Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress.

Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.

Downregulation of Yap1 during limb regeneration results in defective bone formation in axolotl.

The Hippo pathway plays an imperative role in cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Transcription coregulator component of Hippo pathway, YAP1, promotes transcription of genes involved in cell proliferation, migration, differentiation, and suppressing apoptosis. However, its role in epimorphic regeneration has not been fully explored. The axolotl is a well-established model organism for developmental biology and regeneration studies. By exploiting its remarkable regenerative capacity, we investigated the role of Yap1 in the early blastema stage of limb regeneration. Depleting Yap1 using gene-specific morpholinos attenuated the competence of axolotl limb regeneration evident in bone formation defects. To explore the affected downstream pathways from Yap1 down-regulation, the gene expression profile was examined by employing LC-MS/MS technology. Based on the generated data, we provided a new layer of evidence on the putative roles of increased protease inhibition and immune system activities and altered ECM composition in diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. We believe that new insights into the roles of the Hippo pathway in complex structure regeneration were granted in this study.

Development of a Chemogenetic Approach to Manipulate Intracellular pH.

Chemogenetic Operation of iNTRacellular prOton Levels (pH-Control) is a novel substrate-based enzymatic method that enables precise spatiotemporal control of ultralocal acidification in cultured cell lines and primary neurons. The genetically encoded biosensor SypHer3s showed that pH-Control effectively acidifies cytosolic, mitochondrial, and nuclear pH exclusively in the presence of ß-chloro-d-alanine in living cells in a concentration-dependent manner. The pH-Control approach is promising for investigating the ultralocal pH imbalance associated with many diseases.

High-resolution mapping of sensory fibers at the healthy and post-myocardial infarct whole transgenic hearts.

The sensory nervous system is critical to maintain cardiac function. As opposed to efferent innervation, less is known about cardiac afferents. For this, we mapped the VGLUT2-expressing cardiac afferent fibers of spinal and vagal origin by using the VGLUT2::tdTomato double transgenic mouse as an approach to visualize the whole hearts both at the dorsal and ventral sides. For comparison, we colabeled mixed-sex transgenic hearts with either TUJ1 protein for global cardiac innervation or tyrosine hydroxylase for the sympathetic network at the healthy state or following ischemic injury. Interestingly, the nerve density for global and VGLUT2-expressing afferents was found significantly higher on the dorsal side compared to the ventral side. From the global nerve innervation detected by TUJ1 immunoreactivity, VGLUT2 afferent innervation was detected to be 15-25% of the total network. The detailed characterization of both the atria and the ventricles revealed a remarkable diversity of spinal afferent nerve ending morphologies of flower sprays, intramuscular endings, and end-net branches that innervate distinct anatomical parts of the heart. Using this integrative approach in a chronic myocardial infarct model, we showed a significant increase in hyperinnervation in the form of axonal sprouts for cardiac afferents at the infarct border zone, as well as denervation at distal sites of the ischemic area. The functional and physiological consequences of the abnormal sensory innervation remodeling post-ischemic injury should be further evaluated in future studies regarding their potential contribution to cardiac dysfunction.

An improved platform for cultured neuronal network electrophysiology: multichannel optogenetics integrated with MEAs.

Cultured neuronal networks (CNNs) are powerful tools for studying how neuronal representation and adaptation emerge in networks of controlled populations of neurons. To ensure the interaction of a CNN and an artificial setting, reliable operation in both open and closed loops should be provided. In this study, we integrated optogenetic stimulation with microelectrode array (MEA) recordings using a digital micromirror device and developed an improved research tool with a 64-channel interface for neuronal network control and data acquisition. We determined the ideal stimulation parameters including light intensity, frequency, and duty cycle for our configuration. This resulted in robust and reproducible neuronal responses. We also demonstrated both open and closed loop configurations in the new platform involving multiple bidirectional channels. Unlike previous approaches that combined optogenetic stimulation and MEA recordings, we did not use binary grid patterns, but assigned an adjustable-size, non-binary optical spot to each electrode. This approach allowed simultaneous use of multiple input-output channels and facilitated adaptation of the stimulation parameters. Hence, we advanced a 64-channel interface in that each channel can be controlled individually in both directions simultaneously without any interference or interrupts. The presented setup meets the requirements of research in neuronal plasticity, network encoding and representation, closed-loop control of firing rate and synchronization. Researchers who develop closed-loop control techniques and adaptive stimulation strategies for network activity will benefit much from this novel setup.

Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl.

The axolotl (Ambystoma mexicanum) salamander, a urodele amphibian, has an exceptional regenerative capacity to fully restore an amputated limb throughout the life-long lasting neoteny. By contrast, when axolotls are experimentally induced to metamorphosis, attenuation of the limb's regenerative competence is noticeable. Here, we sought to discern the proteomic profiles of the early stages of blastema formation of neotenic and metamorphic axolotls after limb amputation by employing LC-MS/MS technology. We quantified a total of 714 proteins and qRT-PCR for selected genes was performed to validate the proteomics results and provide evidence for the putative link between immune system activity and regenerative potential. This study provides new insights for examination of common and distinct molecular mechanisms in regeneration-permissive neotenic and regeneration-deficient metamorphic stages at the proteome level.

An In Vitro Model for Conditioning Lesion Effect.

Axons of a peripheral nerve grow faster after an axotomy if it attains a prior injury a few days earlier. This is called conditioning lesion effect (CLE) and very much valued since it may provide new insights into neuron biology and axonal regeneration. There are established in vivo experimental paradigms to study CLE, however, there is a need to have an in vitro conditioning technique where CLE occurs in a maximally controlled environment. Mouse primary sensory neurons were isolated from lumbar 4-5 dorsal root ganglia and incubated at 37 °C on a silicon-coated watch glass that prevents cell attachment. After this conditioning period they were transferred to laminin coated culture dishes. Similar cultures were set up with freshly isolated neurons from control animals and from the animals that received a sciatic nerve cut 3 days earlier. All preparations were placed on a live cell imaging microscopy providing physiological conditions and photographed for 48 h. Axonal regeneration and neuronal survival was assessed. During the conditioning incubation period neurons remained in suspended aggregates and did not grow axons. The regeneration rate of the in vitro conditioned neurons was much higher than the in vivo conditioned and control preparations during the first day of normal incubation. However, higher regeneration rates were compromised by progressive substantial neuronal death in both types of conditioned cultures but not in the control preparations. By using neutralizing antibodies, we demonstrated that activity of endogenous leukemia inhibitory factor is essential for induction of CLE in this model.

Modulation of Excitability of Stellate Neurons in the Ventral Cochlear Nucleus of Mice by ATP-Sensitive Potassium Channels.

Major voltage-activated ionic channels of stellate cells in the ventral part of cochlear nucleus (CN) were largely characterized previously. However, it is not known if these cells are equipped with other ion channels apart from the voltage-sensitive ones. In the current study, it was aimed to study subunit composition and function of ATP-sensitive potassium channels (KATP) in stellate cells of the ventral cochlear nucleus. Subunits of KATP channels, Kir6.1, Kir6.2, SUR1, and SUR2, were expressed at the mRNA level and at the protein level in the mouse VCN tissue. The specific and clearly visible bands for all subunits but that for Kir6.1 were seen in Western blot. Using immunohistochemical staining technique, stellate cells were strongly labeled with SUR1 and Kir6.2 antibodies and moderately labeled with SUR2 antibody, whereas the labeling signals for Kir6.1 were too weak. In patch clamp recordings, KATP agonists including cromakalim (50 µM), diazoxide (0.2 mM), 3-Amino-1,2,4-triazole (ATZ) (1 mM), 2,2-Dithiobis (5-nitro pyridine) (DTNP) (330 µM), 6-Chloro-3-isopropylamino- 4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) (1 µM), 6-chloro-3-(methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414) (1 µM), and H2O2 (0.88 mM) induced marked responses in stellate cells, characterized by membrane hyperpolarization which were blocked by KATP antagonists. Blockers of KATP channels, glibenclamide (0.2 mM), tolbutamide (0.1 mM) as well as 5-hydroxydecanoic acid (1 mM), and catalase (500 IU/ml) caused depolarization of stellate cells, increasing spontaneous action potential firing. In conclusion, KATP channels seemed to be composed dominantly of Kir 6.2 subunit and SUR1 and SUR2 and activation or inhibition of KATP channels regulates firing properties of stellate cells by means of influencing resting membrane potential and input resistance.

