Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells

Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4–mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4–mediated phosphoproteome, including construction of kinase–substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.Breast cancer is the most common cancer in women, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Tumor metastasis is the major source of cancer lethality. Cancer stem cells (CSCs) are small-percentage subpopulation within tumors, which are essential for tumor reoccurrence and metastasis (1). G protein-coupled receptor CXCR4 is critical for tumor growth and metastasis and plays important roles in CSC migration, invasion, and proliferation (2). Chemokine stromal cell-derived factor 1 (SDF-1) (CXCL-12) binds to chemokine (C-X-C motif) receptor 4 (CXCR4) and induces SDF-1/CXCR4 signaling. SDF-1 or CXCR4 knockout mice are embryonic lethal. SDF-1 and CXCR4 are vital for tumor angiogenesis and metastasis (3). The large-scale signal transduction and the feedback regulation downstream of SDF-1/CXCR4 signaling in breast CSCs are unknown but critical to understanding the cellular physiology of breast tumor regrowth and metastasis.Protein phosphorylation and dephosphorylation are essential for cellular signal processing (4). Dynamic regulation of reversible, site-specific protein phosphorylation is critical to the signaling networks that regulate CSC self-renewal, differentiation, and metastasis. Protein-reversible phosphorylation has been extensively analyzed in examining one or a few protein phosphorylation events that affect CSC signaling (1). However, the phosphoproteome composed by protein kinase-driven and phosphatase-regulated signaling networks largely controls CSC fate. Therefore, large-scale analysis of differentially regulated protein phosphorylation is central to understanding complex cellular events, such as CSC maintenance and dissemination.To unveil the signal transduction downstream of SDF-1/CXCR4 signaling in CSCs, in this study we have carried out isotope reductive dimethylation and large-scale liquid chromatography tandem mass spectrometry (LC-MS/MS)-based phosphoproteomic profiling and quantification in human breast CSCs upon SDF-1/CXCR4 stimulation. The phosphorylation events presented here include SDF-1/CXCR4–mediated phosphorylation sites in several key kinases and phosphatases, and several important signaling pathways in breast CSCs.     

Recognition of Pain as Another Deficit in Young Males with High Callous-Unemotional Traits

Prior research on callous-unemotional (CU) traits supports a deficit in recognizing fear in faces and body postures. Difficulties recognising others’ emotions may impair the typical behavioural inhibition for violent behaviour. However, recent research has begun to examine other distress cues such as pain. The present study examined emotion recognition skills, including pain, of school-excluded boys aged 11–16 years (N = 50). Using dynamic faces and body poses, we examined the relation between emotion recognition and CU traits using the youth psychopathic traits inventory (YPI) and the inventory of CU traits. Violent delinquency was covaried in regression analyses. Although fearful facial and fearful bodily expressions were unrelated to CU traits, recognition of dynamic pain facial expressions was negatively related to CU traits using the YPI. The failure to replicate a fear and sad deficit are discussed in relation to previous research. Also, findings are discussed in support of a general empathy deficit for distress cues which may underlie the problem behaviour of young males with CU traits.     

Electrocardiographic manifestation of the middle fibers/septal fascicle block: a consensus report

