An O-GlcNAcylomic Approach Reveals ACLY as a Potential Target in Sepsis in the Young Rat

Sepsis in the young population, which is particularly at risk, is rarely studied. O-GlcNAcylation is a post-translational modification involved in cell survival, stress response and metabolic regulation. O-GlcNAc stimulation is beneficial in adult septic rats. This modification is physiologically higher in the young rat, potentially limiting the therapeutic potential of O-GlcNAc stimulation in young septic rats. The aim is to evaluate whether O-GlcNAc stimulation can improve sepsis outcome in young rats. Endotoxemic challenge was induced in 28-day-old rats by lipopolysaccharide injection (E. Coli O111:B4, 20 mg·kg⁻¹) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharide injection, rats were randomly assigned to no therapy, fluidotherapy (NaCl 0.9%, 10 mL·kg⁻¹) ± NButGT (10 mg·kg⁻¹) to increase O-GlcNAcylation levels. Physiological parameters and plasmatic markers were evaluated 2h later. Finally, untargeted mass spectrometry was performed to map cardiac O-GlcNAcylated proteins. Lipopolysaccharide injection induced shock with a decrease in mean arterial pressure and alteration of biological parameters (p < 0.05). NButGT, contrary to fluidotherapy, was associated with an improvement of arterial pressure (p < 0.05). ATP citrate lyase was identified among the O-GlcNAcylated proteins. In conclusion, O-GlcNAc stimulation improves outcomes in young septic rats. Interestingly, identified O-GlcNAcylated proteins are mainly involved in cellular metabolism.     

Does Melatonin Homeostasis Play a Role in Continuous Epigastric Pain Syndrome?

Two clinical forms of functional dyspepsia (FD) are listed in the Rome III criteria: postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS), differing in the recurrence of ailments depending on the diet. Continuous EPS (CEPS) is observed in some EPS patients, also at night, but its cause is still unknown. We showed previously that melatonin (MEL) homeostasis may be associated with FD. In the present work we evaluated selected components of melatonin homeostasis in patients with CEPS. The study included 30 patients with CEPS, 21 women and nine men, aged 21–49 years and 30 control subjects (EPS excluded); organic and mental diseases, as well as Helicobacter pylori infection, were excluded in both groups. The average severity of abdominal pain in the last three months was estimated in a 10-point scale (Visual Analog Scale). The levels of mRNA expression of arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), the main components of MEL homeostasis, were determined in gastric mucosa with real time PCR. The fasting serum level of MEL (at 09:00 a.m.) and circadian urine excretion of 6-sulfatoxymelatonin (6-HMS) were determined with ELISA. AANAT expression in antral mucosa of control subjects was 1.76 ± 0.41, in the gastric body 1.35 ± 0.38, and in the dyspeptic group 1.42 ± 0.38 (p < 0.05) and 0.92 ± 0.55 (p < 0.05), respectively. HIOMT expression in the control was 2.05 ± 0.70 in the antrum and 1.57 ± 0.69 in the body and in the CEPS group, it was: 1.51 ± 0.57 (p < 0.05) and 0.74 ± 0.31 (p < 0.001), respectively. MEL concentration (pg/mL) was 9.41 ± 3.09 in the control group and 5.62 ± 1.34 (p < 0.01) in the CEPS group. Urinary 6-HMS excretion (μg/24 h) was 11.40 ± 4.46 in the controls and 7.68 ± 2.88 (p < 0.05) in the CEPS. Moreover, a negative correlation was found between the tested parameters and severity of epigastric pain. These results indicate that patients with CEPS may display low level of AANAT and HIOMT expression in gastric mucosa, resulting in decreased MEL synthesis.     

