Enhancer looping protein LDB1 modulates MYB expression in T-ALL cell lines in vitro by cooperating with master transcription factors.

BACKGROUND: Despite significant progress in the prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) in recent decades, a notable portion of children still confronts challenges such as treatment resistance and recurrence, leading to limited options and a poor prognosis. LIM domain-binding protein 1 (LDB1) has been confirmed to exert a crucial role in various physiological and pathological processes. In our research, we aim to elucidate the underlying function and mechanisms of LDB1 within the background of T-ALL. METHODS: Employing short hairpin RNA (shRNA) techniques, we delineated the functional impact of LDB1 in T-ALL cell lines. Through the application of RNA-Seq, CUT&Tag, and immunoprecipitation assays, we scrutinized master transcription factors cooperating with LDB1 and identified downstream targets under LDB1 regulation. RESULTS: LDB1 emerges as a critical transcription factor co-activator in cell lines derived from T-ALL. It primarily collaborates with master transcription factors (ERG, ETV6, IRF1) to cooperatively regulate the transcription of downstream target genes. Both in vitro and in vivo experiments affirm the essential fuction of LDB1 in the proliferation and survival of cell lines derived from T-ALL, with MYB identified as a significant downstream target of LDB1. CONCLUSIONS: To sum up, our research establishes the pivotal fuction of LDB1 in the tumorigenesis and progression of T-ALL cell lines. Mechanistic insights reveal that LDB1 cooperates with ERG, ETV6, and IRF1 to modulate the expression of downstream effector genes. Furthermore, LDB1 controls MYB through remote enhancer modulation, providing valuable mechanistic insights into its involvement in the progression of T-ALL.

Super-enhancer-driven IRF2BP2 enhances ALK activity and promotes neuroblastoma cell proliferation.

BACKGROUND: Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB). METHODS: The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing. RESULTS: The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2, and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB. CONCLUSIONS: Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of the NB susceptibility gene ALK.

CDK5RAP3 is a novel super-enhancer-driven gene activated by master TFs and regulates ER-Phagy in neuroblastoma.

Super enhancers (SEs) are genomic regions comprising multiple closely spaced enhancers, typically occupied by a high density of cell-type-specific master transcription factors (TFs) and frequently enriched in key oncogenes in various tumors, including neuroblastoma (NB), one of the most prevalent malignant solid tumors in children originating from the neural crest. Cyclin-dependent kinase 5 regulatory subunit-associated protein 3 (CDK5RAP3) is a newly identified super-enhancer-driven gene regulated by master TFs in NB; however, its function in NB remains unclear. Through an integrated study of publicly available datasets and microarrays, we observed a significantly elevated CDK5RAP3 expression level in NB, associated with poor patient prognosis. Further research demonstrated that CDK5RAP3 promotes the growth of NB cells, both in vitro and in vivo. Mechanistically, defective CDK5RAP3 interfered with the UFMylation system, thereby triggering endoplasmic reticulum (ER) phagy. Additionally, we provide evidence that CDK5RAP3 maintains the stability of MEIS2, a master TF in NB, and in turn, contributes to the high expression of CDK5RAP3. Overall, our findings shed light on the molecular mechanisms by which CDK5RAP3 promotes tumor progression and suggest that its inhibition may represent a novel therapeutic strategy for NB.