Evaluation of liver function by means of serum cytokeratin 18 and hepatocyte growth factor levels in patients with obstructive jaundice.

OBJECTIVE: To evaluate the serum levels of cytokeratin 18 (CK18) and hepatocyte growth factor (HGF) in obstructive jaundice patients before and after treatment and thereby to detect the possible role of CK18 and HGF in patients with obstructive jaundice. PATIENTS AND METHODS: Forty patients who had obstructive jaundice and 40 healthy control subjects were included in the study. Patients were treated using percutaneous, endoscopic or surgical approaches. Blood samples were obtained at the day before and 7 days after the intervention for obstructive jaundice. Serum HGF and CK18 concentrations were determined by ELISA method. RESULTS: There were statistically significant decreases in HGF, CK18, total bilirubin and direct bilirubin serum levels, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, and alkaline phosphatase activities and white blood cell count when compared with pre-treatment levels. CONCLUSION: Evaluating pre- and post-treatment serum HGF and CK18 levels suggested that there was an apoptosis in obstructive jaundice patients and this apoptosis decreased after the decompression of the biliary tract. We also demonstrated that HGF levels were altered at biliary obstruction compared to healthy controls and the levels of this biomarker also decreased after decompression of biliary obstruction. We concluded that these biomarkers can be used as predictors of liver injury in biliary obstruction.

Detailed tail proteomic analysis of axolotl (Ambystoma mexicanum) using an mRNA-seq reference database.

Salamander axolotl has been emerging as an important model for stem cell research due to its powerful regenerative capacity. Several advantages, such as the high capability of advanced tissue, organ, and appendages regeneration, promote axolotl as an ideal model system to extend our current understanding on the mechanisms of regeneration. Acknowledging the common molecular pathways between amphibians and mammals, there is a great potential to translate the messages from axolotl research to mammalian studies. However, the utilization of axolotl is hindered due to the lack of reference databases of genomic, transcriptomic, and proteomic data. Here, we introduce the proteome analysis of the axolotl tail section searched against an mRNA-seq database. We translated axolotl mRNA sequences to protein sequences and annotated these to process the LC-MS/MS data and identified 1001 nonredundant proteins. Functional classification of identified proteins was performed by gene ontology searches. The presence of some of the identified proteins was validated by in situ antibody labeling. Furthermore, we have analyzed the proteome expressional changes postamputation at three time points to evaluate the underlying mechanisms of the regeneration process. Taken together, this work expands the proteomics data of axolotl to contribute to its establishment as a fully utilized model.

Functionalized gold nanoparticles manifested as potent carriers for nucleolar targeting.

It is generally known that gold nanoparticles are localised in the cytoplasm and, if synthesised in small sizes or functionalized with specific proteins, they enter the cell nucleus. However, there is no report emphasising the importance of surface functionalization in their accumulation in the nucleolus. Here, for the first time in the literature, it is proposed that functionalization of gold nanoparticles with a thin layer of polyethyleneimine (PEI) spearheads them to the nucleolus of hard-to-transfect post-mitotic dorsal root ganglion neurones in a size-independent manner. As a potential for theranostic applications, it was found that functionalization with a thin layer of PEI affected the emission signal intensity of gold nanoparticles so that the cellular biodistribution of nanoparticles was visualised clearly under both confocal and two-photon microscopes.