Effect of Neprilysin Inhibition for Ischemic Mitral Regurgitation after Myocardial Injury

Angiotensin receptor neprilysin inhibitor (ARNI) treatment reduces functional mitral regurgitation (MR) to a greater extent than angiotensin receptor blocker (ARB) treatment alone, but the mechanism is unclear. We evaluated the mechanisms of how ARNI has an effect on functional MR. After inducing functional MR by left circumflex coronary artery occlusion, male Sprague Dawley rats (n = 31) were randomly assigned to receive the ARNI LCZ696, the ARB valsartan, or corn oil only (MR control). Excised mitral leaflets and left ventricle (LV) were analyzed, and valvular endothelial cells were evaluated focusing on molecular changes. LCZ696 significantly attenuated LV dilatation after 6 weeks when compared with the control group (LV end-diastolic volume, 461.3 ± 13.8 µL versus 525.1 ± 23.6 µL; p < 0.05), while valsartan did not (471.2 ± 8.9 µL; p > 0.05 to control). Histopathological analysis of mitral leaflets showed that LCZ696 strongly reduced fibrotic thickness compared to the control group (28.2 ± 2.7 µm vs. 48.8 ± 7.5 µm; p < 0.05). Transforming growth factor-β and downstream phosphorylated extracellular-signal regulated kinase were also significantly lower in the LCZ696 group. Consequently, excessive endothelial-to-mesenchymal transition (EndoMT) was mitigated in the LCZ696 group compared to the control group and leaflet area was higher (11%) in the LCZ696 group than in the valsartan group. Finally, the MR extent was significantly lower in the LCZ696 group and functional improvement was observed. In conclusion, neprilysin inhibitor has positive effects on LV reverse remodeling and also attenuates fibrosis in MV leaflets and restores adaptive growth by directly modulating EndoMT.     

Newly Developed Recombinant Antithrombin Protects the Endothelial Glycocalyx in an Endotoxin-Induced Rat Model of Sepsis

(1) Background: The endothelial glycocalyx is a primary target during the early phase of sepsis. We previously reported a newly developed recombinant non-fucosylated antithrombin has protective effects in vitro. We further evaluated the effects of this recombinant antithrombin on the glycocalyx damage in an animal model of sepsis. (2) Methods: Following endotoxin injection, in Wistar rats, circulating levels of hyaluronan, syndecan-1 and other biomarkers were evaluated in low-dose or high-dose recombinant antithrombin-treated animals and a control group (n = 7 per group). Leukocyte adhesion and blood flow were evaluated with intravital microscopy. The glycocalyx was also examined using side-stream dark-field imaging. (3) Results: The activation of coagulation was inhibited by recombinant antithrombin, leukocyte adhesion was significantly decreased, and flow was better maintained in the high-dose group (both p < 0.05). Circulating levels of syndecan-1 (p < 0.01, high-dose group) and hyaluronan (p < 0.05, low-dose group; p < 0.01, high-dose group) were significantly reduced by recombinant antithrombin treatment. Increases in lactate and decreases in albumin levels were significantly attenuated in the high-dose group (p < 0.05, respectively). The glycocalyx thickness was reduced over time in control animals, but the derangement was attenuated and microvascular perfusion was better maintained in the high-dose group recombinant antithrombin group (p < 0.05). (4) Conclusions: Recombinant antithrombin maintained vascular integrity and the microcirculation by preserving the glycocalyx in this sepsis model, effects that were more prominent with high-dose therapy.     

Deep Transcranial Magnetic Stimulation Affects Gut Microbiota Composition in Obesity: Results of Randomized Clinical Trial

