Detection of the HIV-1 Accessory Proteins Nef and Vpu by Flow Cytometry Represents a New Tool to Study Their Functional Interplay within a Single Infected CD4+ T Cell.

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.

Investigation of Early-Stage Non-Small Cell Lung Cancer Patients with Different T2 Descriptors: Real Word Data From a Large Database.

INTRODUCTION: The current study evaluated a large cohort of T2N0M0 NSCLC patients with different T2 descriptors to investigate the prognostic disparities and further externally validate the T category of these patients. METHODS: The Kaplan-Meier Method with the log-rank test was used to plot survival curves. The propensity score matching (PSM) method was used to reduce bias. Univariable and multivariable Cox analyses were used to determine prognostic factors. RESULTS: A total of 13,015 eligible T2N0M0 NSCLC patients were included. There were 5,287, 2,577 and 5,151 patients in the T2a, T2b and non-sized determined T2N0M0 (T2non-sized) groups, respectively. Before PSM, the survival of T2non-sized patients was comparable to that of T2a patients (P = 0.080) but was superior to that of T2b patients (P < 0.001). After PSM, the survival of T2non-sized patients was inferior to that of T2a patients (P = 0.028) but was similar to that of T2b patients (P = 0.325). The T category was further subdivided based on the specific non-sized T2 descriptors and tumor size. The results of the multivariate Cox analysis found that the prognosis of T2 tumors with visceral pleural invasion (size: 0-30 mm) was better than that of T2a tumors, and the prognosis of T2 tumors with visceral pleural invasion (size: 30-40 mm) was inferior to that of T2a tumors but comparable to that of T2b tumors. CONCLUSION: T2 tumors with visceral pleural invasion (size: 30-40 mm) should be assigned to the T2b category, and those with a size interval of 0-30 mm should be assigned to a better prognostic T2a category.

Loss of Oxidation Resistance 1, OXR1, Is Associated with an Autosomal-Recessive Neurological Disease with Cerebellar Atrophy and Lysosomal Dysfunction.

We report an early-onset autosomal-recessive neurological disease with cerebellar atrophy and lysosomal dysfunction. We identified bi-allelic loss-of-function (LoF) variants in Oxidative Resistance 1 (OXR1) in five individuals from three families; these individuals presented with a history of severe global developmental delay, current intellectual disability, language delay, cerebellar atrophy, and seizures. While OXR1 is known to play a role in oxidative stress resistance, its molecular functions are not well established. OXR1 contains three conserved domains: LysM, GRAM, and TLDc. The gene encodes at least six transcripts, including some that only consist of the C-terminal TLDc domain. We utilized Drosophila to assess the phenotypes associated with loss of mustard (mtd), the fly homolog of OXR1. Strong LoF mutants exhibit late pupal lethality or pupal eclosion defects. Interestingly, although mtd encodes 26 transcripts, severe LoF and null mutations can be rescued by a single short human OXR1 cDNA that only contains the TLDc domain. Similar rescue is observed with the TLDc domain of NCOA7, another human homolog of mtd. Loss of mtd in neurons leads to massive cell loss, early death, and an accumulation of aberrant lysosomal structures, similar to what we observe in fibroblasts of affected individuals. Our data indicate that mtd and OXR1 are required for proper lysosomal function; this is consistent with observations that NCOA7 is required for lysosomal acidification.

Cost effectiveness of intermediate-risk squamous cell carcinoma treated with Mohs micrographic surgery compared with wide local excision.

BACKGROUND: The efficacy of Mohs micrographic surgery (MMS) in treating cutaneous squamous cell carcinoma has been demonstrated. The cost effectiveness of MMS has rarely been studied to support the perceived higher cost. OBJECTIVE: Perform a cost-effectiveness analysis to determine whether MMS is cost effective over wide local excision (WLE) for Brigham and Women's Hospital tumor stage T2a cutaneous squamous cell carcinoma over a 5-year period. METHODS: A Markov model with a 5-year time horizon was created using variables from published data. Costs in United States dollars and quality-adjusted life-years (QALY) were calculated. RESULTS: MMS was $333.83 less expensive ($4365.57 [95% CI, $3664.68-$6901.66] vs $4699.41 [95% CI, $3782.94-$10,019.31]) than WLE. MMS gained 2.22 weeks of perfect health (3.776 QALY [95% CI, 3.774-3.777] for MMS and 3.733 QALY [95% CI, 3.728-3.777]) over 5 years. The incremental cost-effectiveness ratio was -$7,822.19. MMS had a 99.9% probability of being more cost effective than WLE. Annualized savings of choosing MMS over WLE would be $200 million and over 25,000 QALY. MMS could cost 3.1 times its current rate and remain cost effective. LIMITATIONS: Relied on data from external retrospective sources. CONCLUSION: MMS is less costly and more effective than WLE and should be strongly considered for stage T2a cSCC, given improvements in costs and QALY.

