Targeted integration in CHO cells using CRIS-PITCh/Bxb1 recombinase-mediated cassette exchange hybrid system.

Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.

Hyperspectral Infrared Observations of Arctic Snow, Sea Ice, and Non-Frozen Ocean from the RV Polarstern during the MOSAiC Expedition October 2019 to September 2020.

This study highlights hyperspectral infrared observations from the Marine-Atmospheric Emitted Radiance Interferometer (M-AERI) collected as part of the Department of Energy (DOE) Atmospheric Radiation Measurement (ARM) Mobile Facility (AMF) deployment on the icebreaker RV Polarstern during the Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) expedition from October 2019 to September 2020. The ARM M-AERI directly measures the infrared radiance emission spectrum between 520 cm-1 and 3000 cm-1 (19.2-3.3 µm) at 0.5 cm-1 spectral resolution. These ship-based observations provide a valuable set of radiance data for the modeling of snow/ice infrared emission as well as validation data for the assessment of satellite soundings. Remote sensing using hyperspectral infrared observations provides valuable information on sea surface properties (skin temperature and infrared emissivity), near-surface air temperature, and temperature lapse rate in the lowest kilometer. Comparison of the M-AERI observations with those from the DOE ARM meteorological tower and downlooking infrared thermometer are generally in good agreement with some notable differences. Operational satellite soundings from the NOAA-20 satellite were also assessed using ARM radiosondes launched from the RV Polarstern and measurements of the infrared snow surface emission from the M-AERI showing reasonable agreement.

classifieR a flexible interactive cloud-application for functional annotation of cancer transcriptomes.

BACKGROUND: Transcriptionally informed predictions are increasingly important for sub-typing cancer patients, understanding underlying biology and to inform novel treatment strategies. For instance, colorectal cancers (CRCs) can be classified into four CRC consensus molecular subgroups (CMS) or five intrinsic (CRIS) sub-types that have prognostic and predictive value. Breast cancer (BRCA) has five PAM50 molecular subgroups with similar value, and the OncotypeDX test provides transcriptomic based clinically actionable treatment-risk stratification. However, assigning samples to these subtypes and other transcriptionally inferred predictions is time consuming and requires significant bioinformatics experience. There is no "universal" method of using data from diverse assay/sequencing platforms to provide subgroup classification using the established classifier sets of genes (CMS, CRIS, PAM50, OncotypeDX), nor one which in provides additional useful functional annotations such as cellular composition, single-sample Gene Set Enrichment Analysis, or prediction of transcription factor activity. RESULTS: To address this bottleneck, we developed classifieR, an easy-to-use R-Shiny based web application that supports flexible rapid single sample annotation of transcriptional profiles derived from cancer patient samples form diverse platforms. We demonstrate the utility of the " classifieR" framework to applications focused on the analysis of transcriptional profiles from colorectal (classifieRc) and breast (classifieRb). Samples are annotated with disease relevant transcriptional subgroups (CMS/CRIS sub-types in classifieRc and PAM50/inferred OncotypeDX in classifieRb), estimation of cellular composition using MCP-counter and xCell, single-sample Gene Set Enrichment Analysis (ssGSEA) and transcription factor activity predictions with Discriminant Regulon Expression Analysis (DoRothEA). CONCLUSIONS: classifieR provides a framework which enables labs without access to a dedicated bioinformation can get information on the molecular makeup of their samples, providing an insight into patient prognosis, druggability and also as a tool for analysis and discovery. Applications are hosted online at https://generatr.qub.ac.uk/app/classifieRc and https://generatr.qub.ac.uk/app/classifieRb after signing up for an account on https://generatr.qub.ac.uk .

Current UK clinical practice in diagnosing dementia in younger adults: compliance with quality indicators in electronic health records from mental health trusts.