There are fibers in the left ventricle (LV) (LV middle network) that in around one third of cases may be considered a true septal fascicle that arises from the common left bundle. Its presence and the evidence that there are 3 points of activation onset in the LV favor the quadrifascicular theory of the intraventricular activation of both ventricles. Since the 70s, different authors have suggested that the block of the left middle fibers (MS)/left septal fascicle may explain different electrocardiographic (ECG) patterns. The 2 hypothetically based criteria that are in some sense contradictory include: a) the lack of septal "q" wave due to first left and later posteriorly shifting of the horizontal plane loop and b) the presence of RS in lead V(2) (V(1)-V(2)) due to some anterior shifting of the horizontal plane vectorcardiogram loop. However, there are many evidence that the lack of septal q waves can be also explained by predivisional first-degree left bundle-branch block and that the RS pattern in the right precordial leads may be also explained by first-degree right bundle-branch block. The transient nature of these patterns favor the concept that some type of intraventricular conduction disturbance exists but a doubt remains about its location. Furthermore, the RS pattern could be explained by many different normal variants. To improve our understanding whether these patterns are due to MF/left septal fascicle block or other ventricular conduction disturbances (or both), it would be advisable: 1) To perform more histologic studies (heart transplant and necropsy) of the ventricular conduction system; 2) To repeat prior experimental studies using new methodology/technology to isolate the MF; and 3) To change the paradigm: do not try to demonstrate if the block of the fibers produces an ECG change but to study with new electroanatomical imaging techniques, if these ECG criteria previously described correlate or not with a delay of activation in the zone of the LV that receives the activation through these fibers or in other zones.     

Electrocardiographic changes of ST-elevation myocardial infarction in patients with complete occlusion of the left main trunk without collateral circulation: Differential diagnosis and clinical considerations

Acute coronary syndromes due to involvement of the left main trunk usually present with subtotal occlusion and electrocardiographic pattern with predominant ST depression (non-ST-elevation myocardial infarction). The cases with complete occlusion frequently present an ST-elevation myocardial infarction pattern, but these patients usually die before reaching the hospital. We present a series of 7 patients with total left main trunk occlusion without collateral circulation showing ST-elevation myocardial infarction pattern. The electrocardiographic pattern is similar to left anterior descending coronary artery proximal occlusion to first septal and first diagonal but without ST elevation in V(1) and aVR because of left circumflex coronary artery compromise. In 4 (60%) of 7 of cases, there is also advanced right bundle-branch block plus superoanterior hemiblock. Despite severe clinical state at entrance (5/7 presented cardiac arrest/cardiogenic shock), 3 patients (43%) survived after percutaneous coronary intervention.     

Analysis of mutational status, SNP rs16754, and expression levels of Wilms tumor 1 (WT1) gene in acute promyelocytic leukemia

Overexpression, polymorphisms, and mutations of the WT1 gene have been reported in several human tumors including acute myeloid leukemia (AML) and variably correlated with prognosis. Acute promyelocytic leukemia (APL) represents the AML subset disclosing higher WT1 expression levels; however, no WT1 studies specifically focused on APL have been conducted. We screened for the presence of mutations, SNP rs16754, and expression levels of WT1 gene in 103 adult patients with newly diagnosed APL. Fms-like tyrosine kinase (FLT3) mutations were analyzed as well. WT1 mutations were identified in four (4?%) patients. At least one copy of the minor SNP rs16754 allele (WT1 ^AG or WT1 ^GG) was detected in 30 (29?%) patients. Six patients (6?%) were homozygous for the minor allele (WT1 ^GG ) and this genotype was associated with higher WT1 mRNA copies (p?=?0.018). FLT3 mutations were found in 37?% of patients and correlated with high WT1 mRNA expression (p?=?0.004). Patients heterozygous or homozygous for the minor allele and patients homozygous for major (WT1 ^AA) allele did not differ in terms of presenting features. In adult APL, WT1 gene mutational and polymorphic profile shows similarities with pediatric AML rather than with adult AML.     

Sodium Channel Blockers Modulate Abnormal Activity of Regenerating Nociceptive Corneal Nerves After Surgical Lesion