A Well-Controlled BioID Design for Endogenous Bait Proteins.

The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate the overexpression of bait proteins. Control cell lines are typically nonengineered cells or engineered clones, implying a considerable risk for artifacts because of clonal variation. Genome engineering can also transform BioID, a proximity labeling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, resulting in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins wherein we introduce a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering, leading to bicistronic expression of both p53 and BirA* under control of the endogenous promoter. By targeting a Cas9-cytidine deaminase base editor to the T2A autocleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. Using quantitative proteomics we show significant benefits over the classical ectopic expression of p53-BirA*, and we provide a first well-controlled view of the proximal proteins of endogenous p53 in colon carcinoma cells. This novel application for base editors expands the CRISPR/Cas9 toolbox and can be a valuable addition for synthetic biology.

An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast.

OBJECTIVES: Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences. RESULTS: Here, we have constructed a T2A-Leu2 system for selection of yeast surface display libraries that blocks amplification of "stop codon" plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A-Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast. CONCLUSIONS: The method highlights the utility of the T2A-Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.

Impairment of a membrane-targeting protein translated from a downstream gene of a "self-cleaving" T2A peptide conjunction.

The requirement for reliable bicistronic or multicistronic vectors in gene delivery systems is at the forefront of bio/biomedical technology. A method that provides an efficient co-expression of multiple heterologous proteins would be valuable for many applications, especially in medical science for treating various types of disease. In this study, we designed and constructed a bicistronic expression vector using a self-cleaving 2A peptide derived from a virus of the insect Thosea asigna (T2A). This exhibited the most efficient cleavage of the 2A sequence. Two versions of the T2A-based vector were constructed by switching the DNA sequences encoding the proteins of interest, the N-myristoylated protein and the nuclear-homing protein, upstream and downstream of the 2A linker, respectively. Our results showed that similar levels of mRNA expression were found and 100% of cleavage efficiency of T2A was observed. Nevertheless, we also reported the cleared evidence that the N-myristoylated protein cannot be placed downstream of the 2A sequence. Since the protein product fails to translocate to the plasma membrane due to altered myristoylation process, the gene position of the T2A-based vector is meaningful for the subcellular localization of the N-myristoylated protein. Therefore, the observation was marked as a precaution for using the 2A peptide. To adopt the 2A peptide technology for generating the bicistronic or multicistronic expression, the vector design should be carefully considered for the transgene position, signal sequences, and post-translational modifications of each individual protein.

Robot-assisted partial nephrectomy for large renal masses: a multi-institutional series.

OBJECTIVES: To compare peri-operative outcomes after robot-assisted partial nephrectomy (RAPN) for cT2a (7 to <10 cm) to cT1 tumours. MATERIALS AND METHODS: Patients with a cT1a (n = 1 358, 76.4%), cT1b (n = 379, 21.3%) or cT2a (n = 41, 2.3%) renal mass were identified from a multi-institutional RAPN database. Intra- and postoperative outcomes were compared for cT2a masses vs cT1a and cT1b masses using multivariable regression models (linear, logistic, Poisson etc.), adjusting for operating surgeon and a modified R.E.N.A.L. nephrometry score that excluded the radius component. RESULTS: The median sizes for cT1a, cT1b and cT2a tumours were 2.5, 5.0 and 8.0 cm, respectively (P < 0.001) with modified R.E.N.A.L. nephrometry scores being 6.0, 6.5 and 7.0, respectively (cT1a, P < 0.001; cT1b, P = 0.105). RAPN for cT2a vs cT1a masses was associated with a 12% increase in operating time (P < 0.001), a 32% increase in estimated blood loss (P < 0.001), a 7% increase in ischaemia time (P = 0.008), a 3.93 higher odds of acute kidney injury at discharge (95% confidence interval [CI] 1.33, 8.76; P = 0.009) and a higher risk of recurrence (hazard ratio [HR] 10.9, 95% CI 1.31, 92.2; P = 0.027). RAPN for cT2a vs cT1b masses was associated with a 12% increase in blood loss (P = 0.036), a 5% increase in operating time (P = 0.062) and a marginally higher risk of recurrence (HR 11.2, 95% CI 0.77, 11.5; P = 0.059). RAPN for cT2a tumours was not associated with differences in complications (cT1a, P = 0.535; cT1b, P = 0.382), positive margins (cT1a, P = 0.972; cT1b, P = 0.681), length of stay (cT1a, P = 0.507; cT1b, P = 0.513) or renal function decline up to 24 months post-RAPN (cT1a, P = 0.124; cT1b, P = 0.467). CONCLUSION: For T2a tumours RAPN is a feasible treatment option in a select patient population when performed by experienced surgeons in institutions equipped to manage postoperative complications. Although RAPN was associated with greater blood loss and longer operating and ischaemia time in T2a tumours, it was not associated with greater complication or positive surgical margin rates compared with T1 tumours. Renal function preservation rates were equivalent for up to 24 months postoperatively; however, 12-month recurrence-free survival was significantly lower in the T2a group. Extended follow-up is required to further evaluate long-term survival.