OBJECTIVES: To examine current UK practice in diagnosis of patients under 65 with young onset dementia, within 5 years of date of diagnosis, identified from electronic health records of 8 NHS mental health trusts. METHODS: Patients diagnosed with young onset dementia were assembled from the UK-Clinical Record Interactive System, (UK-CRIS) using diagnosis of dementia as the index date. A pre-designed proforma, derived by international Delphi consensus from experts in the field in previous work, was used to assess components of the diagnostic assessment in 402 electronic health records across 8 NHS sites. Information was extracted on key aspects of clinical and physical examination according to both a minimum and gold standard. RESULTS: Percentage compliance rates analysed by NHS site and statement, including compliance for site for minimum standard (11 statements), the additional 20 statements required for Gold standard, and the complete Gold standard set (31 statements) show that the additional 20 statements in the Gold standard had consistently higher compliance rates for every site compared to the minimum set. CONCLUSION: Findings confirmed variation in clinical practice and identified commonly missed items in examination and enquiry compared to expert consensus. This suggests that a template proforma, which contains the key indicators for comprehensive assessment of dementia in young adults according to a quality standard could help support clinicians to improve record keeping and reduce gaps in knowledge.

How to measure the need for transition to adult services among young people with Attention Deficit Hyperactivity Disorder (ADHD): a comparison of surveillance versus case note review methods.

BACKGROUND: Health services have not provided adequate support for young people with long term health conditions to transfer from child to adult services. National Institute of Health and Care (NICE) guidance on transition has been issued to address these gaps. However, data are often sparse about the number of young adults who might need to transition. Using Attention Deficit Hyperactivity Disorder (ADHD) as an exemplar, this study used an existing surveillance system and a case note review to capture the incidence of the transition process, and compared and contrasted the findings. METHODS: The Child and Adolescent Psychiatry Surveillance System (CAPSS) was used to estimate the incident transition of young people with Attention Deficit Hyperactivity Disorder (ADHD) from child to adult services. This involves consultant child and adolescent psychiatrists from the United Kingdom (UK) and Republic of Ireland (ROI) reporting relevant young people as they are seen in clinics. In parallel, a case note review was conducted using the Maudsley Biomedical Research Centre (BRC) Clinical Records Interactive Search (CRIS). The study period ran for twelve months with a nine month follow up to see how the transition proceeded. RESULTS: CRIS identified 76 cases in the study period, compared to 18 identified using surveillance via CAPSS. Methodological issues were experienced using both methods. Surveillance issues; eligibility criteria confusion, reporting errors, incomplete questionnaires, difficulties contacting clinicians, and surveillance systems do not cover non-doctors and psychiatrists who are not consultants. Case note review issues using CRIS included the need for researchers to interpret clinical notes, the availability and completeness of data in the notes, and data limited to the catchment of one particular mental health trust. CONCLUSIONS: Both methods demonstrate strengths and weaknesses; the combination of both methods in the absence of strong routinely collected data, allowed a more robust estimate of the level of need for service planning and commissioning.

Services for people at high risk improve outcomes in patients with first episode psychosis.

OBJECTIVE: About one-third of patients referred to services for people at high risk for psychosis may have already developed a first episode of psychosis (FEP). We compared clinical outcomes in FEP patients who presented to either high risk or conventional mental health services. METHOD: Retrospective study comparing duration of hospital admission, referral-to-diagnosis time, need for compulsory hospital admission and frequency of admission in patients with FEP who initially presented to a high-risk service (n = 164) to patients with FEP who initially presented to conventional mental health services (n = 2779). Regression models were performed, controlling for several confounders. RESULTS: FEP patients who had presented to a high-risk service spent 17 fewer days in hospital [95% CI: -33.7 to (-0.3)], had a shorter referral-to-diagnosis time [B coefficient -74.5 days, 95% CI: -101.9 to -(47.1)], a lower frequency of admission [IRR: 0.49 (95% CI: 0.39-0.61)] and a lower likelihood of compulsory admission [OR: 0.52 (95% CI: 0.34-0.81)] in the 24 months following referral, as compared to FEP patients who were first diagnosed at conventional services. CONCLUSION: Services for people at high risk for psychosis are associated with better clinical outcomes in patients who are already psychotic.

Constraining East Asia ammonia emissions through satellite observations and iterative Finite Difference Mass Balance (iFDMB) and investigating its impact on inorganic fine particulate matter.