Growing evidence highlights the crucial role of gut microbiota in affecting different aspects of obesity. Considering the ability of deep transcranial magnetic stimulation (dTMS) to modulate the cortical excitability, the reward system, and, indirectly, the autonomic nervous system (ANS), we hypothesized a potential role of dTMS in affecting the brain-gut communication pathways, and the gut microbiota composition in obesity. In a hospital setting, 22 subjects with obesity (5 M, 17 F; 44.9 ± 2.2 years; BMI 37.5 ± 1.0 kg/m²) were randomized into three groups receiving 15 sessions (3 per week for 5 weeks) of high frequency (HF), low frequency (LF) dTMS, or sham stimulation. Fecal samples were collected at baseline and after 5 weeks of treatment. Total bacterial DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Italy) and analyzed by a metagenomics approach (Ion Torrent Personal Genome Machine). After 5 weeks, a significant weight loss was found in HF (HF: −4.1 ± 0.8%, LF: −1.9 ± 0.8%, sham: −1.3 ± 0.6%, p = 0.042) compared to LF and sham groups, associated with a decrease in norepinephrine compared to baseline (HF: −61.5 ± 15.2%, p < 0.01; LF: −31.8 ± 17.1%, p < 0.05; sham: −35.8 ± 21.0%, p > 0.05). Furthermore, an increase in Faecalibacterium (+154.3% vs. baseline, p < 0.05) and Alistipes (+153.4% vs. baseline, p < 0.05) genera, and a significant decrease in Lactobacillus (−77.1% vs. baseline, p < 0.05) were found in HF. Faecalibacterium variations were not significant compared to baseline in the other two groups (LF: +106.6%, sham: +27.6%; p > 0.05) as well as Alistipes (LF: −54.9%, sham: −15.1%; p > 0.05) and Lactobacillus (LF: −26.0%, sham: +228.3%; p > 0.05) variations. Norepinephrine change significantly correlated with Bacteroides (r² = 0.734; p < 0.05), Eubacterium (r² = 0.734; p < 0.05), and Parasutterella (r² = 0.618; p < 0.05) abundance variations in HF. In conclusion, HF dTMS treatment revealed to be effective in modulating gut microbiota composition in subjects with obesity, reversing obesity-associated microbiota variations, and promoting bacterial species representative of healthy subjects with anti-inflammatory properties.     

Proliferation to Apoptosis Tumor Cell Ratio as a Biomarker to Improve Clinical Management of Pre-Malignant and Symptomatic Plasma Cell Neoplasms

Proliferation and apoptosis of neoplastic cells are prognostic biomarkers in plasma cell neoplasms (PCNs). The prognostic capacity of proliferation to apoptosis ratio (Ratio-PA) in the era of immunomodulatory treatments is re-evaluated in 316 gammopathy of undetermined significance (MGUS), 57 smoldering multiple myeloma (SMM), and 266 multiple myeloma (MM) patients. Ratio-PA of 0.77 ± 0.12, 1.94 ± 0.52, and 11.2 ± 0.7 (p < 0.0001) were observed in MGUS, SMM, and MM patients. Ten-year overall survival (10y-OS) rates for patients with low/high Ratio-PA were 93.5%/77.3% p < 0.0001) for MGUS, 82.5%/64.7% (p < 0.05) for SMM, and 62.3%/47.0% (p < 0.05) for MM. For patients with low, intermediate, and high risk, 10y-OS for low/high Ratio-PA were 95.5%/72.9% (p < 0.0001), 74.2%/50.4% (p < 0.0001), and 35.3%/20.0% (p = 0.836), respectively. Ratio-PA was an independent prognostic factor for OS (HR = 2.119, p < 0.0001, Harrell-C-statistic = 0.7440 ± 0.0194) when co-analyzed with sex, age, and standard risk. In patients with Ratio-PA^high, only first-line therapy with VRd/VTd, but not PAD/VCD, coupled with ASCT was associated with high 10y-OS (82.7%). Tumor cell Ratio-PA estimated at diagnosis offers a prognostic biomarker that complements standard risk stratification and helps to guide the clinical management of pre-malignant and symptomatic PCNs. Every effort should be made to provide first-line therapies including VTd or VRd associated with ASCT to patients with Ratio-PA^high at higher risk of progression and death.     