The BET inhibitor GNE-987 effectively induces anti-cancer effects in T-cell acute lymphoblastic leukemia by targeting enhancer regulated genes.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy originating from T progenitor cells. It accounts for 15% of childhood and 25% of adult ALL cases. GNE-987 is a novel chimeric molecule developed using proteolysis-targeting chimeras (PROTAC) technology for targeted therapy. It consists of a potent inhibitor of the bromodomain and extraterminal (BET) protein, as well as the E3 ubiquitin ligase Von Hippel-Lindau (VHL), which enables the effective induction of proteasomal degradation of BRD4. Although GNE-987 has shown persistent inhibition of cell proliferation and apoptosis, its specific antitumor activity in T-ALL remains unclear. In this study, we aimed to investigate the molecular mechanisms underlying the antitumor effect of GNE-987 in T-ALL. To achieve this, we employed technologies including RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and CUT&Tag. The degradation of BET proteins, specifically BRD4, by GNE-987 has a profound impact on T-ALL cell. In in vivo experiments, sh-BRD4 lentivirus reduced T-ALL cell proliferation and invasion, extending the survival time of mice. The RNA-seq and CUT&Tag analyses provided further insights into the mechanism of action of GNE-987 in T-ALL. These analyses revealed that GNE-987 possesses the ability to suppress the expression of various genes associated with super-enhancers (SEs), including lymphoblastic leukemia 1 (LCK). By targeting these SE-associated genes, GNE-987 effectively inhibits the progression of T-ALL. Importantly, SE-related oncogenes like LCK were identified as critical targets of GNE-987. Based on these findings, GNE-987 holds promise as a potential novel candidate drug for the treatment of T-ALL.

Transcranial direct current stimulation of the primary motor cortex on postoperative pain and spontaneous oscillatory electroencephalographic activity following lumbar spine surgery: A pilot study.

BACKGROUND: Transcranial direct current stimulation (tDCS) on primary motor cortex (M1) provides a new way to relieve postoperative pain. Previous studies only found postoperative analgesia dosage significantly reduced in tDCS group while the patient-controlled analgesia (PCA) was applied. However, there lacks the study about the effect of M1-tDCS on pain intensity and brain activity while the analgesia dosage is the same for both groups. OBJECTIVE: To investigate whether M1-tDCS can (1) reduce pain intensity and (2) change spontaneous electroencephalography (EEG) oscillations in prefrontal cortex, in patients with postoperative pain, after taking the constant dosage of analgesics. METHODS: A prospective, single-blind, randomized, sham-controlled study was conducted. 32 patients with postoperative pain after lumbar spine surgery were recruited. All patients received same dosage of dezocine before intervention. In the morning of the first day after surgery and before dezocine injection, a single 20-minute session of anodal M1-tDCS was applied to 'tDCS' group while sham stimulation to 'sham' group. Numeric rating scale (NRS) and resting-state EEG with eyes-closed were measured and analyzed. EEG spectral powers were analyzed using repeated measures analysis of variance (ANOVA). Correlation analysis was conducted between the change of NRS and the change of spectral power. RESULTS: The NRS in "tDCS" group significantly decreased (p < 0.01) while not in "sham" group after intervention. Only spectral power within alpha2 band (10-13 Hz) in Fp1 and beta1 band (13-20 Hz) in Fp1 showed significant Time×Intervention interaction effect. These changes of the spectral power also showed significant correlation with the change of NRS. CONCLUSIONS: The postoperative pain intensity in patients receiving surgery could reduce after a single session of anodal M1-tDCS compared to sham M1-tDCS. The effect to the top-down dimension of postoperative pain might account for the analgesic effect of M1-tDCS, which reflecting slow oscillations in left prefrontal EEG.

Time course of EEG activities in continuous tracking task: a pilot study.

Motor learning is crucial to surgical skills enhancement, but its neural mechanism has been investigated only using some simple tasks with limited motor involvement. This study aimed to gain more understanding of the neural dynamics during motor learning by investigating the time course of electroencephalogram (EEG) activities in the continuous tracking task that involves more motor components. One participant performed 16 trials of the continuous tracking task on Day1 and Day2 respectively. The 16-channel EEG signals were recorded and analyzed in both the resting and active states. Results showed that the tracking performance improved from Day1 to Day2. Regarding the EEG, it was found that the relative amplitude in the individual alpha band (IAB) decreased locally over primary motor cortex from the resting state to the active state on both days, and this reduction was more focused on the left primary motor cortex on Day2 compared to Day1. Moreover, in the active state the alpha coherence between central and frontal areas also significantly increased from Day1 to Day2. Time course of alpha activities may explain the tracking performance enhancement from Day1 to Day2. Future work will include more participants to extend the validity of current results.

