symptoms before the ship implemented quarantine and a further two-thirds had symptoms within one incubation period of quarantine commencing. Prior to ship-based quarantine, exposure to a close contact or cabin mate later confirmed SARS-CoV-2 positive was associated with a 3.78 fold (95% CI, 2.24-6.37) higher risk of COVID-19 acquisition compared to non-exposed passengers. Exposure to a positive cabin mate during the ship's quarantine carried a relative risk of 6.18 (95% CI, 1.96-19.46) of developing COVID-19. Persistently asymptomatic cases represented 29% of total cases. The median time to the first of two consecutive negative PCR-based SARS-CoV-2 assays was 13 days for asymptomatic cases and 19 days for symptomatic cases (p = 0.002). CONCLUSION: Ship based quarantine was effective at reducing transmission of SARS-CoV-2 amongst Australian passengers, but the risk of infection was higher if an individual shared a cabin or was a close contact of a confirmed case. Managing COVID-19 in cruise ship passengers is challenging and requires enhanced health measures and access to onshore quarantine and isolation facilities.

Selecting the right anticoagulant for stroke prevention in atrial fibrillation.

OBJECTIVE: The embolization of thrombi formed within the atria can occur in any form of atrial fibrillation (AF), i.e., paroxysmal, persistent, or permanent. Although ischemic stroke is the most frequent embolic event associated with AF, embolization to other sites in the pulmonary and systemic circulations may occasionally occur. To avert the risk of embolization, long-term oral anticoagulation therapy is recommended for all AF patients if the CHA2DS2-VASC score is at least 1 for men and at least 2 for women. Since anticoagulant therapy is associated with an increased risk of bleeding, the choice of oral anticoagulant agent should be made by careful consideration of the benefit-to-risk ratio. The use of a newer class of direct oral anticoagulants (DOACs) as an alternative to the anti-vitamin K (AVK) anticoagulants (warfarin, acenocumarol, etc.) can help mitigate the need for periodic monitoring of International Normalized Ratio (INR) and adverse bleeding events that are commonly associated with the use of AVK anticoagulants. Though the use of DOACs (dabigatran, rivaroxaban, edoxaban, apixaban, etc.) is gaining ground due to their relative safety profile and the low overall cost, quite a few clinicians remain skeptical about their use. PATIENTS AND METHODS: Our objective was to evaluate the risk of thromboembolism, stroke, neuropsychiatric illness, depression, and dementia, in patients with non-valvular atrial fibrillation who have been treated with either acenocumarol or apixaban, as well as to see the inflammatory status (ESR) and levels of fibrinogen. Our team at Municipal Emergency University Hospital, Timisoara, Romania, conducted a retrospective study using the medical records of AF patients who were treated with either apixaban or acenocumarol between 2016-2019. We divided the patients into two groups and compared the groups for the aforementioned outcomes. RESULTS: AF patients who were prescribed apixaban had a lower rate of stroke and psychiatric illness compared to those on acenocumarol. No significant correlation was found in terms of risk of developing depression or dementia between the groups. CONCLUSIONS: Non-valvular AF patients on apixaban had lower rates of thromboembolic events than the patients on acenocumarol. This article will serve as a reminder of the positive health and financial outcomes of apixaban use, especially to those healthcare systems that are still oblivious to the decrease in economic burden and gain in quality-adjusted life years (QALY) by the long-term use of NOACS/ DOACS instead of the AVK anticoagulants.

The Healthy Crew, Clean Vessel, and Set Departure Date Triad: Successful Control of Outbreaks of COVID-19 On Board Four Cargo Vessels.

BACKGROUND: A variety of infectious diseases can cause outbreaks on board vessels, with both health and economic effects. Internationally, Coronavirus Disease 2019 (COVID-19) outbreaks have occurred on numerous cruise and cargo vessels and the containment measures, travel restrictions, and border closures continue to make it increasingly difficult for ship operators world-wide to be granted pratique, effect crew changes, and conduct trade. An effective outbreak management strategy is essential to achieve the outcome triad - healthy crew, clean vessel, and set departure date - while maintaining the safety of the on-shore workers and broader community and minimizing disruption to trade. This report describes the principles of COVID-19 outbreak responses on four cargo vessels, including the successful use of one vessel as a quarantine facility. METHODS: Established principles of management and the experiences of COVID-19 outbreaks on cruise ships elsewhere informed a health-lead, multi-agency, strict 14-day quarantine (Q) regime based on: population density reduction on board; crew segregation; vessel cleaning and sanitation; infection risk zones, access, and control measures; health monitoring; case identification and management; food preparation and delivery; waste management control; communication; and welfare and security. FINDINGS: Sixty-five crew were diagnosed with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection (range 2-25; attack rate 10%-81%; 15 asymptomatic). No deaths were recorded, and only one crew was hospitalized for COVID-19-related symptoms but did not require intensive care support. Catering crew were among the cases on three vessels. All non-essential crew (n-EC) and most of the cases were disembarked. During the vessel's Q period, no further cases were diagnosed on board, and no crew became symptomatic after completion of Q. The outbreak response duration was 15-17 days from initial decision.No serious health issues were reported, no response staff became infected, and only two Q protocol breaches occurred among crew. INTERPRETATION: Despite increasing risk of outbreaks on cargo vessels, maritime trade and crew exchanges must continue. The potential consequences of COVID-19 outbreaks to human life and to trade necessitate a balanced response. The principles described can offer health, financial, operational, and safety advantages.

