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.

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.

A Comparison of Laparoscopic Surgery and Open Surgery in Liver Resections: A Single-Center Experience.

OBJECTIVE: With the developments in patient management and the increase in surgical experience, the use of laparoscopy in liver resections has become widespread. However, with the consensus meetings and international recommendations, laparoscopic liver resections have been tried to be standardized. We aimed to present this laparoscopic liver resection experience by comparing open and laparoscopic techniques. MATERIALS AND METHODS: Patients who underwent liver resections between 2015 and 2022 were retrospectively screened and divided into 2 groups as laparoscopic liver resections and patients who underwent liver resection with open surgery. Indications, resection techniques, operative times, length of hospital stay, early hospital mortality, and complications were compared between both groups using statistical methods. RESULTS: Laparoscopic surgery was performed in 31 (14%) patients, and open surgery was performed in 189 (86%). The mean operation time was 316 ± 168.2 minutes in patients who underwent laparoscopic liver resection. It was 329.4 ± 123.6 in the open surgery group. The length of hospital stay was 11.6 ± 4.9 days in patients who underwent laparoscopic liver resection, while it was 19.7 ± 12.1 days in patients who underwent open surgery. The difference between the length of hospital stay was statistically significant (Mann-Whitney U-test, P=.00). There was no difference between the 2 groups in terms of complications and early mortality. CONCLUSION: Laparoscopic liver resections are a safe method that can be applied in 3 or less segment resections. As the experience of the surgical team increases, it can be safely applied for major hepatectomies.

Significance of the Neutrophil-to-Lymphocyte Ratio, Platelet-to-Lymphocyte Ratio, and Preoperative Nutritional Index as Predictors of Morbidity in Patients Who Underwent Liver Resection for Alveolar Echinococcosis.

AIM: We aimed to evaluate the significance of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and preoperative nutritional index (PNI) as predictors of morbidity in patients who underwent liver resection for alveolar echinococcosis. MATERIAL AND METHODS: This single-center study was designed as a retrospective study after obtaining ethical committee approval. The files of patients hospitalized at Ataturk University Faculty of Medicine, Erzurum, Turkey, between 2010 and 2019 and who underwent resection or liver transplantation for liver alveolar cysts were reviewed. Demographic features, laboratory parameters (complete blood count and biochemical parameters), lesion localizations and characteristics, type of surgery, intraoperative and postoperative complications (morbidity), and mortality status were evaluated by scanning patients' files. Preoperative blood samples were taken the day before the surgery, which is the period farthest from surgical stress, to have more accurate results. By contrast, postoperative blood samples were taken on the first postoperative day when surgical stress was the highest. The differences between the morbidity groups, including NLR, PLR, and PNI, were compared. RESULTS: Of the 172 patients in the study, 96 (55.8%) were female. The mean age of all patients was 48.51±15.57 (18-90). Perioperative complications were seen in 30 (17.4%) patients, while the morbidity and mortality rates of the study were 28.5% and 19.2%, respectively. Age, gender of patients, and preoperative laboratory parameters, including NLR, PLR, and PNI, did not affect morbidity. However, the presence of perioperative vascular injury (P=0.040) and complications (P=0.047), low postoperative lymphocyte rates (P=0.038), and high postoperative NLR were associated with increased morbidity. In addition, the mortality rate was significantly increased in patients with morbidity (P<0.001). CONCLUSION: From the results of the present study, it was found that preoperative parameters did not affect morbidity, while increased postoperative NLR levels and decreased lymphocyte rates increased morbidity.

Active shrinkage protects neurons following axonal transection.

Trauma, vascular events, or neurodegenerative processes can lead to axonal injury and eventual transection (axotomy). Neurons can survive axotomy, yet the underlying mechanisms are not fully understood. Excessive water entry into injured neurons poses a particular risk due to swelling and subsequent death. Using in vitro and in vivo neurotrauma model systems based on laser transection and surgical nerve cut, we demonstrated that axotomy triggers actomyosin contraction coupled with calpain activity. As a consequence, neurons shrink acutely to force water out through aquaporin channels preventing swelling and bursting. Inhibiting shrinkage increased the probability of neuronal cell death by about 3-fold. These studies reveal a previously unrecognized cytoprotective response mechanism to neurotrauma and offer a fresh perspective on pathophysiological processes in the nervous system.