Liver transplantation for alveolar echinococcosis in an endemic region.

Alveolar echinococcosis (AE) is a chronic disease caused by ingestion of the eggs of the parasitic cestode Echinococcosis multilocularis (EM). In severe cases, liver transplantation (LT) may represent the only possibility of survival and cure. Patients undergoing LT associated with hepatic AE at our institution between April 2011 and October 2014 were investigated retrospectively. The clinical findings of the 27 patients who participated in the study were noted. Kaplan-Meier and chi-square tests were used to investigate the effect of these characteristics on survival and mortality. Living donor LT was performed on 20 patients (74.1%), and deceased donor LT was performed on 7 patients (25.9%). Hilar invasion was the most common indication (14 patients, 51.9%) for transplantation. The patient follow-up was 16.1 ± 11.4 months, and the overall survival rate was 77.8%. Primary nonfunction developed only in 2 patients in the posttransplantation period. Six patients died during monitoring, the most common cause of death being sepsis (3 patients). The relationship between the mortality rate of the patients and the invasion of the bile duct and/or portal vein by alveolar lesions was found to be statistically significant (P = 0.024 and P = 0.043, respectively). According to PNM staging, when the AE disease exceeds the resectability limits, the only alternative for the treatment of the disease is LT. However, different from LT due to cirrhosis, it is extremely difficult to perform a transplantation for AE disease because of the invasive characteristics of it. In order to decrease the difficulty of the operation and the postoperative mortality, the intracystic abscess and cholangitis which occur because of AE must be treated via medical and percutaneous methods before transplantation.

The efficiency of Gd-EOB-DTPA-enhanced magnetic resonance cholangiography in living donor liver transplantation: a preliminary study.

The aim of this study was to evaluate utility of gadoxetic acid disodium (Gd-EOB-DTPA)-enhanced magnetic resonance cholangiography (MRC) for the detection of biliary complications after living donor liver transplantation (LDLT). A total of 18 patients with suspected biliary complications underwent MRC. T2-weighted MRC and contrast-enhanced MRC (CE-MRC) were used to identify the biliary complications. MRC included routine breath-hold T2-weighted MRC using half-Fourier acquisition single-shot turbo spin-echo (HASTE) sequences and Gd-EOB-DTPA-enhanced MRC T1-weighted volumetric interpolated breath-hold examination (VIBE) sequences. Before confirming the biliary complications, one observer reviewed the MRC images and the CE-MRC images separately. The verification procedures and MRC findings were compared, and the sensitivity, specificity, and accuracy of both techniques were calculated for the identification of biliary complications. The observer found six of seven biliary complications using CE-MRC. The sensitivity was 85.7% and the accuracy was 94.4%. Using MRC alone, sensitivity was 57.1% and accuracy was 55.5%. The accuracy of Gd-EOB-DTPA-enhanced MRC was superior to MRC in locating biliary leaks (p < 0.05). The usage of Gd-EOB-DTPA-enhanced MRC yields information that complements the MRC findings that improve the identification of biliary complications. We recommend the use of MRC in addition to Gd-EOB-DTPA-enhanced MRC to increase the preoperative accuracy when assessing the biliary complications after LDLT.

The clinical importance of different localizations of the papilla associated with juxtapapillary duodenal diverticula.