Uncertainty in ammonia (NH3) emissions causes the inaccuracy of fine particulate matter simulations, which is associated with human health. To address this uncertainty, in this work, we employ the iterative finite difference mass balance (iFDMB) technique to revise NH3 emissions over East Asia using the Cross-track Infrared Sounder (CRIS) satellite for July, August, and September 2019. Compared to the emissions, the revised NH3 emissions show an increase in China, particularly in the North China Plain (NCP) region, corresponding to agricultural land use in July, August, and September and a decrease in South Korea in September. The enhancement in NH3 emissions resulted in a remarkable increase in concentrations of NH3 by 5 ppb. in July and September, there is an increase in ammonium (NH4+) and nitrate (NO3-) concentrations by 5 µg/m3, particularly in the NCP region, while in August, both NH4+ and NO3- concentrations exhibit a decrease. For sulfate (SO42-), in August and September, the concentrations decreased over most regions of China and Taiwan, as a result of the production of ammonium sulfate; increased concentrations of SO42-, however, were simulated over South Korea, Japan, and the southern region of Chengdu, caused by higher relative humidity (RH). In contrast, during the month of July, our simulations showed an increase in SO42- concentrations over most regions of China. To gain a more comprehensive understanding, we defined a sulfur conversion ratio ( [Formula: see text] ), which explains how changes in sulfur in the gas phase affect changes in sulfate concentrations. A subsequent sensitivity analysis performed in this study indicated the same relationship between changes in ammonia and its effect on inorganic fine particulate matter (PM2.5). This study highlights the challenge of controlling and managing inorganic PM2.5 and indicates that reducing the emissions of air pollutants do not necessarily lead to a reduction in their concentrations.

Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.

Peroxisomes are multifunctional, ubiquitous, and dynamic organelles. They are responsible for diverse metabolic and physiological functions and communicate with other organelles, including the ER, mitochondria, lipid droplets, and lysosomes, through membrane contact sites. However, despite their importance for healthy cell function, remarkably, little is known about how peroxisomes and peroxisomal proteins are regulated under physiological conditions in human cells. Here, we present a method to generate reporter cell lines to measure endogenous expression of peroxisomal proteins of interest. By CRISPR-mediated knock-in of an easily detectable protein-coding tag in-frame into the relevant genomic loci, endogenous levels of the protein of interest in a cell population can be quantified in a high-throughput manner under different conditions. This has important implications for the fundamental understanding of how peroxisomal proteins are regulated and may reveal the therapeutic potential of modulating peroxisomal protein expression to improve cell performance.

High-Throughput Analysis of CRISPR-Cas9 Editing Outcomes in Cell and Animal Models Using CRIS.py.

Genome editing using the CRISPR-Cas9 platform creates precise modifications in cells and whole organisms. Although knockout (KO) mutations can occur at high frequencies, determining the editing rates in a pool of cells or selecting clones that contain only KO alleles can be a challenge. User-defined knock-in (KI) modifications are achieved at much lower rates, making the identification of correctly modified clones even more challenging. The high-throughput format of targeted next-generation sequencing (NGS) provides a platform allowing sequence information to be gathered from a one to thousands of samples. However, it also poses a challenge in terms of analyzing the large amount of data that is generated. In this chapter, we present and discuss CRIS.py, a simple and highly versatile Python-based program for analyzing NGS data for genome-editing outcomes. CRIS.py can be used to analyze sequencing results for any kind of modification or multiplex modifications specified by the user. Moreover, CRIS.py runs on all fastq files found in a directory, thereby concurrently analyzing all uniquely indexed samples. CRIS.py results are consolidated into two summary files, which allows users to sort and filter results and quickly identify the clones (or animals) of greatest interest.

Comparison between two patient-reported outcome measures for patients with cervical radiculopathy: A think-aloud study.