Improved estimation of myelin water fraction using complex model fitting.

In gradient echo (GRE) imaging, three compartment water modeling (myelin water, axonal water and extracellular water) in white matter has been demonstrated to show different frequency shifts that depend on the relative orientation of fibers and the B0 field. This finding suggests that in GRE-based myelin water imaging, a signal model may need to incorporate frequency offset terms and become a complex-valued model. In the current study, three different signal models and fitting approaches (a magnitude model fitted to magnitude data, a complex model fitted to magnitude data, and a complex model fitted to complex data) were investigated to address the reliability of each model in the estimation of the myelin water signal. For the complex model fitted to complex data, a new fitting approach that does not require background phase removal was proposed. When the three models were compared, the results from the new complex model fitting showed the most stable parameter estimation.

Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease.

BACKGROUND & AIMS: With the increasing prevalence of liver disease worldwide, there is an urgent clinical need for reliable methods to diagnose and stage liver pathology. Liver biopsy, the current gold standard, is invasive and limited by sampling and observer dependent variability. In this study, we aimed to assess the diagnostic accuracy of a novel magnetic resonance protocol for liver tissue characterisation. METHODS: We conducted a prospective study comparing our magnetic resonance technique against liver biopsy. The individual components of the scanning protocol were T1 mapping, proton spectroscopy and T2* mapping, which quantified liver fibrosis, steatosis and haemosiderosis, respectively. Unselected adult patients referred for liver biopsy as part of their routine care were recruited. Scans performed prior to liver biopsy were analysed by physicians blinded to the histology results. The associations between magnetic resonance and histology variables were assessed. Receiver-operating characteristic analyses were also carried out. RESULTS: Paired magnetic resonance and biopsy data were obtained in 79 patients. Magnetic resonance measures correlated strongly with histology (r(s)=0.68 p<0.0001 for fibrosis; r(s)=0.89 p<0.001 for steatosis; r(s)=-0.69 p<0.0001 for haemosiderosis). The area under the receiver operating characteristic curve was 0.94, 0.93, and 0.94 for the diagnosis of any degree of fibrosis, steatosis and haemosiderosis respectively. CONCLUSION: The novel scanning method described here provides high diagnostic accuracy for the assessment of liver fibrosis, steatosis and haemosiderosis and could potentially replace liver biopsy for many indications. This is the first demonstration of a non-invasive test to differentiate early stages of fibrosis from normal liver.

Dual MRI T1 and T2(*) contrast with size-controlled iron oxide nanoparticles.

Contrast-enhancing magnetic resonance mechanism, employing either positive or negative signal changes, has contrast-specific signal characteristics. Although highly sensitive, negative contrast typically decreases the resolution and spatial specificity of MRI, whereas positive contrast lacks a high contrast-to-noise ratio but offers high spatial accuracy. To overcome these individual limitations, dual-contrast acquisitions were performed using iron oxide nanoparticles and a pair of MRI acquisitions. Specifically, vascular signals in MR angiography were positively enhanced using ultrashort echo (UTE) acquisition, which provided highly resolved vessel structures with increased vessel/tissue contrast. In addition, fast low angle shot (FLASH) acquisition yielded strong negative vessel contrast, resulting in the higher number of discernible vessel branches than those obtained from the UTE method. Taken together, the high sensitivity of the negative contrast delineated ambiguous vessel regions, whereas the positive contrast effectively eliminated the false negative contrast areas (e.g., airways and bones), demonstrating the benefits of the dual-contrast method. FROM THE CLINICAL EDITOR: In this study, the MRI properties of iron oxide nanoparticles were studied in an animal model. These contrast agents are typically considered negative contrast materials, leading to signal loss on T2* weighted images, but they also have known T1 effects as well, which is lower than that of standard positive contrast agents (like gadolinium or manganese) but is still detectable. This dual property was utilized in this study, demonstrating high sensitivity of the negative contrast in delineating ambiguous vessel regions, whereas the positive contrast eliminated false negative contrast areas (areas giving rise to susceptibility effects).