Toxicity evaluation of zinc aluminium levodopa nanocomposite via oral route in repeated dose study

Nanotechnology, through nanomedicine, allowed drugs to be manipulated into nanoscale sizes for delivery to the different parts of the body, at the same time, retaining the valuable pharmacological properties of the drugs. However, efficient drug delivery and excellent release potential of these delivery systems may be hindered by possible untoward side effects. In this study, the sub-acute toxicity of oral zinc aluminium nanocomposite with and without levodopa was assessed using the Organization for Economic Co-operation and Development guidelines. No sign or symptom of toxicity was observed in orally treated rats with the nanocomposite at 5 and 500 mg/kg concentrations. Body weight gain, feeding, water intake, general survival and organosomatic index were not significantly different between control and treatment groups. Aspartate aminotransferase (AST) in 500 mg/kg levodopa nanocomposite (169 ± 30 U/L), 5 mg/kg levodopa nanocomposite (172 ± 49 U/L), and 500 mg/kg layered double hydroxides (LDH) nanocomposite (175 ± 25 U/L) were notably elevated compared to controls (143 ± 05 U/L); but the difference were not significant (p > 0.05). However, the differences in aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio of 500 mg/kg levodopa nanocomposite (0.32 ± 0.12) and 500 mg/kg LDH nanocomposite (0.34 ± 0.12) were statistically significant (p < 0.05) compared to the control (0.51 ± 0.07). Histology of the liver, spleen and brain was found to be of similar morphology in both control and experimental groups. The kidneys of 500-mg/kg-treated rats with levodopa nanocomposite and LDH nanocomposite were found to have slight inflammatory changes, notably leukocyte infiltration around the glomeruli. The ultra-structure of the neurons from the substantia nigra of nanocomposite-exposed group was similar to those receiving only normal saline. The observed result has suggested possible liver and renal toxicity in orally administered levodopa intercalated nanocomposite; it is also dose-dependent that needs further assessment.     

Adjuvant Effect of Quillaja saponaria Saponin (QSS) on Protective Efficacy and IgM Generation in Turbot (Scophthalmus maximus) upon Immersion Vaccination

The adjuvant effect of Quillaja saponaria saponin (QSS) on protection of turbot fry was investigated with immersion vaccination of formalin-killed Vibrio anguillarum O1 and various concentrations of QSS (5, 25, 45 and 65 mg/L). Fish were challenged at days 7, 14 and 28 post-vaccination. Significantly high relative percent of survival (RPS) ((59.1 ± 13.6)%, (81.7 ± 8.2)%, (77.8 ± 9.6)%) were recorded in the fish that received bacterins immersion with QSS at 45 mg/L, which is comparable to the positive control group vaccinated by intraperitoneal injection (IP). Moreover, a remarkably higher serum antibody titer was also demonstrated after 28 days in the vaccinated fish with QSS (45 mg/L) than those vaccinated fish without QSS (p < 0.05), but lower than the IP immunized fish (p < 0.05). Significant upregulation of IgM gene expression has also been identified in the tissues of skin, gill, spleen and kidney from the immunized fish in comparison to the control fish. Taken together, the present study indicated that QSS was able to dramatically evoke systemic and mucosal immune responses in immunized fish. Therefore, QSS might be a promising adjuvant candidate for fish vaccination via an immersion administering route.     

Platelet Inhibition Prevents NLRP3 Inflammasome Activation and Sepsis-Induced Kidney Injury

Platelets, cellular mediators of thrombosis, are activated during sepsis and are increasingly recognized as mediators of the immune response. Platelet activation is significantly increased in sepsis patients compared to ICU control patients. Despite this correlation, the role of activated platelets in contributing to sepsis pathophysiology remains unclear. We previously demonstrated NOD-like receptor protein 3 inflammasome (NLRP3) inflammasome activation in sepsis-induced platelets from cecal-ligation puncture (CLP) rats. Activated platelets were associated with increased pulmonary edema and glomerular injury in CLP vs. SHAM controls. In this study, we investigated whether inhibition of platelet activation would attenuate NLRP3 activation and renal and pulmonary injury in response to CLP. CLP was performed in male and female Sprague Dawley (SD) rats (n = 10/group) to induce abdominal sepsis and SHAM rats served as controls. A subset of CLP animals was treated with Clopidogrel (10 mg/kg/day, CLP + CLOP) to inhibit platelet activation. At 72 h post-CLP, platelet activation and NLRP3 inflammasome assembly were evaluated, IL-1β and IL-18 were measured in plasma, and tissues, renal and pulmonary pathology, and renal function were assessed. Activated platelets were 7.8 ± 3.6% in Sham, 22 ± 6% in CLP and significantly decreased to 14.5 ± 0.6% in CLP + CLOP (n = 8–10/group, p < 0.05). NLRP3 inflammasome assembly was inhibited in platelets of CLP + CLOP animals vs. CLP. Significant increases in plasma and kidney IL-1β and IL-18 in response to CLP were decreased with Clopidogrel treatment. Renal injury, but not lung histology or renal function was improved in CLP + CLOP vs. CLP. These data provide evidence that activated platelets may contribute to sepsis-induced renal injury, possibly via NLRP3 activation in platelets. Platelets may be a therapeutic target to decrease renal injury in septic patients.     

