TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation.

Background/aim: Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Materials and methods: Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. Results: In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. Conclusion: TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.

Low C3 in a 4-month-old baby: is it a problem?

A 4-month-old male baby was admitted because his father and uncles had chronic kidney disease. His father was diagnosed with membranoproliferative glomerulonephritis at the age of 5, underwent a kidney transplant at the age of 22, and lost the graft due to recurrence of the disease. In contrast, the young uncle was diagnosed with C3 glomerulopathy and mycophenolate mofetil and eculizumab were initiated early. It was remarkable that our patient had normal kidney function and urine analyses but low serum C3 level (0.56 g/L; N, 0.9-1.8 g/L). In the disease-associated clinical exome analysis, a heterozygous change in the CFH gene was found. The same mutation was found homozygous in the uncle. In genetically inherited diseases, findings may occur sequentially; early screening of at-risk individuals contributes to kidney survival.

Zinc (Zn) and adipose-derived mesenchymal stem cells (AD-MSCs) on MPTP-induced Parkinson's disease model: A comparative evaluation of behavioral and immunohistochemical results.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons and sustained neuroinflammation due to microglial activation. Adipose tissue-derived mesenchymal stem cells (AD-MSCs) secrete neuroprotective factors to prevent neuronal damage. Furthermore, Zn regulates stem cell proliferation and differentiation and has immunomodulatory functions. Our in vivo study aimed to investigate whether Zn affects the activities of AD-MSCs in the MPTP-induced mouse model. Male C57BL/6 mice were randomly divided into six groups (n = 6): Control, Zn, PD, PD+Zn, PD+ (AD-MSC), PD+ (AD-MSC)+Zn. MPTP toxin (20 mg/kg) was dissolved in saline and intraperitoneally injected into experimental groups for two days with 12 h intervals. On the 3rd day, AD-MSCs were given to the right lateral ventricle of the PD+ (AD-MSC) and PD+ (AD-MSC)+Zn groups by stereotaxic surgery. Then, ZnSO4H2O was administered intraperitoneally for 4 days at 2 mg/kg. Seven days post MPTP injection, the motor activities of the mouse were evaluated. Then immunohistochemical analyzes were performed in SNpc. Our results showed that motor activity was lower in Group PD. AD-MSC and Zn administration have improved this impairment. MPTP caused a decrease in TH and BDNF expressions in dopaminergic neurons in Group PD. However, TH and BDNF expressions were more intense in the other groups. MCP-1, TGF-ß, and IL-10 expressions increased in administered groups compared to the Group PD. The present study indicates that Zn's individual and combined administration with AD-MSCs reduces neuronal damage in the MPTP-induced mouse model. In addition, anti-inflammatory responses that emerge with Zn and AD-MSCs may have a neuroprotective effect.

Investigation of the relationship between reproductive disorders and chromosomal abnormalities in a large-scale, single-center 10-year retrospective study.

OBJECTIVE: Chromosomal changes are an important cause of reproductive disorders. This study investigated the chromosomal changes and prevalence of pathologies in individuals admitted to our Genetic Evaluation Center over a 10-year period due to a reproductive disorder. MATERIALS & METHODS: The chromosomal findings of 4345 individuals with reproductive disorders who applied to our Genetic Evaluation Center at Akdeniz University in Antalya, Turkey between 2011 and 2021 were retrospectively evaluated. RESULTS: In this study, an abnormal karyotype was found in a total of 138 individuals (87 males and 51 females). Although the incidence of this abnormal karyotype varied among the diseases in the reproductive disorder subgroups, it was most frequently seen in azoospermia (17.0%). Of the 138 abnormalities, 75 were numerical and 54 were structural. The remaining 9 abnormalities consisted of 6 sex reversals and 3 patients with both numerical and structural anomalies. Additionally, the X chromosome was the chromosome most frequently involved in these abnormalities, being observed in 40.6% of patients. CONCLUSION: This 10-year, single-center study involved one of the largest case series in the literature to investigate the subtypes of reproductive disorders and their chromosomal relationship. Although the importance of chromosome analysis has been deemphasized, it is still recommended for use by the guidelines and, as the results of this study demonstrate, is still a highly effective method in the investigation of reproductive disorders. Furthermore, chromosome analysis of individuals diagnosed with a reproductive disorder is also very important in the practice of the increasingly utilized preimplantation genetic diagnosis (PGD).