The acute effect of different NAD+ precursors included in the combined metabolic activators.

NAD+ and glutathione precursors are currently used as metabolic modulators for improving the metabolic conditions associated with various human diseases, including non-alcoholic fatty liver disease, neurodegenerative diseases, mitochondrial myopathy, and age-induced diabetes. Here, we performed a one-day double blinded, placebo-controlled human clinical study to assess the safety and acute effects of six different Combined Metabolic Activators (CMAs) with 1 g of different NAD+ precursors based on global metabolomics analysis. Our integrative analysis showed that the NAD+ salvage pathway is the main source for boosting the NAD+ levels with the administration of CMAs without NAD+ precursors. We observed that incorporation of nicotinamide (Nam) in the CMAs can boost the NAD+ products, followed by niacin (NA), nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), but not flush free niacin (FFN). In addition, the NA administration led to a flushing reaction, accompanied by decreased phospholipids and increased bilirubin and bilirubin derivatives, which could be potentially risky. In conclusion, this study provided a plasma metabolomic landscape of different CMA formulations, and proposed that CMAs with Nam, NMN as well as NR can be administered for boosting NAD+ levels to improve altered metabolic conditions.

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.

Longitudinal metabolomics analysis reveals the acute effect of cysteine and NAC included in the combined metabolic activators.

Growing evidence suggests that the depletion of plasma NAD+ and glutathione (GSH) may play an important role in the development of metabolic disorders. The administration of Combined Metabolic Activators (CMA), consisting of GSH and NAD+ precursors, has been explored as a promising therapeutic strategy to target multiple altered pathways associated with the pathogenesis of the diseases. Although studies have examined the therapeutic effect of CMA that contains N-acetyl-l-cysteine (NAC) as a metabolic activator, a system-wide comparison of the metabolic response to the administration of CMA with NAC and cysteine remains lacking. In this placebo-controlled study, we studied the acute effect of the CMA administration with different metabolic activators, including NAC or cysteine with/without nicotinamide or flush free niacin, and performed longitudinal untargeted-metabolomics profiling of plasma obtained from 70 well-characterized healthy volunteers. The time-series metabolomics data revealed the metabolic pathways affected after the administration of CMAs showed high similarity between CMA containing nicotinamide and NAC or cysteine as metabolic activators. Our analysis also showed that CMA with cysteine is well-tolerated and safe in healthy individuals throughout the study. Last, our study systematically provided insights into a complex and dynamics landscape involved in amino acid, lipid and nicotinamide metabolism, reflecting the metabolic responses to CMA administration containing different metabolic activators.

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.

Turkish HPB Surgery Association consensus report on hepatic cystic Echinococcosis (HCE).

Objectives: Cystic Echinococcosis (CE) is one of the important problems of the Eurasian region. We aimed to prepare a consensus report in order to update the treatment approaches of this disease. This study was conducted by Turkish HPB Surgery Association. Material and Methods: This study was conducted with the modified Delphi model. For this purpose, we conducted a three-stage consensus-building approach. Results: Six topics, including diagnosis, medical treatment, percutaneous treatment, surgical treatment, management of complications and posttreatment follow-up and recurrences in HCE were discussed. Conclusion: The expert panel made recommendations for every topic.

External Iliac Artery Anastomosis and Internal Iliac Artery Anastomosis for Artery anastomosis in Deceased-donor Kidney Transplantation and Multifactorial Analysis of Graft Survival.