Effect of Platelet-Rich Plasma and Bioactive Glass Powder for the Improvement of Rotator Cuff Tendon-to-Bone Healing in a Rabbit Model

To test the hypothesis that a platelet-rich plasma (PRP) plus bioactive glass (BG) mixture could shorten the tendon-bone healing process in rotator cuff tendon repair, thirty mature male New Zealand white rabbits were randomly divided into three groups, Control, PRP, and PRP + BG. All groups underwent a surgical procedure to establish a rotator cuff tendon healing model. Mechanical examinations and histological assays were taken to verify the adhesion of the tendon-bone. Real-time PCR was adopted to analyze Bone Morphogenetic Protein-2 (BMP-2). The maximum load-to-failure value in mechanical examinations was significantly higher in the PRP + BG group than that in the control group after six weeks (Control 38.73 ± 8.58, PRP 54.49 ± 8.72, PRP + BG 79.15 ± 7.62, p < 0.001), but it was not significantly different at 12 weeks (PRP 74.27 ± 7.74, PRP + BG 82.57 ± 6.63, p = 0.145). In histological assays, H&E (hematoxylin-eosin) staining showed that the interface between the tendon-bone integration was much sturdier in the PRP + BG group compared to the other two groups at each time point, and more ordered arranged tendon fibers can be seen at 12 weeks. At six weeks, the mRNA expression levels of BMP-2 in the PRP + BG group were higher than those in the other groups (PRP + BG 0.65 ± 0.11, PRP 2.284 ± 0.07, Control 0.12 ± 0.05, p < 0.05). However, there was no significant difference in the mRNA expression levels of BMP-2 among the three groups at 12 weeks (p = 0.922, 0.067, 0.056). BMP-2 levels in PRP and PRP+BG groups were significantly lower at 12 weeks compared to six weeks (p = 0.006, <0.001).We found that the PRP + BG mixture could enhance tendon-bone healing in rotator cuff tendon repair.     

Induction of Tertiary Phase Epileptiform Discharges after Postasphyxial Infusion of a Toll-Like Receptor 7 Agonist in Preterm Fetal Sheep

Background: Toll-like receptor (TLR) agonists are key immunomodulatory factors that can markedly ameliorate or exacerbate hypoxic–ischemic brain injury. We recently demonstrated that central infusion of the TLR7 agonist Gardiquimod (GDQ) following asphyxia was highly neuroprotective after 3 days but not 7 days of recovery. We hypothesize that this apparent transient neuroprotection is associated with modulation of seizure-genic processes and hemodynamic control. Methods: Fetuses received sham asphyxia or asphyxia induced by umbilical cord occlusion (20.9 ± 0.5 min) and were monitored continuously for 7 days. GDQ 3.34 mg or vehicle were infused intracerebroventricularly from 1 to 4 h after asphyxia. Results: GDQ infusion was associated with sustained moderate hypertension that resolved after 72 h recovery. Electrophysiologically, GDQ infusion was associated with reduced number and burden of postasphyxial seizures in the first 18 h of recovery (p < 0.05). Subsequently, GDQ was associated with induction of slow rhythmic epileptiform discharges (EDs) from 72 to 96 h of recovery (p < 0.05 vs asphyxia + vehicle). The total burden of EDs was associated with reduced numbers of neurons in the caudate nucleus (r² = 0.61, p < 0.05) and CA1/2 hippocampal region (r² = 0.66, p < 0.05). Conclusion: These data demonstrate that TLR7 activation by GDQ modulated blood pressure and suppressed seizures in the early phase of postasphyxial recovery, with subsequent prolonged induction of epileptiform activity. Speculatively, this may reflect delayed loss of early protection or contribute to differential neuronal survival in subcortical regions.     