An Enhanced Retroviral Vector for Efficient Genetic Manipulation and Selection in Mammalian Cells.

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies.

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics.

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.

Tanshinone T1/T2A facilitate let-7 to suppress non-small cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. Tanshinones are a group of compounds in Salvia miltiorrhiza. Although the effects of tanshinone Ⅰ (T1) and tanshinone ⅡA (T2A) are widely concerned, the mechanism of T1 and T2A in lung cancer is rarely studied. EXPERIMENTAL PROCEDURE: Xenograft tumor growth was performed to detect the role of T1/T2A in vivo. Next-generation sequencing of miRNA expression profiles in T1/T2A-treated A549 cells showed that T1/T2A upregulated the expression of the let-7 family. Then, let-7a-5p and its downstream target gene BORA were identified as the research objects in this paper. Mechanistically, we examined the interplay between miR-let-7 and BORA through the dual-luciferase reporter assay. Finally, the potential regulatory role of T1/T2A on Lin28B or MYC was explored. RESULTS: This study found that the let-7 family was significantly up-regulated via "Next-generation" sequencing (NGS) in the T1/T2A-treated A549 cell line, while BORA was downregulated. BORA was confirmed as a direct target of let-7. LncRNA MYCLo-5 was up-regulated after treatment with tanshinones. Knockdown of MYCLo-5 promoted the cell cycle and proliferation of non-small cell lung cancer (NSCLC). CONCLUSIONS: This study explored the effect of tanshinone T1 and T2A on NSCLC in vitro and in vivo, revealing the T1/T2A- let-7/ BORA /MYCLo-5 regulatory pathway, which provided new ideas for lung cancer treatment.

T2 and T2⁎ mapping and weighted imaging in cardiac MRI.

Cardiac imaging is progressing from simple imaging of heart structure and function to techniques visualizing and measuring underlying tissue biological changes that can potentially define disease and therapeutic options. These techniques exploit underlying tissue magnetic relaxation times: T1, T2 and T2*. Initial weighting methods showed myocardial heterogeneity, detecting regional disease. Current methods are now fully quantitative generating intuitive color maps that do not only expose regionality, but also diffuse changes - meaning that between-scan comparisons can be made to define disease (compared to normal) and to monitor interval change (compared to old scans). T1 is now familiar and used clinically in multiple scenarios, yet some technical challenges remain. T2 is elevated with increased tissue water - edema. Should there also be blood troponin elevation, this edema likely reflects inflammation, a key biological process. T2* falls in the presence of magnetic/paramagnetic materials - practically, this means it measures tissue iron, either after myocardial hemorrhage or in myocardial iron overload. This review discusses how T2 and T2⁎ imaging work (underlying physics, innovations, dependencies, performance), current and emerging use cases, quality assurance processes for global delivery and future research directions.

Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases.

Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through "humanization" rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Overall Survival of Biopsy-confirmed T1B and T2A Kidney Cancers Managed With Observation: Prognostic Value of Tumor Histology.

INTRODUCTION: The natural history of T1b (4-7 cm) or T2a (> 7-10 cm) kidney cancers managed with observation is not well-understood. The aim of our study was to determine if the addition of histologic subtype to a predictive model of overall survival (OS) that includes covariates for competing risks in observed, biopsy-proven, T1b and T2a renal cell carcinomas (RCCs) improves the model's performance. MATERIALS AND METHODS: We queried the National Cancer Database for patients with biopsy-proven stage T1b or T2a RCC and managed nonoperatively between 2004 and 2015. OS was estimated by Kaplan-Meier curves based on histologic subtype. The concordance index (c-index) from a Cox proportional hazards model was used to estimate the extent to which histologic subtypes predict survival for each stage when included in a model along with competing risks of age, gender, race/ethnicity, insurance status, area-level socioeconomic indicators, Charlson-Deyo index, and tumor grade. RESULTS: A total of 937 patients (754 with T1b and 185 with T2a) with biopsy-proven RCC were identified. Kaplan-Meier analysis suggested differences in OS by histologic subtype where sarcomatoid, followed by clear cell, papillary, and chromophobe, had the highest mortality risk at 1, 3, and 5 years. However, there was marginal improvement in the multivariable model of OS using competing risks and histology (c-index, 0.64 and 0.697) compared with competing risks alone (c-index, 0.631 and 0.671) for T1b and T2a RCCs, respectively. CONCLUSIONS: In patients with T1b or T2a RCC managed with observation, incorporation of histologic subtype into a risk-stratification model to determine prognostic OS did not improve modeling of OS compared with variables representing competing risks. Histologic subtype of observed T1b and T2a RCC appears to have prognostic OS value when not considering competing risks. These findings may impact the usefulness of renal biopsy to inform decision-making when managing patients with T1b and T2a renal tumors with observation.