The relationship between COVID-19 and HLA in kidney transplant recipients, an evaluation of predictive and prognostic factors.

INTRODUCTION: The purpose of this study was to determine the predictive and prognostic factors for COVID-19 infection and its relationship with human leukocyte antigen (HLA) in kidney transplant recipients. MATERIAL AND METHOD: Three hundred fifty kidney transplant recipients were included in the study. Recipients were divided into two groups: COVID-19(+) (n = 100) and control (n = 250). The relationships between HLA frequencies, COVID-19 infection, and prognostic factors (age, donor type, immunosuppression protocol, etc.) were then evaluated. Logistic regression analysis, heatmap, and decision tree methods were used to determine predictive and prognostic factors. The study was performed retrospectively. RESULTS: Advanced age and deceased transplantation emerged as predictive of SARS-CoV-2 infection, while the presence of HLA-A*11, the HLA match ratio, and high-dose tacrolimus were identified as prognostic factors in kidney transplant recipients. HLA-A10, HLA-B*13, HLA-B22, and HLA-B*55 were shown to be associated with SARS-CoV-2 infection at univariate analysis, and HLA-B*57, HLA-DRB1*11, and HLA-DRB1*13 at logistic regression analysis. CONCLUSION: HLA-A10, HLA-B*13, HLA-B*55, HLA-B*57, HLA-DRB1*11, and HLA-DRB1*13 were identified for the first time in the literature associated with SARS-CoV-2 infection in kidney transplant recipients.

Correlation of miRNAs With Prognosis in Composite Tissue Allotransplantation.

OBJECTIVES: The number of composite tissue allotransplant procedures is increasing and has gained popularity. As with other transplant procedures, early detection of possible pathologies is as important as clinical follow-up. The present study investigated the correlation between microRNA expression levels and clinical follow-up of individuals undergoing composite tissue transplant. MATERIALS AND METHODS: Whole microRNA expression levels were analyzed from peripheral blood mononuclear cells obtained from preoperative and postoperative blood of patients who underwent facial transplant. Analyses were performed using microRNA levels from patients' preoperative blood samples. RESULTS: The clinical findings of patients with facial transplant were correlated with individual miRNA expression level changes. The expression of miR-31, the high expression of which has been linked to rejection, was significantly low in our patients. No expression changes were observed in other rejection-related microRNAs. Grade 1 rejection was generally seen in our patients, and these findings are consistent with the degree and frequency of rejection episodes in our cases. In addition, immunosuppression-associated diseases such as squamous cell carcinoma, posttransplant lymphoproliferative disorders, and aspergillosis, which are encountered clinically, were found to correlate with expression changes in microRNAs such as miR-150-5p, miR-21-5p, miR-17-5p, miR-20a-5p, and miR-3607-5p. CONCLUSIONS: Defining the clinical findings and immunosuppression-associated pathologies encountered in composite tissue transplant using biomarkers such as microRNA can play an important role in the improvement of these transplant procedures and in predicting patient morbidity. Therefore, the use of microRNAs may be useful in the clinical follow-up of patients who have received composite tissue allotransplant.

Effects of Different Tacrolimus Doses on Adipose-Derived Stem Cells.

OBJECTIVES: Tacrolimus is currently one of the most commonly used immunosuppressant agents to prevent rejection in organ transplant. Although modern immunosuppressive agents have been successful, rejection cannot be completely prevented. Therefore, in organ transplant research, additional treatment methods are being investigated, with the most important one being stem cell therapy. In addition to tacrolimus therapy, stem cell therapy is now clinically applicable, with frequency of concomitant use seeming to expand in the future. In this study, the effects of tacrolimus on stem cells were investigated. MATERIALS AND METHODS: Adipose-derived stem cells were treated with tacrolimus at different doses and time points. We analyzed the effects of changes in stem cell proliferation using MTT analysis. Sox2, Oct3/4, and Nanog protein levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure stem cell pluripotency capabilities. RESULTS: Our observations showed that tacrolimus causes changes in stem cell proliferation and pluripotency, with changes dependent on time and dose. CONCLUSIONS: The dose of tacrolimus and the time of application of stem cells are important in the planning of stem cell therapy in organ transplant.