Morphometric and Nanomechanical Features of Platelets from Women with Early Pregnancy Loss Provide New Evidence of the Impact of Inherited Thrombophilia

Pregnancy is associated with hypercoagulation states and increased thrombotic risk, especially in women with thrombophilia. We combine atomic force microscopy (AFM) and flow cytometry to examine the morphology and nanomechanics of platelets derived from women with early pregnancy loss (EPL) and control pregnant (CP) and non-pregnant (CNP) women. Both control groups exhibit similar morphometric parameters (height and surface roughness) and membrane stiffness of platelets. EPL patients’ platelets, on the other hand, are more activated than the control groups, with prominent cytoskeletal rearrangement. In particular, reduced membrane roughness (22.9 ± 6 nm vs. 39.1 ± 8 nm) (p < 0.05) and height (692 ± 128 nm vs. 1090 ± 131 nm) (p < 0.05), strong alteration in the membrane Young modulus, increased production of platelets’ microparticles, and higher expression of procoagulant surface markers, as well as increased occurrence of thrombophilia (FVL, FII20210A, PLA1/A2, MTHFR C677T or 4G/5G PAI-1) polymorphisms were found. We suggest that the carriage of thrombophilic mutations triggers structural and nanomechanical abnormalities in platelets, resulting in their increased activation. The activation state of platelets can be well characterized by AFM, and the morphometric and nanomechanical characteristics might serve as a new criterion for evaluation of the cause of miscarriage and offer the prospect of an innovative approach serving for diagnostic purposes.     

Cardioprotective Properties of Mannitol—Involvement of Mitochondrial Potassium Channels

Cardiac preconditioning (PC) and postconditioning (PoC) are powerful measures against the consequences of myocardial ischemia and reperfusion (I/R) injury. Mannitol—a hyperosmolar solution—is clinically used for treatment of intracranial and intraocular pressure or promotion of diuresis in renal failure. Next to these clinical indications, different organ-protective properties—e.g., perioperative neuroprotection—are described. However, whether Mannitol also confers cardioprotection via a pre- and/or postconditioning stimulus, possibly reducing consequences of I/R injury, remains to be seen. Therefore, in the present study we investigated whether (1) Mannitol-induced pre- and/or postconditioning induces myocardial infarct size reduction and (2) activation of mitochondrial ATP-sensitive potassium (mK_ATP) channels is involved in cardioprotection by Mannitol. Experiments were performed on isolated hearts of male Wistar rats via a pressure controlled Langendorff system, randomized into 7 groups. Each heart underwent 33 min of global ischemia and 60 min of reperfusion. Control hearts (Con) received Krebs–Henseleit buffer as vehicle only. Pre- and postconditioning was achieved by administration of 11 mmol/L Mannitol for 10 min before ischemia (Man-PC) or immediately at the onset of reperfusion (Man-PoC), respectively. In further groups, the mK_ATP channel blocker 5HD, was applied with and without Mannitol, to determine the potential underlying cardioprotective mechanisms. Primary endpoint was infarct size, determined by triphenyltetrazolium chloride staining. Mannitol significantly reduced infarct size both as a pre- (Man-PC) and postconditioning (Man-PoC) stimulus compared to control hearts (Man-PC: 31 ± 4%; Man-PoC: 35 ± 6%, each p < 0.05 vs. Con: 57 ± 9%). The mK_ATP channel inhibitor completely abrogated the cardioprotective effect of Mannitol-induced pre- (5HD-PC-Man-PC: 59 ± 8%, p < 0.05 vs. Man-PC) and postconditioning (5HD-PoC-Man-PoC: 59 ± 10% vs. p < 0.05 Man-PoC). Infarct size was not influenced by 5HD itself (5HD-PC: 60 ± 14%; 5HD-PoC: 54 ± 14%, each ns vs. Con). This study demonstrates that Mannitol (1) induces myocardial pre- and postconditioning and (2) confers cardioprotection via activation of mK_ATP channels.     