Simplifying the Detection of Surface Presentation Levels in Yeast Surface Display by Intracellular tGFP Expression.

Yeast surface display (YSD) is an ultra-high throughput method used in protein engineering. Protein-protein interactions as well as surface presentation on the yeast cell surface are verified through fluorophore-conjugated labeling agents.In this chapter we describe an improved setup for full-length surface presentation detection. To this end, we used a single open reading frame (ORF) encoding for the protein to be displayed and a 2A sequence and tGFP for an intracellular fluorescence signal. The 2A sequence allows the simultaneous generation of two separate proteins from the same ORF through ribosomal skipping. The entangled expression of the POI on the yeast surface and intracellular tGFP obviates the need for fluorescent staining steps.

Cas9 Protein Triggers Differential Expression of Inherent Genes Especially NGFR Expression in 293T Cells.

INTRODUCTION: CRISPR/CAS9 systems, which can be utilized in vitro biological experiments, have recently captured much attention for their important roles and benefits. However, full realization of the potential of CRISPR/CAS9 approaches requires addressing many challenges and side effects. The expression of genes and potential side effects of CRISPR/CAS9 in human cells remains to be elucidated. The aim of our study was to explore the effect of CRISPR/CAS9 on gene expression in 293T cells. METHODS: A Cas9-expressing PX458 plasmid and Cas9-deactivated PX458-T2A plasmid were used to study the role of CRISPR/CAS9 on regulating gene expression in 293T cells. Gene expression in 293T cells after transfection of the PX458 plasmid or PX458-T2A plasmid was detected by RNA sequencing and correlative statistical analysis. Differential gene expression in both PX458 transfected 293T cells and PX458-T2A transfected 293T cells compared with normal 293T cells was detected using quantitative reverse transcription polymerase chain reaction (RT qPCR). The mRNA and protein levels were measured using reverse transcription PCR and Western blot. Co-IP assay combined with shotgun LC-MS/MS were used to investigate the differences of NGFR-interaction proteins between PX458 transfected 293T cells and PX458-T2A transfected 293T cells. RESULTS: In this study, we observed that PX458 plasmid transfection and Cas9 expression can affect the expression of different genes, including FOSB (FBJ murine osteosarcoma viral oncogene homolog B), IL-11 (Interleukin-11), MMP1 (matrix metalloproteinase), CYP2D6 (CytochromeP4502D6), and NGFR (matrix metalloproteinase 1). Downregulation of NGFR after PX458 transfection was confirmed by RT qPCR and western blot analysis. NGFR expression was significantly lower in PX458 transfected 293T cells than in normal 293T cells and PX458-T2A transfected 293T cells. The co-IP dilutions analyzed by shotgun LC-MS/MS showed a total of 183 proteins interact with NGFR in PX458 transfected 293T cells while 221 proteins interact with NGFR were identified in PX458-T2A transfected 293T cells using the MASCOT engine. CONCLUSIONS: Cas9 expression by transfection of the PX458 plasmid was negatively correlated with the NGFR mRNA level and NGFR protein expression in 293T cells, while PX458-T2A, in which Cas9 is deactivated, did not affect NGFR expression. The decrease in NGFR expression also affects the amount of proteins that interact with NGFR. These results suggest that the effect of Cas9 on NGFR expression and the expression of other genes should be noticed when developing cell-based studies and therapies utilizing CRISPR/CAS9 systems.

A kinase-dependent feedforward loop affects CREBB stability and long term memory formation.

In Drosophila, long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, we integrated Trojan exons encoding T2A-GAL4 into genes encoding putative kinases and selected for genes expressed in MB. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. We identified a novel, conserved kinase, Meng-Po (MP, CG11221, SBK1 in human), the loss of which severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. We show that PKA phosphorylates MP and that both proteins synergize in a feedforward loop to control CREBB levels and LTM. key words: Drosophila, Mushroom bodies, SBK1, deGradFP, T2A-GAL4, MiMIC.