BACKGROUND: Previous studies have evaluated the presence of juxtapapillary duodenal diverticula (JPDD) and the association with pancreatobiliary disease, but not the association of the papilla with an existing JPDD. We investigated the association of different localizations of the papilla with JPDD. METHODS: We studied patients in whom JPDD was detected during endoscopic retrograde cholangiopancreatography. Patients were classified into 3 groups: 1) papilla located inside the diverticulum, 2) papilla located at the edge of the diverticulum and 3) papilla located closer than 3 cm to the diverticulum. The patients were examined with respect to localization of papilla-diverticula and to the association of the localization with pancreaticobiliary disease. RESULTS: We enrolled 274 patients in our study. Biliary stone disease more frequently existed in group 3. The number of patients presenting with obstructive jaundice was higher in groups 2 (83.6%) and 3 (83.3%) than group 1 (66%). Cholangitis was more common in group 1 (21.3%) than in groups 2 (6.7%) and 3 (2.3%). The presence of biliary stone disease among patients presenting with pancreatitis was significantly different between groups 1 and 3 (p = 0.013) and between groups 2 and 3 (p = 0.017). The common bile duct more frequently contained stones or sludge in group 3 than in groups 1 and 2. CONCLUSION: When the papilla is located close to the JPDD, the incidence of biliary stone disease decreases, and pancreatobiliary diseases are caused mostly in the absence of biliary stone disease.

CONTEXTE: Des études antérieures ont évalué la présence de diverticules duodénaux juxtapapillaires (DDJP) et leur lien avec la maladie pancréatobiliaire, mais n'ont pas analysé le lien entre la papille et les DDJP existants. Nous avons analysé le lien entre diverses localisations de la papille et les DDJP. MÉTHODES: Nous avons étudié des patients chez qui des DDJP ont été détectés lors d'une cholangiopancréatographie endoscopique rétrograde. Les patients ont été classés en 3 groupes : 1) papille à l'intérieur du diverticule, 2) papille à l'extrémité du diverticule et 3) papille à moins de 3 cm du diverticule. L'examen a donc porté sur la localisation de la papille par rapport aux diverticules et sur le lien entre sa localisation et la maladie pancréatobiliaire. RÉSULTATS: Nous avons inscrit 274 patients à notre étude. La cholélithiase s'observait davantage dans le groupe 3. Le nombre de patients souffrant d'ictère obstructif était plus élevé dans les groupes 2 (83,6 %) et 3 (83,3 %) que dans le groupe 1 (66 %). La cholangite était plus fréquente dans le groupe 1 (21,3 %) que dans les groupes 2 (6,7 %) et 3 (2,3 %). Le taux de cholélithiase chez les patients souffrant de pancréatite était significativement différent entre les groupes 1 et 3 (p = 0,013) et entre les groupes 2 et 3 (p = 0,017). Il y avait plus de calculs ou de boue biliaires dans le canal cholédoque des patients du groupe 3 que dans ceux des groupes 1 et 2. CONCLUSION: Lorsque la papille est située près des DDJP, l'incidence de la cholélithiase diminue, et les maladies pancréatobiliaires sont pour la plupart causées en l'absence de cholélithiase.

Completion thyroidectomy in differentiated thyroid cancer: When to perform?

OBJECTIVE: Completion thyroidectomy is recommended in patients who have been diagnosed with differentiated thyroid cancer on histopathological evaluation, if their first operation was a conservative approach. The critical issue is when to do the second operation. MATERIAL AND METHODS: The medical records of 66 patients who underwent completion thyroidectomy for the treatment of differentiated thyroid cancer in our clinic between 2006-2013 were retrospectively analyzed. All data were compared after patients were divided into two groups according to the interval between the first surgery and completion thyroidectomy. RESULTS: Fifty-two patients (78.8%) were women and 14 patients (21.2%) were male. Completion thyroidectomy was performed 10-90 days after the initial surgery (group 1) in 26 patients, whereas it was performed later than 90 days in 40 patients (group 2). Temporary hypoparathyroidism occurred in two patients (7.7%) in group 1, and in 3 patients (7.5%) in group 2. Transient recurrent laryngeal nerve palsy was observed in 1 patient (3.9%) in group 1, and in 1 patient (2.5%) in group 2. There were no permanent morbidities in both groups. Residual tumor rate after completion thyroidectomy was 45.5%. There was no statistically significant difference between the two groups in terms of complications after completion thyroidectomy. CONCLUSION: Although in some studies it is recommended that completion thyroidectomy should be performed either before scar tissue development or after clinical remission of scar tissue, edema and inflammation, we believe that timing of surgery has no effect on morbidity.