Retrospective Study of Metastatic Melanoma and Renal Cell Carcinoma to the Brain with Multivariate Analysis of Prognostic Pre-Treatment Clinical Factors

Patients with brain metastasis from renal cell carcinoma (RCC) or melanoma have historically had very poor prognoses of less than one year. Stereotactic radiosurgery (SRS) can be an effective treatment for patients with these tumors. This study analyzes the effect of pretreatment prognostic factors on overall survival (OS) for RCC and melanoma patients with metastasis to the brain treated with SRS. A total of 122 patients with brain metastases from either RCC or melanoma were grouped by age at brain metastasis diagnosis, whether they received whole brain radiation therapy (WBRT) in addition to SRS, or they underwent surgical resection, Karnofsky Performance Score (KPS), number of brain metastases, and primary tumor. Median survival times for melanoma patients and RCC patients were 8.20 ± 3.06 and 12.70 ± 2.63 months, respectively. Patients with >5 metastases had a significantly shorter median survival time (6.60 ± 2.45 months) than the reference group (1 metastasis, 10.70 ± 13.40 months, p = 0.024). Patients with KPS ≤ 60 experienced significantly shorter survival than the reference group (KPS = 90–100), with median survival times of 5.80 ± 2.46 months (p < 0.001) and 45.20 ± 43.52 months, respectively. We found a median overall survival time of 12.7 and 8.2 months for RCC and melanoma, respectively. Our study determined that a higher number of brain metastases (>5) and lower KPS were statistically significant predictors of a lower OS prognosis.     

Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3β (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading.     

Low-Dose Empagliflozin Improves Systolic Heart Function after Myocardial Infarction in Rats: Regulation of MMP9, NHE1, and SERCA2a

The effects of the selective sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin in low dose on cardiac function were investigated in normoglycemic rats. Cardiac parameters were measured by intracardiac catheterization 30 min after intravenous application of empagliflozin to healthy animals. Empagliflozin increased the ventricular systolic pressure, mean pressure, and the max dP/dt (p < 0.05). Similarly, treatment with empagliflozin (1 mg/kg, p.o.) for one week increased the cardiac output, stroke volume, and fractional shortening (p < 0.05). Myocardial infarction (MI) was induced by ligation of the left coronary artery. On day 7 post MI, empagliflozin (1 mg/kg, p.o.) improved the systolic heart function as shown by the global longitudinal strain (−21.0 ± 1.1% vs. −16.6 ± 0.7% in vehicle; p < 0.05). In peri-infarct tissues, empagliflozin decreased the protein expression of matrix metalloproteinase 9 (MMP9) and favorably regulated the cardiac transporters sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and sodium hydrogen exchanger 1 (NHE1). In H9c2 cardiac cells, empagliflozin decreased the MMP2,9 activity and prevented apoptosis. Empagliflozin did not alter the arterial stiffness, blood pressure, markers of fibrosis, and necroptosis. Altogether, short-term treatment with low-dose empagliflozin increased the cardiac contractility in normoglycemic rats and improved the systolic heart function in the early phase after MI. These effects are attributed to a down-regulation of MMP9 and NHE1, and an up-regulation of SERCA2a. This study is of clinical importance because it suggests that a low-dose treatment option with empagliflozin may improve cardiovascular outcomes post-MI. Down-regulation of MMPs could be relevant to many remodeling processes including cancer disease.     