OBJECTIVE: To determine the effects of surgical techniques applied to arterial anastomosis for kidney transplantation on the graft outcome. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Organ Transplantation Center, Ataturk University Research Hospital and School of Medicine, Erzurum, Turkey, from January 2010 to January 2020. METHODOLOGY: In total, 143 consecutive patients who underwent deceased-donor-donor kidney transplantation during a 10-years period were retrospectively analysed. All patients were divided into two groups according to the vascular anastomosis techniques (end-to side external iliac and end-to-end internal iliac). The two groups were compared in terms of urine volume on postoperative days 1 and 7; blood creatinine levels on postoperative days 1, 2, and 7; complications; and graft survival. RESULTS: The mean patient age was 42.04 ± 11.1 years. No significant difference was observed between creatinine values ​​and urine amounts for both surgical techniques (p >0.05). Only the amount of urine on the postoperative 7th day had a significant effect on graft survival (p <0.05). There was no significant difference between the two anastomosis techniques in terms of graft survival (p >0.05). CONCLUSION: Both surgical techniques can be used safely in renal transplantation and arterial anastomosis. Also, decreased urine volume during follow-up can be considered as an early indicator of graft loss in the long-term. KEY WORDS: Kidney transplantation, Surgical anastomosis, Delayed graft function, Graft survival.

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.

Development of diaphragmatic hernia following hepatectomy.

BACKGROUND: The majority of the diaphragmatic hernias developing on the right side are hernias developing after right hepatectomy. We aimed to present the clinical presentation at the time of admission, surgical treatment, and postoperative course of patients that developed DH following a major liver resection in our center. PATIENTS AND METHODS: Liver surgeries performed in Ataturk University Organ Transplant Center and Ataturk University Research Hospital General surgery Hepatopancreatobiliary ward between 2012 and 2021 were analysed retrospectively. Demographic information, admission types (emergency or elective), admission clinics, the process of diaphragmatic hernia formation following hepatectomy, imaging methods used for diagnosis, and surgical methods performed were recorded for the patients with diaphragmatic hernia. Qualitative values were tabulated and their percentages were calculated. RESULTS: Six hundred and sixty patients who underwent major liver surgery in our center between 2012 and 2021 were analysed. It was found that diaphragmatic hernia developed in 9 (1.4%) of those patients. The incidence of diaphragmatic hernia after donor hepatectomy was 3.04% in our study. The mean time from the first surgery until the DH diagnosis was 47.33 ± 38.16 months. 1 (11.1%) patient had small intestine perforation and 1 patient had both small intestine and colon perforation. One patient died before to the surgery. CONCLUSION: DH following liver resection can cause fatal complications and it should be intervened when diagnosed. Although it is rare, centers particularly focusing on hepatobiliary surgery should determine a follow-up protocol to detect DH that develops after major liver resections.

Giant Incisional Hernia Repair Using Open Intraperitoneal Dual Mesh.

Aim Giant incisional herniae are larger than 15 cm and are typically treated with an open approach. Our aim was to highlight the outcomes of treating giant incisional hernia using open intraperitoneal dual mesh. Methods Between January 2015 and December 2021, 25 patients with giant incisional hernias, where fascial defects were 15-30 cm, were evaluated retrospectively. Intraperitoneal dual mesh was used in all patients. The patients were evaluated in terms of age, gender, body mass index (BMI), previous abdominal surgeries, defect diameter, anesthesia method, length of hospital stay, drain application, complications, and recurrence. Results Eleven of the patients were male and 14 were female. The mean age was 62±13.5 years (29-82 years). The average BMI was 32 kg/m2 (20-52 kg/m2). The mean size of the fascial defect was 22±5.5 cm (15-30). The mean operation time was 90 minutes (70-130 minutes). Six patients had type I and II complications according to the Clavien-Dindo classification, specifically superficial skin infections, skin erosion, subcutaneous bleeding, and temporary ileus due to intestinal adhesion. During the average follow-up period of 36 months (6-70 months), no major complications were observed related to the recurrence and use of dual mesh. Conclusion In the treatment of giant incisional hernia, open intraperitoneal dual mesh application should be kept in mind as an effective treatment option with low complication and recurrence rates.