Regulation of E2F1 activity via PKA-mediated phosphorylations.

E2F1 becomes activated during the G1 phase of the cell cycle, and posttranslational modifications modulate its activity. Activation of G-protein coupled receptors (GPCR) by many ligands induces the activation of adenylate cyclases and the production of cAMP, which activates the PKA enzyme. Activated PKA elicits its biological effect by phosphorylating the target proteins containing serine or threonine amino acids in the RxxS/T motif. Since PKA activation negatively regulates cell proliferation, we thought that activated PKA would negatively affect the activity of E2F1. In line with this, when we analyzed the amino acid sequence of E2F1, we found 3 hypothetical consensus PKA phosphorylation sites located at 127-130, 232-235, and 361-364 positions and RYET, RLLS, and RMGS sequences. After showing the binding and phosphorylation of E2F1 by PKA, we converted the codons of Threonine-130, Serine-235, and Serine-364 to Alanine and Glutamic acid codons on the eukaryotic E2F1 expression vector we had previously created. We confirmed the phosphorylation of T130, S235, and S364 by developing monoclonal antibodies against phospho-specific forms of these sites and showed that their phosphorylation is cell cycle-dependent. According to our results, PKA-mediated phosphorylation of E2F1 by PKA inhibits proliferation and glucose uptake and induces caspase-3 activation and senescence.

AKT-mediated phosphorylation of TWIST1 is essential for breast cancer cell metastasis.

Previously, it was shown that human TWIST1 (basic helix-loop-helix (b-HLH) is phosphorylated by Akt kinase at S42, T121, and S123. To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. We hypothesized that alanine mutants would inhibit the metastatic ability of 4T1 cells while glutamic acid mutants would convert nonmetastatic 67NR cells into metastatic phenotype. To confirm this hypothesis, we created metastatic 4T1 and nonmetastatic 67NR cells expressing alanine mutants and glutamic acid mutants mouse TWIST1, respectively. Then, we injected 1 × 106 67NR and 1 × 105 4T1 cells overexpressing mutants of TWIST1 into the breast tissue of BALB/c mice. At the end of the 4th week, we sacrificed the animals, determined the numbers of tumors at lungs and liver. Although 67NR cells overexpressing wild-type TWIST1 did not show any metastasis, cells overexpressing S42E and T125E mutants showed 15-30 macroscopic metastasis to liver and lungs. Parallel to this, 4T1 cells expressing S42A and T125A mutants of TWIST1 showed no macroscopic metastasis. Our results indicate that phosphorylation of S42 and T125 by AKT is essential for TWIST1-mediated tumor growth and metastasis.

Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1.

Tumor necrosis factor alpha (TNF-α) plays a paramount role in homeostasis by inducing tumor cytotoxicity and activating immune system. The signaling complexes formed by TNFR1 to activate JNK, p38, and nuclear factor-kappa B pathways and to subsequently induce apoptosis and necroptosis are well known. However, this "canonical TNF-α signaling" does not explain how ERK, AKT, and STAT3 can be activated by TNF-α. In addition, little to nothing is known about negative regulation of TNFR1 signaling. Because cyclic AMP-activated kinase (PKA) shows anti-TNF and anti-inflammatory activities, we postulated that PKA might affect TNF-α signaling by directly phosphorylating TNFR1. In line with this, we identified 2 putative PKA-phosphorylation motifs RRRT411 and REAT417 within the death domain of TNFR1, and investigated whether "canonical" and "noncanonical" TNFR1 signaling is regulated by modifications of T411 and T417. In this study, we demonstrate for the first time that PKA directly binds to and phosphorylates TNFR1 after TNF-α stimulation. To further support our hypothesis, we generated alanine and phosphomimetic (aspartic acid) mutants of TNFR1 at positions T411 and T417, ectopically expressed these mutants, and determined their influence on TNF-α-induced activations of ERKs, AKT, STAT3, p38α, and JNK1/2. Our results clearly showed that phosphomimetic mutants significantly suppressed and alanine mutants augmented TNF-α-induced phosphorylations of ERKs, AKT, Stat3, p38α, and JNKs. These findings strongly suggest that PKA-mediated phosphorylation of T411 and T417 of TNFR1 interferes with both "canonical" and "noncanonical" TNF-α signaling. [Figure: see text].