VLDL from Metabolic Syndrome Individuals Enhanced Lipid Accumulation in Atria with Association of Susceptibility to Atrial Fibrillation

Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL was therefore separated from normal (normal-VLDL) and MetS individuals. Wild type C57BL/6 male mice were divided into control, normal-VLDL (nVLDL), and MetS-VLDL (msVLDL) groups. VLDL (15 µg/g) and equivalent volumes of saline were injected via tail vein three times a week for six consecutive weeks. Cardiac chamber size and function were measured by echocardiography. MetS-VLDL significantly caused left atrial dilation (control, n = 10, 1.64 ± 0.23 mm; nVLDL, n = 7, 1.84 ± 0.13 mm; msVLDL, n = 10, 2.18 ± 0.24 mm; p < 0.0001) at week 6, associated with decreased ejection fraction (control, n = 10, 62.5% ± 7.7%, vs. msVLDL, n = 10, 52.9% ± 9.6%; p < 0.05). Isoproterenol-challenge experiment resulted in AF in young msVLDL mice. Unprovoked AF occurred only in elderly msVLDL mice. Immunohistochemistry showed excess lipid accumulation and apoptosis in msVLDL mice atria. These findings suggest a pivotal role of VLDL in AF pathogenesis for MetS individuals.     

Resistance Training Regulates Cardiac Function through Modulation of miRNA-214

Aims: To determine the effects of resistance training (RT) on the expression of microRNA (miRNA)-214 and its target in sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a), and on the morphological and mechanical properties of isolated left ventricular myocytes. Main methods: Male Wistar rats were divided into two groups (n = 7/group): Control (CO) or trained (TR). The exercise-training protocol consisted of: 4 × 12 bouts, 5×/week during 8 weeks, with 80% of one repetition maximum. Key findings: RT increased the left ventricular myocyte width by 15% and volume by 12%, compared with control animals (p < 0.05). The time to half relaxation and time to peak were 8.4% and 4.4% lower, respectively, in cells from TR group as compared to CO group (p < 0.05). RT decreased miRNA-214 level by 18.5% while its target SERCA2a expression were 18.5% higher (p < 0.05). Significance: Our findings showed that RT increases single left ventricular myocyte dimensions and also leads to faster cell contraction and relaxation. These mechanical adaptations may be related to the augmented expression of SERCA2a which, in turn, may be associated with the epigenetic modification of decreased miRNA-214 expression.     

A Disease Marker for Aspirin-Induced Chronic Urticaria

There are currently no diagnostic methods in vitro for aspirin-induced chronic urticaria (AICU) except for the provocation test in vivo. To identify disease markers for AICU, we investigated the single nucleotide polymorphism (SNP) of the promoter loci of high-affinity IgE receptor (FcεRIα) and CD203c expression level in Chinese patients with AICU. We studied two genotypic and allelic frequencies of rs2427827 (–344C/T) and rs2251746 (–66T/C) gene polymorphisms of FcεRIα in 20 patients with AICU, 52 subjects with airway hypersensitivity without aspirin intolerance, and 50 controls in a Chinese population. The results showed that the frequencies of two SNPs (–344C>T, –66C>T) were similar to the normal controls. The allele frequency of –344CC was significantly higher in the patients with AICU compared to those with airway sensitivity (p = 0.019). We also studied both histamine release and CD203c expression on KU812 cells to assess aspirin-induced basophil activation. We found that the activity of basophil activation of AICU was significantly higher in the patients with AICU compared to those with airway hypersensitivity without aspirin intolerance. The mean fluorescence intensity of the CD203c expression were 122.5 ± 5.2 vs. 103.3 ± 3.3 respectively, (p < 0.05), and the percentages of histamine release were 31.3% ± 7.4% vs. −24.0% ± 17.5%, (p < 0.05) respectively. Although the mean fluorescence intensity of CD203c expression and the percentage of histamine release were significantly up-regulated by aspirin, they were not affected by anti-IgE antibodies. These results suggest that a single SNP of FcεRIα (–344C>T) is less likely to develop AICU and the basophil activation activity in the sera by measuring CD203c expression can be applicable to confirm the diagnosis of AICU.