BACKGROUND: The Cervical Radiculopathy Impact Scale (CRIS) and Patient Specific Functional Scale 2.0 (PSFS 2.0) are patient-reported outcome measures (PROMs) used to assess activity limitations in patients with cervical radiculopathy (CR). This study a) compared the CRIS subscale 3 and the PSFS 2.0 in patients with CR with respect to completeness and patient preference, b) established the correlation between both PROMs in assessing the individual patient's level of functional limitations and c) assessed the frequency of reported functional limitations. METHODS: Participants with CR participated in semi-structured, individual, face-to-face interviews as part of a "think-aloud" process; verbalising their thoughts while completing both PROMs. Sessions were digitally recorded and transcribed verbatim for analysis. RESULTS: Twenty-two patients were recruited. The most frequently reported functional limitation on the CRIS was: 'working at a computer' (n = 17) and overhead activities' (n = 10) for the PSFS 2.0. There was significant moderate positive correlation between the scores on the PSFS 2.0 and the CRIS (Spearman's r = 0.55, n = 22 p = .008). Most patients (n = 18; 82%) preferred the ability to present their own individual functional limitations of the PSFS 2.0. Eleven participants (50%) preferred the 11-point scale of the PSFS 2.0 over the 5-point Likert scale scoring option of the CRIS. CONCLUSION: Both easy to complete PROMs capture functional limitations in patients with CR. Most patients prefer the PSFS 2.0 over the CRIS. The wording and layout of both PROMs need refinement to enhance user-friendliness and avoid misinterpretation.

ORCID coverage in research institutions-Readiness for partially automated research reporting.

Reporting and presentation of research activities and outcome for research institutions in official, normative standards are more and more important and are the basis to comply with reporting duties. Institutional Current Research Information Systems (CRIS) serve as important databases or data sources for external and internal reporting, which should ideally be connected with interfaces to the operational systems for automated loading routines to extract relevant research information. This investigation evaluates whether (semi-) automated reporting using open, public research information collected via persistent identifiers (PIDs) for organizations (ROR), persons (ORCID), and research outputs (DOI) can reduce effort of reporting. For this purpose, internally maintained lists of persons to whom an ORCID record could be assigned (internal ORCID person lists) of two different German research institutions-Osnabrück University (UOS) and the non-university research institution TIB-Leibniz Information Center for Science and Technology Hannover-are used to investigate ORCID coverage in external open data sources like FREYA PID Graph (developed by DataCite), OpenAlex and ORCID itself. Additionally, for UOS a detailed analysis of discipline specific ORCID coverage is conducted. Substantial differences can be found for ORCID coverage between both institutions and for each institution regarding the various external data sources. A more detailed analysis of ORCID distribution by discipline for UOS reveals disparities by research area-internally and in external data sources. Recommendations for future actions can be derived from our results: Although the current level of coverage of researcher IDs which could automatically be mapped is still not sufficient to use persistent identifier-based extraction for standard (automated) reporting, it can already be a valuable input for institutional CRIS.

4D-Var Inversion of European NH3 Emissions Using CrIS NH3 Measurements and GEOS-Chem Adjoint With Bi-Directional and Uni-Directional Flux Schemes.

We conduct the first 4D-Var inversion of NH3 accounting for NH3 bi-directional flux, using CrIS satellite NH3 observations over Europe in 2016. We find posterior NH3 emissions peak more in springtime than prior emissions at continental to national scales, and annually they are generally smaller than the prior emissions over central Europe, but larger over most of the rest of Europe. Annual posterior anthropogenic NH3 emissions for 25 European Union members (EU25) are 25% higher than the prior emissions and very close (<2% difference) to other inventories. Our posterior annual anthropogenic emissions for EU25, the UK, the Netherlands, and Switzerland are generally 10%-20% smaller than when treating NH3 fluxes as uni-directional emissions, while the monthly regional difference can be up to 34% (Switzerland in July). Compared to monthly mean in-situ observations, our posterior NH3 emissions from both schemes generally improve the magnitude and seasonality of simulated surface NH3 and bulk NH x wet deposition throughout most of Europe, whereas evaluation against hourly measurements at a background site shows the bi-directional scheme better captures observed diurnal variability of surface NH3. This contrast highlights the need for accurately simulating diurnal variability of NH3 in assimilation of sun-synchronous observations and also the potential value of future geostationary satellite observations. Overall, our top-down ammonia emissions can help to examine the effectiveness of air pollution control policies to facilitate future air pollution management, as well as helping us understand the uncertainty in top-down NH3 emissions estimates associated with treatment of NH3 surface exchange.

