Low Protein Expression Research Articles

Heart failure in mice induces a dysfunction of the sinus node associated with reduced CaMKII signaling.

Dysfunction of the sinoatrial node (SAN), the natural heart pacemaker, is common in heart failure (HF) patients. SAN spontaneous activity relies on various ion currents in the plasma membrane (voltage clock), but intracellular Ca2+ ([Ca2+]i) release via ryanodine receptor 2 (RYR2; Ca2+ clock) plays an important synergetic role. Whereas remodeling of voltage-clock components has been revealed in HF, less is known about possible alterations to the Ca2+ clock. Here, we analyzed [Ca2+]i handling in SAN from a mouse HF model after transverse aortic constriction (TAC) and compared it with sham-operated animals. ECG data from awake animals showed slower heart rate in HF mice upon autonomic nervous system blockade, indicating intrinsic sinus node dysfunction. Confocal microscopy analyses of SAN cells within whole tissue showed slower and less frequent [Ca2+]i transients in HF. This correlated with fewer and smaller spontaneous Ca2+ sparks in HF SAN cells, which associated with lower RYR2 protein expression level and reduced phosphorylation at the CaMKII site. Moreover, PLB phosphorylation at the CaMKII site was also decreased in HF, which could lead to reduced sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) function and lower sarcoplasmic reticulum Ca2+ content, further depressing the Ca2+ clock. The inhibition of CaMKII with KN93 slowed [Ca2+]i transient rate in both groups, but this effect was smaller in HF SAN, consistent with less CaMKII activation. In conclusion, our data uncover that the mechanism of intrinsic pacemaker dysfunction in HF involves reduced CaMKII activation.

Microvascular remodeling and altered angiogenic signaling in human kidneys distal to occlusive atherosclerotic renal artery stenosis.

Renal artery stenosis (RAS) is an important cause of chronic kidney disease and secondary hypertension. In animal models, renal ischemia leads to downregulation of growth factor expression and loss of intrarenal microcirculation. However, little is known about the sequelae of large-vessel occlusive disease on the microcirculation within human kidneys. This study included five patients who underwent nephrectomy due to renovascular occlusion and seven nonstenotic discarded donor kidneys (four deceased donors). Micro-computed tomography was performed to assess microvascular spatial densities and tortuosity, an index of microvascular immaturity. Renal protein expression, gene expression and histology were studied in vitro using immunoblotting, polymerase chain reaction and staining. RAS demonstrated a loss of medium-sized vessels (0.2-0.3 mm)comparedwithdonorkidneys (P=0.037) and increased microvascular tortuosity. RAS kidneys had greater protein expression of angiopoietin-1, hypoxia-inducible factor-1α and thrombospondin-1 but lower protein expression of vascular endothelial growth factor (VEGF) than donor kidneys. Renal fibrosis, loss of peritubular capillaries (PTCs) and pericyte detachment were greater in RAS, yet they had more newly formed PTCs than donor kidneys. Therefore, our study quantified significant microvascular remodeling in the poststenotic human kidney. RAS induced renal microvascular loss, vascular remodeling and fibrosis. Despite downregulated VEGF, stenotic kidneys upregulated compensatory angiogenic pathways related to angiopoietin-1. These observations underscore the nature of human RAS as a microvascular disease distal to main vessel stenosis and support therapeutic strategies directly targeting the poststenotic kidney microcirculation in patients with RAS.

Overexpressing CrePAPS Polyadenylate Activity Enhances Protein Translation and Accumulation in Chlamydomonas reinhardtii.

The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3′-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform.

Calcitriol Supplementation Protects Against Apoptosis and Alleviates the Severity of Abdominal Aortic Aneurysm Induced by Angiotensin II and Anti-TGFβ.

The present study aims to assess the effect of vitamin D deficiency (VDD) and its supplementation on the severity of AAA in mice. AAA was induced by AngII and anti-TGF-β administration. Animals were divided into four groups: Sham, mice with AAA, mice with AAA, and VDD, and mice with AAA supplemented with calcitriol. Blood pressure, echocardiography, abdominal aortic tissues, and plasma samples were monitored for all groups. VDD was associated with enhanced activity of cleaved MMP-9 and elastin degradation and positively correlated with the severity of AAA. Calcitriol supplementation decreased the INFγ/IL-10 ratio and enhanced the Nrf2 pathway. Moreover, Cu/Zn-superoxide dismutase expression and catalase and neutral sphingomyelinase activity were exacerbated in AAA and VDD groups. Furthermore, calcitriol supplementation showed a significantly lower protein expression of caspase-8, caspase-3, Bid, and t-Bid, and prevented the apoptosis of VSMCs treated by AngII and anti-TGF-β. Calcitriol supplementation may alleviate AAA severity and could be of great interest in the clinical management of AAA. VDD enhances antioxidant enzymes activity and expression, whereas calcitriol supplementation alleviates AAA severity by re-activating Nrf2 and inhibiting apoptotic pathways.

Identifying factors contributing to increased susceptibility to COVID-19 risk: a systematic review of Mendelian randomization studies.