Anaesthetic Management for Face Transplantations: The Experience of Akdeniz University.

OBJECTIVE: Solid organ transplantation is a rescue therapy, whereas face transplantation, as well as other composite tissue allotransplantations, offers treatment options to enhance the quality of life. Difficult airway, severe haemorrhage and prolonged operative length are among the frequently encountered complications of anaesthetic management in patients with a history of multiple reconstructive surgeries. METHODS: Five face allotransplants that were performed in our institute, arising from four full and one partial face transplantations, were reviewed. The pitfalls encountered before, during and following surgery were then summarised. RESULTS: Two of our patients (patients 3 and 4) underwent permanent tracheostomy preoperatively. Transplantation was initiated after surgical tracheostomy under local anaesthesia and under sedation in cases 2 and 5. Patient 1 was orally intubated without difficulty under general anaesthesia and was operated following tracheal cannulation via surgical tracheostomy. Thirteen units of red blood cells were transfused each for patients 2 and 4. Two other patients (patients 1 and 4) each received 5 units of red blood cells. Patient 5, who underwent mid-face transplantation lasting for 7.5 h, was not transfused. No major life-threatening complications were observed intraoperatively or following surgery. CONCLUSION: Face transplantation is a surgical procedure in which anaesthetic management may be problematic. The anaesthetist may encounter difficulties, such as difficult airway, severe bleeding, a prolonged operative time and postoperative complications. Side effects and complications may be reduced by strict follow-up and haemodynamic monitoring of patients.

The regulation of circadian clock by tumor necrosis factor alpha.

All organisms display circadian rhythms which are under the control of the circadian clock located in the hypothalamus at the suprachiasmatic nucleus, (SCN). The circadian rhythms allow individuals to adjust their physiological activities and daily behavior for the diurnal changes in the living environment. To achieve these, all metabolic processes are aligned with the sleep/wake and fasting/feeding cycles. Subtle changes of daily behavior or food intake can result in misalignment of circadian rhythms. This can cause development of variety of metabolic diseases and even cancer. Although light plays a pivotal role for the activation of the master clock in SCN, the peripheral secondary clocks (or non-SCN), such as melatonin, growth hormone (GH), insulin, adiponectin and Ghrelin also are important in maintaining the circadian rhythms in the brain and peripheral organs. In recent years, growing body of evidence strongly suggest that CA2+ signaling, tumor necrosis factor alpha (TNFα) and transforming growth factor beta (TGFß) also play very important roles in the regulation of circadian rhythms by regulating the transcription of the clock genes.

E2F1 transcription factor and its impact on growth factor and cytokine signaling.

E2F1 is a transcription factor involved in cell cycle regulation and apoptosis. The transactivation capacity of E2F1 is regulated by pRb. In its hypophosphorylated form, pRb binds and inactivates DNA binding and transactivating functions of E2F1. The growth factor stimulation of cells leads to activation of CDKs (cyclin dependent kinases), which in turn phosphorylate Rb and hyperphosphorylated Rb is released from E2F1 or E2F1/DP complex, and free E2F1 can induce transcription of several genes involved in cell cycle entry, induction or inhibition of apoptosis. Thus, growth factors and cytokines generally utilize E2F1 to direct cells to either fate. Furthermore, E2F1 regulates expressions of various cytokines and growth factor receptors, establishing positive or negative feedback mechanisms. This review focuses on the relationship between E2F1 transcription factor and cytokines (IL-1, IL-2, IL-3, IL-6, TGF-beta, G-CSF, LIF), growth factors (EGF, KGF, VEGF, IGF, FGF, PDGF, HGF, NGF), and interferons (IFN-α, IFN-ß and IFN-γ).