Multidrug-Resistant Pseudomonas aeruginosa Infection in a Child with Cystic Fibrosis.

We describe a pediatric cystic fibrosis patient who developed a pulmonary exacerbation due to two multidrug-resistant (MDR) Pseudomonas aeruginosa isolates. In addition to these MDR organisms, the case was further complicated by ß-lactam allergy. Despite the MDR phenotype, both isolates were susceptible to an antimicrobial combination.

Multitask training promotes automaticity of a fundamental laparoscopic skill without compromising the rate of skill learning.

BACKGROUND: A defining characteristic of expertise is automated performance of skills, which frees attentional capacity to better cope with some common intraoperative stressors. There is a paucity of research on how best to foster automated performance by surgical trainees. This study examined the use of a multitask training approach to promote automated, robust laparoscopic skills. METHODS: Eighty-one medical students completed training of a fundamental laparoscopic task in either a traditional single-task training condition or a novel multitask training condition. Following training, participants' laparoscopic performance was tested in a retention test, two stress transfer tests (distraction and time pressure) and a secondary task test, which was included to evaluate automaticity of performance. The laparoscopic task was also performed as part of a formal clinical examination (OSCE). RESULTS: The training groups did not differ in the number of trials required to reach task proficiency (p = .72), retention of skill (ps > .45), or performance in the clinical examination (p = .14); however, the groups did differ with respect to the secondary task (p = .016). The movement efficiency (number of hand movements) of single-task trainees, but not multitask trainees, was negatively affected during the secondary task test. The two stress transfer tests had no discernable impact on the performance of either training group. CONCLUSION: Multitask training was not detrimental to the rate of learning of a fundamental laparoscopic skill and added value by providing resilience in the face of a secondary task load, indicative of skill automaticity. Further work is needed to determine the extent of the clinical utility afforded by multitask training.

Cathodal Transcranial Direct Current Stimulation Over Left Dorsolateral Prefrontal Cortex Area Promotes Implicit Motor Learning in a Golf Putting Task.

BACKGROUND: Implicit motor learning is characterized by low dependence on working memory and stable performance despite stress, fatigue, or multi-tasking. However, current paradigms for implicit motor learning are based on behavioral interventions that are often task-specific and limited when applied in practice. OBJECTIVE: To investigate whether cathodal transcranial direct current stimulation (tDCS) over the left dorsolateral prefrontal cortex (DLPFC) area during motor learning suppressed working memory activity and reduced explicit verbal-analytical involvement in movement control, thereby promoting implicit motor learning. METHODS: Twenty-seven healthy individuals practiced a golf putting task during a Training Phase while receiving either real cathodal tDCS stimulation over the left DLPFC area or sham stimulation. Their performance was assessed during a Test phase on another day. Verbal working memory capacity was assessed before and after the Training Phase, and before the Test Phase. RESULTS: Compared to sham stimulation, real stimulation suppressed verbal working memory activity after the Training Phase, but enhanced golf putting performance during the Training Phase and the Test Phase, especially when participants were required to multi-task. CONCLUSION: Cathodal tDCS over the left DLPFC may foster implicit motor learning and performance in complex real-life motor tasks that occur during sports, surgery or motor rehabilitation.

Exploring personality dimensions that influence practice and performance of a simulated laparoscopic task in the objective structured clinical examination.