Genetically Encoded Biosensors Unveil Neuronal Injury Dynamics via Multichromatic ATP and Calcium Imaging.

When a cell sustains damage, it liberates cytosolic ATP, which can serve as an injury signal, affecting neighboring cells. This study presents a methodological approach that employs in vitro axotomy and in vivo laser ablation to simulate cellular injury. Specially tailored biosensors are employed to monitor ATP dynamics and calcium transients in injured cells and their surroundings. To simultaneously visualize extracellular and cytosolic ATP, we developed bicistronic constructs featuring GRABATP1.0 and MaLionR biosensors alongside the calcium sensor RCaMP, enabling multiparametric imaging. In addition to transducing primary neuron cultures, we developed another method where we cocultured dorsal root ganglion neurons together with specialized "sniffer" cell lines expressing the bicistronic biosensors. Exploiting these approaches, we successfully demonstrated the release of ATP from the injured neurons and its extracellular diffusion in response to cellular injury in vitro and in vivo. Axotomy triggered intracellular calcium mobilization not only in the injured neuron but also in the intact neighboring cells, providing new insights into ATP's role as an injury signal. The tools developed in this study have demonstrated remarkable efficiency in unraveling the intricacies of ATP-mediated injury signaling.

Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions.

The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.

Development of a Dual Reporter System to Simultaneously Visualize Ca2+ Signals and AMPK Activity.

In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.

Dual activity of Minnelide chemosensitize basal/triple negative breast cancer stem cells and reprograms immunosuppressive tumor microenvironment.

Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.

Non-invasive detection of biliary leaks using Gd-EOB-DTPA-enhanced MR cholangiography: comparison with T2-weighted MR cholangiography.

OBJECTIVE: To evaluate the added role of T1-weighted (T1w) gadolinium ethoxybenzyl diethylenetriamine penta-acetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance cholangiography (MRC) compared with T2-weighted MRC (T2w-MRC) in the detection of biliary leaks. METHODS: Ninety-nine patients with suspected biliary complications underwent routine T2w-MRC and T1w contrast-enhanced (CE) MRC using Gd-EOB-DTPA to identify biliary leaks. Two observers reviewed the image sets separately and together. MRC findings were compared with those of surgery and percutaneous transhepatic cholangiopancreatography. The sensitivity, specificity and accuracy of the techniques in identifying biliary leaks were calculated. RESULTS: Accuracy of locating biliary leaks was superior with the combination of Gd-EOB-DTPA-enhanced MRC and T2w-MRC (P < 0.05).The mean sensitivities were 79 % vs 59 %, and the mean accuracy rates were 84 % vs 58 % for combined CE-MRC and T2w-MRC vs sole T2w-MRC. Nineteen out of 21 patients with biliary-cyst communication, 90.4 %, and 12/15 patients with post-traumatic biliary extravasations, 80 %, were detected by the combination of Gd-EOB-DTPA-enhanced MRC and T2w-MRC images, P < 0.05. CONCLUSIONS: Gd-EOB-DTPA-enhanced MRC yields information that complements T2w-MRC findings and improves the identification and localisation of the bile extravasations (84 % accuracy, 100 % specificity, P < 0.05). We recommend Gd-EOB-DTPA-enhanced MRC in addition to T2w-MRC to increase the preoperative accuracy of identifying and locating extravasations of bile. KEY POINTS: • Magnetic resonance cholangiography (MRC) does not always detect bile leakage and cysto-biliary communications. • Gd-EOB-DTPA-enhanced MRC helps by demonstrating extravasation of contrast material into fluid collections. • Gd-EOB-DTPA-enhanced MRC also demonstrates the leakage site and bile duct injury type. • Combined Gd-EOB-DTPA-enhanced and T2w-MRC can provide comprehensive information about biliary system. • Gd-EOB-DTPA-enhanced MRC is non-invasive and does not use ionising radiation.