PurposeTo test the effect of different sodium channel blockers on the electrical activity of corneal nociceptors in intact and surgically injured corneas.MethodsIn anesthetized guinea pigs, a 4-mm diameter corneal flap was performed in one eye at a midstromal depth using a custom-made microkeratome. At different times after surgery (3 hours to 15 days), the electrical activity of corneal nociceptor fibers was recorded from ciliary nerve filaments in the superfused eye in vitro. Mechanical threshold was measured using calibrated von Frey hairs; chemical stimulation was performed applying 30-second CO₂ gas pulses. The characteristics of the spontaneous and stimulus-evoked activity of corneal nociceptors recorded from intact and lesioned corneas, before and after treatment with the sodium channel blockers lidocaine, carbamazepine, and amitriptyline, were compared.ResultsNo spontaneous or stimulus-evoked impulse activity was detected inside the flap at any of the studied time points. However, both were recorded from mechanonociceptor and polymodal nociceptors fibers in the surrounding corneal tissue, being significantly higher (sensitization) 24 to 48 hours after surgery. In these fibers, none of the tested drugs affected mechanical threshold, but they significantly reduced the CO₂ response of polymodal nociceptors of intact and injured corneas. Likewise, they diminished significantly the transient increase in spontaneous and stimulus-evoked activity of sensitized polymodal nociceptors.ConclusionsNa⁺ channel blockers decrease the excitability of intact and sensitized corneal nociceptor fibers, thus acting as potential tools to attenuate their abnormal activity, which underlies the spontaneous pain, hyperalgesia, and allodynia often accompanying surgical corneal lesions, as occurs after photorefractive surgery.     

Impact of comorbidity on outcomes and overall survival after open and minimally invasive esophagectomy for locally advanced esophageal cancer

Background

The aim of this study was to examine the impact of the Charlson Comorbidity Index-Grade (CCI-G) on predicting outcomes and overall survival after open and minimally invasive esophagectomy (MIE).

Methods

One hundred and forty-six patients who underwent esophagectomy between 1995 and 2011 for stage II and III cancer were selected and separated into open esophagectomy (Open) and MIE groups. Risk adjustment was performed using the CCI-G. The outcomes of interest were operative time, estimated blood loss (EBL), lymph node harvest, length of hospital stay (LOS), major complications, 30-day mortality, and overall survival.

Results

Sixty-four patients (44 %) underwent Open while 71 (49 %) had MIE. An additional (7 %) were converted and classified with MIE. There was no significant difference between MIE and Open in terms of operative time. MIE had less EBL (mean difference = 234 mL, p < 0.001), higher lymph node harvest (mean = 7.4 nodes, p < 0.001), and shorter LOS (median = 1.5 days, p = 0.02). Atrial arrhythmias were the most frequent complication, occurring in 33 % of patients in both the MIE and the Open group (p = 0.988). Thirty-day mortality was 2 % for MIE and 5 % for Open (p = 0.459). Five-year survival was 41 % for MIE and 33 % for Open (p = 0.513). Operative approach, age, gender, BMI, clinical stage, and neoadjuvant therapy did not have any significant effect on the outcomes or overall survival. CCI-G influenced outcomes with operative time, LOS, cardiovascular complication, and anastomotic leak rate, favoring CCI-G 0 compared to CCI-G 3. Overall survival was worse for CCI-G 1 in comparison with CCI-G 0 [hazard ratio (HR) 1.99, p = 0.027].

Conclusions

MIE is a safe alternative to open esophagectomy for the treatment of locally advanced esophageal cancer. The presence of comorbidities increased operative time, length of hospital stay, and postoperative complications while worsening overall survival.     

Strengths,Weaknesses, Opportunities,and Threats Analysis of Artificial Intelligence and Machine Learning Applications in Radiology

Currently, the use of artificial intelligence (AI) in radiology, particularly machine learning (ML), has become a reality in clinical practice. Since the end of the last century, several ML algorithms have been introduced for a wide range of common imaging tasks, not only for diagnostic purposes but also for image acquisition and postprocessing. AI is now recognized to be a driving initiative in every aspect of radiology. There is growing evidence of the advantages of AI in radiology creating seamless imaging workflows for radiologists or even replacing radiologists. Most of the current AI methods have some internal and external disadvantages that are impeding their ultimate implementation in the clinical arena. As such, AI can be considered a portion of a business trying to be introduced in the health care market. For this reason, this review analyzes the current status of AI, and specifically ML, applied to radiology from the scope of strengths, weaknesses, opportunities, and threats (SWOT) analysis.