Anatomical characterization of vagal nodose afferent innervation and ending morphologies at the murine heart using a transgenic approach.

Heart is an extensively innervated organ and its function is strictly coordinated by autonomic neural circuits. After pathological events such as myocardial infarction (MI), cardiac nerves undergo a structural and functional remodeling contributing to cardiac dysfunction. Although the efferent component of the cardiac nerves has been well described, sensory innervation of the heart has not been defined in detail. Considering its importance, comprehensive description of vagal afferent innervation on the whole heart would enable a better description of autonomic imbalances manifesting as sympathoexcitation and vagal withdrawal in post-ischemic states. To address this issue, we globally mapped the vagal nodose afferent fibers innervating the whole murine heart with unprecedented resolution. By using the Phox2b-Cre::tdTomato transgenic mouse line, we described the detailed distribution and distinct vagal sensory ending morphologies at both the dorsal and ventral sides of the mouse heart. By neural tracing analysis, we quantitated the distribution and prevalence of vagal afferent nerve fibers with varying diameters across dorsal and ventral surfaces of the heart. Moreover, we demonstrated that vagal afferents formed flower spray and end-net-like endings within the atria and ventricles. As distinct from the atria, vagal afferents formed intramuscular array-like endings within the ventricles. Furthermore, we showed that vagal afferents undergo structural remodeling by forming axonal sprouts around the infarct area in post-MI hearts. These findings improve our understanding of the potential effect of vagal afferent remodeling on autonomic imbalance and generation of cardiac arrhythmias and could prospectively contribute to the development of more effective neuromodulatory therapies.

A Retrospective Evaluation of Spleen Hydatid Cyst Treatments: The Spleen-Preserving Conservative Approach is Preferable to Splenectomy.

OBJECTIVE: Cystic echinococcosis (CE) is rarely encountered in the spleen, even in areas where the disease is endemic. There is no consensus in the literature concerning the treatment of splenic cystic echinococcosis. This study was intended to compare the treatment results and factors affecting the selection of the spleen- preserving approach or splenectomy in treatment. MATERIALS AND METHODS: Four hundred and seventy patients operated for cystic echinococcosis at a university clinic between January 2010 and December 2019 were retrospectively evaluated. Splenic cystic echinococ- cosis was identified in 22 patients (4.7%). Demographic features as well as clinical and laboratory findings of patients who underwent splenectomy or spleen-preserving operations were compared between the groups. RESULTS: Spleen-preserving cystotomy was performed on 18 patients (81.8%), and splenectomy on 4 (18.2%). Eleven patients (50%) had isolated cystic echinococcosis in the spleen, while another 11 (50%) had hepatic and splenic cystic echinococcosis. The median cyst size was 10.0 cm (min: 3, max: 20). Splenectomy patients had significantly larger cyst sizes (median, min-max; 8.0 cm, 3-15 cm vs. 15.0 cm, 10-20) (Z=2.071, P=.042). Postoperatively, 1 patient from the splenectomy group (25.0%) developed deep vein thrombosis, and another from the cystotomy group (5.5%) developed pulmonary embolism. No other serious complications, re-operation requirements, or recurrence were observed after a median follow-up period of 27.0 months (min: 5, max: 92). CONCLUSION: Since no collections or recurrence were observed, and splenic functions were preserved in patients who underwent spleen-preserving cystotomy, we recommend the use of spleen-protective cys- totomy. Splenectomy should be employed in patients with cyst diameters exceeding 10 cm, with a central location, and in cases with difficult cavity management and a high risk of developing postoperative collections.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.

Assessment of the Body Posture of Interventional Radiologists.

Physicians in interventional radiology are exposed to high physical stress. To avoid negative long-term effects resulting from unergonomic working conditions, we demonstrated the feasibility of a system that gives feedback about unergonomic situations arising during the intervention based on the Azure Kinect camera. The overall feasibility of the approach could be shown.