Study design for a randomized clinical trial of cognitive-behavioral therapy for posttraumatic headache.

Posttraumatic headache (PTH) is a common debilitating condition arising from head injury and is highly prevalent among military service members and veterans with traumatic brain injury (TBI). Diagnosis and treatment for PTH is still evolving, and surprisingly little is known about the putative mechanisms that drive these headaches. This manuscript describes the design of a randomized clinical trial of two nonpharmacological (i.e., behavioral) interventions for posttraumatic headache. Design of this trial required careful consideration of PTH diagnosis and inclusion criteria, which was challenging due to the lack of standard clinical characteristics in PTH unique from other types of headaches. The treatments under study differed in clinical focus and dose (i.e., number of treatment sessions), but the trial was designed to balance the treatments as well as possible. Finally, while the primary endpoints for pain research can vary from assessments of pain intensity to objective and subjective functional measures, this trial of PTH interventions chose carefully to establish clinically relevant endpoints and to maximize the opportunity to detect significant differences between groups with two primary outcomes. All these issues are discussed in this manuscript.

Cris never left you wondering about where you stood! His motto was: Just do it!

In this commentary, we reflect on our experiences being PhD students of Prof. Cris dos Remedios in the Muscle Research Unit at The University of Sydney at the turn of the new millennium. Cris was/is an example of a fine scientist and a great academic mentor for us and so many others (scientists, academics, surgeons, medical doctors and health professionals) who carry the legacy and traditions of Cris dos Remedios into the future.

The Clinical Efficacy of a Pure Alginate Formulation (Lamina G) for Controlling Symptoms in Individuals with Reflux Symptoms: A Randomized Clinical Study.

Background/Aims: Bicarbonate-containing alginate formulations are reported to be effective for controlling reflux symptoms. However, the efficacy of Lamina G alginate without gas production has not been reported. The aim is to evaluate the efficacy of a non-bicarbonate alginate in individuals with reflux symptoms without reflux esophagitis. Methods: Participants who had experienced heartburn or regurgitation for 7 consecutive days were randomized to one of the following treatment groups: proton pump inhibitors (PPI) plus alginate (combination) or PPI plus placebo (PPI only). In addition, as a reference group, patients received placebo plus alginate (alginate only). The primary endpoint compared the percentage of patients with complete resolution of symptoms for the final 7 days of the treatment. Secondary endpoints compared changes in symptom score, symptom-free days during the treatment period, the Reflux Disease Questionnaire, Patient Assessment of Upper Gastrointestinal Disorders (PAGI)-Quality of Life and PAGI-Symptoms Severity Index scores, the investigator's assessment of symptoms, and incidence of adverse events. Results: Complete resolution of heartburn or regurgitation was not significantly different between the combination and PPI only groups (58.7% vs 57.5%, p=0.903). The secondary endpoints were not significantly different between the two groups. Complete resolution of heartburn or regurgitation, did not differ between the alginate only reference group and the PPI only group (75.0% vs 57.5%, p=0.146). Conclusions: The addition of non-bicarbonate alginate to PPI was no more effective than PPI alone in controlling reflux symptoms.

Outcomes associated with use of the Cancer Risk Intake System among primary care safety-net patients identified as needing colorectal cancer screening.

Despite demonstrated primary and secondary prevention benefits, screening for colorectal cancer (CRC) is sub-optimal. We implemented the Cancer Risk Intake System (CRIS) among a convenience sample of patients presenting for primary care in Dallas County safety-net clinics. CRIS, which assesses individuals' CRC risks and generates guideline-based screening recommendations for them and their providers, had been found in a randomized trial to facilitate risk-based screening, compared to usual care. Here, of 924 patients ages ≥50 who used CRIS, 699 were identified as needing screening, with 39.2% needing colonoscopy rather than FIT. However, following use of CRIS and patients' and providers' receipt of guideline-concordant recommendations, 20.9% elevated-risk patients received no screening orders, only 44.1% received guideline-concordant colonoscopy orders, and less than half of these (48.4%) completed colonoscopy. Guideline-concordant screening orders were more common for average-risk patients (62.5% received orders for FIT and 26.6% for colonoscopy). However, like their elevated-risk counterparts who received screening orders, more than half of average-risk patients in each order group (52.3% for FIT and 52.8% for colonoscopy) did not complete screening. We found no correlates for receiving screening orders, but higher comorbidity scores were associated with less screening completion among the average-risk group. We had hoped CRIS would facilitate risk-based screening but, although orders for and receipt of colonoscopy were more common for elevated- than average-risk patients, they were still suboptimal in this clinical setting with a "FIT-first" strategy. A stronger intervention may be necessary to increase guideline-concordant recommendations and screening among patients 50 and older.