A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A (KMT2A) gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor BCL2 and the proto-oncogene MYC However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses CASP9, as well as KMT2A-AFF1-driven FFLs that regulate BCL2 and MYC through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response.

Delusions of Certainty: Commercial Vessel COVID-19 Risk Stratification.

Since the World Health Organization's (WHO's) pandemic declaration on March 11, 2020, coronavirus disease 2019 (COVID-19) outbreaks have occurred on numerous maritime vessels and the containment measures, travel restrictions, and border closures continue to make it increasingly difficult for ship operators world-wide to be granted pratique, conduct trade, and conduct crew changes.Knowledge of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) circulating on-board a ship prior to its arrival has significant implications for the protection of shore-based maritime workers (ie, pilots, stevedores, and surveyors), the broader community, and trade. A useful approach is a graded assessment of the public health risk. The Western Australia (WA) experience and associated observed pitfalls in implementing the prediction equation for the potential presence of SARS-CoV-2 on-board based on five COVID-19 outbreaks on commercial and cruise vessels during 2020 is described.Despite best efforts, the qualitative and quantitative predictors of SARS-CoV-2 circulating on-board commercial vessels are failing to deliver the required certainty, and to date, the only accepted method of ascertaining the presence of SARS-CoV-2 remains the real-time reverse transcription polymerase chain reaction (rRT-PCR) testing reported by an accredited laboratory.Based on legal or regulatory requirements, germane processes, underpinned by robust and auditable processes and procedures, must be put in place to inform the risk assessment of SARS-CoV-2 circulating on-board vessels.

Successful Control of an Onboard COVID-19 Outbreak Using the Cruise Ship as a Quarantine Facility, Western Australia, Australia.

Onboard quarantining has been only partially effective to control outbreaks of coronavirus disease on cruise ships. We describe the successful use of the ship as a quarantine facility during the response to the outbreak on the MS Artania, which docked in Western Australia, Australia. The health-led 14-day quarantine regime was based on established principles of outbreak management and experiences of coronavirus disease outbreaks on cruise ships elsewhere. The attack rate in the crew was 3.3% (28/832) before quarantine commencement and 4.8% (21/441) during quarantine on board. No crew members became symptomatic after completion of quarantine. Infection surveillance involved telephone correspondence, face-to-face visits, and testing for severe acute respiratory syndrome coronavirus 2. No serious health issues were reported, no response staff became infected, and only 1 quarantine breach occurred among crew. Onboard quarantine could offer financial and operational advantages in outbreak response and provide reassurance to the shore-based wider community regarding risk for infection.

Harnessing tRNA for Processing Ability and Promoter Activity.

Transfer RNA (tRNA) and their associated production and processing machinery can be coopted as a versatile tool for the production of guide RNAs (gRNAs) for Cas9-based genome engineering. Using different tRNA variants enables the production of gRNAs at a variety of steady state levels. Furthermore, engineered tRNAs can be used to process gRNAs from Pol-II transcripts, thus enabling spatial/temporal control of gRNA expression. Here we describe the design, cloning, and testing of tRNA scaffolds for both Pol-III-driven expression of different levels of gRNAs, and for processing gRNAs from Pol-II transcripts.

Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs.

The type-II CRISPR-Cas9 system has been repurposed to create synthetic programmable transcriptional regulators (CRISPR-TRs). Subsequent modifications of the system now allow for spatiotemporal control of CRISPR-mediated gene activation and repression. Among these solutions, the development of inducible spacer-blocking hairpin guide RNAs (iSBH-sgRNAs) provide an easy to implement and versatile way to condition the activation of most CRISPR-TRs on the presence of a user defined inducer. In this chapter, I cover the know-how relating to the design and synthesis of iSBH-sgRNAs, as well as the implementation in mammalian cells of inducible CRISPR-TR strategies based on this technology.

A Gaussian Process Model of Human Electrocorticographic Data.

We present a model-based method for inferring full-brain neural activity at millimeter-scale spatial resolutions and millisecond-scale temporal resolutions using standard human intracranial recordings. Our approach makes the simplifying assumptions that different people's brains exhibit similar correlational structure, and that activity and correlation patterns vary smoothly over space. One can then ask, for an arbitrary individual's brain: given recordings from a limited set of locations in that individual's brain, along with the observed spatial correlations learned from other people's recordings, how much can be inferred about ongoing activity at other locations throughout that individual's brain? We show that our approach generalizes across people and tasks, thereby providing a person- and task-general means of inferring high spatiotemporal resolution full-brain neural dynamics from standard low-density intracranial recordings.