BACKGROUND: Surgical educators have encouraged the investigation of individual differences in aptitude and personality in surgical performance. An individual personality difference that has been shown to influence laparoscopic performance under time pressure is movement specific reinvestment. Movement specific reinvestment has 2 dimensions, movement self-consciousness (MS-C) (i.e., the propensity to consciously monitor movements) and conscious motor processing (CMP) (i.e., the propensity to consciously control movements), which have been shown to differentially influence laparoscopic performance in practice but have yet to be investigated in the context of psychological stress (e.g., the objective structured clinical examination [OSCE]). OBJECTIVE: This study investigated the role of individual differences in propensity for MS-C and CMP in practice of a fundamental laparoscopic skill and in laparoscopic performance during the OSCE. Furthermore, this study examined whether individual differences during practice of a fundamental laparoscopic skill were predictive of laparoscopic performance during the OSCE. METHODS: Overall, 77 final-year undergraduate medical students completed the movement specific reinvestment scale, an assessment tool that quantifies the propensity for MS-C and CMP. Participants were trained to proficiency on a fundamental laparoscopic skill. The number of trials to reach proficiency was measured, and completion times were recorded during early practice, later practice, and the OSCE. RESULTS: There was a trend for CMP to be negatively associated with the number of trials to reach proficiency (p = 0.064). A higher propensity for CMP was associated with fewer trials to reach proficiency (ß = -0.70, p = 0.023). CMP and MS-C did not significantly predict completion times in the OSCE (p > 0.05). Completion times in early practice (ß = 0.05, p = 0.016) and later practice (ß = 0.47, p < 0.001) and number of trials to reach proficiency (ß = 0.23, p = 0.003) significantly predicted completion times in the OSCE. CONCLUSION: It appears that a higher propensity for CMP predicts faster rates of learning of a fundamental laparoscopic skill. Furthermore, laparoscopic performance during practice is indicative of laparoscopic performance in the challenging conditions of the OSCE. The lack of association between the 2 dimensions of movement specific reinvestment and performance during the OSCE is explained using the theory of reinvestment as a framework. Overall, consideration of personality differences and individual differences in ability during practice could help inform the development of individualized surgical training programs.

Implicit motor learning promotes neural efficiency during laparoscopy.

BACKGROUND: An understanding of differences in expert and novice neural behavior can inform surgical skills training. Outside the surgical domain, electroencephalographic (EEG) coherence analyses have shown that during motor performance, experts display less coactivation between the verbal-analytic and motor planning regions than their less skilled counterparts. Reduced involvement of verbal-analytic processes suggests greater neural efficiency. The authors tested the utility of an implicit motor learning intervention specifically devised to promote neural efficiency by reducing verbal-analytic involvement in laparoscopic performance. METHODS: In this study, 18 novices practiced a movement pattern on a laparoscopic trainer with either conscious awareness of the movement pattern (explicit motor learning) or suppressed awareness of the movement pattern (implicit motor learning). In a retention test, movement accuracy was compared between the conditions, and coactivation (EEG coherence) was assessed between the motor planning (Fz) region and both the verbal-analytic (T3) and the visuospatial (T4) cortical regions (T3-Fz and T4-Fz, respectively). RESULTS: Movement accuracy in the conditions was not different in a retention test (P = 0.231). Findings showed that the EEG coherence scores for the T3-Fz regions were lower for the implicit learners than for the explicit learners (P = 0.027), but no differences were apparent for the T4-Fz regions (P = 0.882). CONCLUSIONS: Implicit motor learning reduced EEG coactivation between verbal-analytic and motor planning regions, suggesting that verbal-analytic processes were less involved in laparoscopic performance. The findings imply that training techniques that discourage nonessential coactivation during motor performance may provide surgeons with more neural resources with which to manage other aspects of surgery.

Subcellular-resolution delivery of a cytokine through precisely manipulated nanowires.

Precise delivery of molecular doses of biologically active chemicals to a pre-specified single cell among many, or a specific subcellular location, is still a largely unmet challenge hampering our understanding of cell biology. Overcoming this could allow unprecedented levels of cell manipulation and targeted intervention. Here, we show that gold nanowires conjugated with a cytokine such as tumour-necrosis factor-alpha can be transported along any prescribed trajectory or orientation using electrophoretic and dielectrophoretic forces to a specific location with subcellular resolution. The nanowire, 6 microm long and 300 nm in diameter, delivered the cytokine and activated canonical nuclear factor-kappaB signalling in a single cell. Combined computational modelling and experimentation indicated that cell stimulation was highly localized to the nanowire vicinity. This targeted delivery method has profound implications for controlling signalling events on the single cell level.