Recorded poor insight as a predictor of service use outcomes: cohort study of patients with first-episode psychosis in a large mental healthcare database.

OBJECTIVES: To investigate recorded poor insight in relation to mental health and service use outcomes in a cohort with first-episode psychosis. DESIGN: We developed a natural language processing algorithm to ascertain statements of poor or diminished insight and tested this in a cohort of patients with first-episode psychosis. SETTING: The clinical record text at the South London and Maudsley National Health Service Trust in the UK was used. PARTICIPANTS: We applied the algorithm to characterise a cohort of 2026 patients with first-episode psychosis attending an early intervention service. PRIMARY AND SECONDARY OUTCOME MEASURES: Recorded poor insight within 1 month of registration was investigated in relation to (1) incidence of psychiatric hospitalisation, (2) odds of legally enforced hospitalisation, (3) number of days spent as a mental health inpatient and (4) number of different antipsychotic agents prescribed; outcomes were measured over varying follow-up periods from 12 months to 60 months, adjusting for a range of sociodemographic and clinical covariates. RESULTS: Recorded poor insight, present in 48.9% of the sample, was positively associated with youngest and oldest age groups, unemployment and schizophrenia (compared with bipolar disorder) and was negatively associated with Asian ethnicity, married status, home ownership and recorded cannabis use. It was significantly associated with higher levels of all four outcomes over the succeeding 12 months. Associations with hospitalisation incidence and number of antipsychotics remained independently significant when measured over 60 and 48 months, respectively. CONCLUSIONS: Recorded poor insight in people with recent onset psychosis predicted higher subsequent inpatient mental healthcare use. Improving insight might benefit patients' course of illness as well as reduce mental health service use.

Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis.

The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in KrasG12D-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) ß-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically.

Novel agonist of α4ß2* neuronal nicotinic receptor with antinociceptive efficacy in rodent models of acute and chronic pain.

OBJECTIVE: To demonstrate the antinociceptive and antihypersensitivity mechanisms of Cris-104 (1-{2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl]ethyl}piperidine), a novel selective α4ß2* nicotinic acetylcholine receptor (nAChR) agonist, in rodent acute/inflammatory and chronic pain models. MATERIALS AND METHODS: Hot-plate and formalin tests in mice were used to examine Cris-104-induced antinociceptive effects on thermal/inflammatory pain. Cris-104 effects on hypersensitivity, norepinephrine (NE) release in the spinal dorsal horn, and neuronal activity in the locus coeruleus (LC) were examined in rats with lumbar spinal nerve ligation using behavioral, microdialysis, and extracellular recording methods. Cris-104 effects on spontaneous locomotion were examined in an open-field test. RESULTS: Cris-104 induced dose-dependent antinociception effects in hot-plate and formalin tests, and these effects were blocked by the general nAChR antagonist mecamylamine, the selective α4ß2* nAChR antagonist dihydro-beta-erythroidine, and the α2-adrenoceptor antagonist yohimbine, but not by the α1-adrenoceptor antagonist prazosin. Systemic and spinally perfused Cris-104 increased NE concentrations in microdialysates from the spinal cord in both normal and SNL rats. Systemic Cris-104 increased neuronal activity in the LC of normal rats. Mecamylamine blocked the effects of Cris-104 on spinal NE release and LC neuronal activity. Systemic Cris-104 did not affect locomotor activity significantly. CONCLUSION: The α4ß2 neuronal nAChR agonist, Cris-104, was effective for treatment of pain via descending noradrenergic inhibition of pain signaling.

Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.