BackgroundTo summarize modifiable factors for coronavirus disease 2019 (COVID-19) suggested by Mendelian randomization studies.MethodsIn this systematic review, we searched PubMed, EMBASE and MEDLINE, from inception to 15 November 2021, for Mendelian randomization studies in English. We selected studies that assessed associations of genetically predicted exposures with COVID-19-related outcomes (severity, hospitalization and susceptibility). Risk of bias of the included studies was evaluated based on the consideration of the three main assumptions for instrumental variable analyses.ResultsWe identified 700 studies through systematic search, of which 50 Mendelian randomization studies were included. Included studies have explored a wide range of socio-demographic factors, lifestyle attributes, anthropometrics and biomarkers, predisposition to diseases and druggable targets in COVID-19 risk. Mendelian randomization studies suggested that increases in smoking, obesity and inflammatory factors were associated with higher risk of COVID-19. Predisposition to ischaemic stroke, combined bipolar disorder and schizophrenia, attention-deficit and hyperactivity disorder, chronic kidney disease and idiopathic pulmonary fibrosis was potentially associated with higher COVID-19 risk. Druggable targets, such as higher protein expression of histo-blood group ABO system transferase (ABO), interleukin (IL)-6 and lower protein expression of 2′-5′ oligoadenylate synthetase 1 (OAS1) were associated with higher risk of COVID-19. There was no strong genetic evidence supporting the role of vitamin D, glycaemic traits and predisposition to cardiometabolic diseases in COVID-19 risk.ConclusionThis review summarizes modifiable factors for intervention (e.g. smoking, obesity and inflammatory factors) and proteomic signatures (e.g. OAS1 and IL-6) that could help identify drugs for treating COVID-19.

Correlation of Lipin gene expression with hepatic fat content in rats with intrauterine growth retardation

To study the correlation of the expression of Lipin1 in visceral adipose tissue and Lipin2 in liver tissue with hepatic fat content in rats with intrauterine growth retardation (IUGR). Pregnant rats were given a low-protein (10% protein) diet during pregnancy to establish a model of IUGR in neonatal rats. The pregnant rats in the control group were given a normal-protein (21% protein) diet during pregnancy. The neonatal rats were weighed and liver tissue was collected on day 1 and at weeks 3, 8, and 12 after birth, and visceral adipose tissue was collected at weeks 3, 8, and 12 after birth. The 3.0T 1H-magnetic resonance spectroscopy was used to measure hepatic fat content at weeks 3, 8, and 12 after birth. Real-time PCR was used to measure mRNA expression levels of Lipin2 in liver tissue and Lipin1 in visceral adipose tissue. Western blot was used to measure protein levels of Lipin2 in liver tissue and Lipin1 in visceral adipose tissue. A Pearson correlation analysis was performed to investigate the correlation of mRNA and protein expression of Lipin with hepatic fat content. The IUGR group had significantly higher mRNA and protein expression levels of Lipin1 in visceral adipose tissue than the control group at weeks 3, 8, and 12 after birth (P<0.05). Compared with the control group, the IUGR group had significantly lower mRNA and protein expression levels of Lipin2 in liver tissue on day 1 after birth and significantly higher mRNA and protein expression levels of Lipin2 at weeks 1, 3, 8, and 12 after birth (P<0.05). At week 3 after birth, there was no significant difference in hepatic fat content between the IUGR and control groups (P>0.05), while at weeks 8 and 12 after birth, the IUGR group had a significantly higher hepatic fat content than the control group (P<0.05). The protein and mRNA expression levels of Lipin1 were positively correlated with hepatic fat content (r=0.628 and 0.521 respectively; P<0.05), and the protein and mRNA expression levels of Lipin2 were also positively correlated with hepatic fat content (r=0.601 and 0.524 respectively; P<0.05). Upregulation of the mRNA and protein expression levels of Lipin1 in visceral adipose tissue and Lipin2 in liver tissue can increase hepatic fat content in rats with IUGR and may be associated with obesity in adulthood.

Association of pSTAT3, pSyk with c-Myc, p53, BCL2 proteins expression and survival of patients with diffuse large B-cell lymphoma

Background . Diffuse large B cell lymphoma (DLBCL) is one of the most common non-Hodgkin’s lymphomas. The effectiveness of R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone) therapy was observed in about 60 % of patients, in 20–30 % of cases DLBCL is characterized by a refractory course. Standard clinical prognostic criteria do not accurately determine the prognosis of the disease. In this regard, the problem of developing additional predictors of DLBCL course remains relevant today and is directly related to the molecular pathogenesis of the disease. The objective : to determine the relationship of pSTAT3, pSyk with c-Myc, p53, BCL2 oncoproteins expression and survival of DLBCL patients. Materials and methods . Biopsy specimens of lymph nodes and other organs and tissues obtained from 100 patients with newly diagnosed DLBCL were used for the study. All patients received standard first-line immunochemotherapy according to the R-CHOP regimen. Determination of the relative amount of tumor cells expressing pSTAT3, pSyk was carried out using immunohistochemical and morphometric methods. The threshold value of proteins expression was calculated using the ROC analysis: for pSTAT3 it was 68 % of positive tumor cells, for pSyk – 28 %. The relationship of pSTAT3, pSyk with c-Myc, p53, BCL2 expression was determined using the Pearson χ2 test. Overall (OS) and progression-free (PFS) survival were calculated using the Kaplan–Meier method (log-rank test). Results . The suprathreshold pSTAT3 expression was found to be associated with a high content of c-Myc and p53 transcription factors in tumor cells (p = 0.015 and p = 0.010, respectively). In the group of patients with isolated overexpression of pSTAT3, the 5-year OS and PFS were lower, and the risk of death and disease progression was almost 1.5 times higher than in patients with low protein expression (p = 0.015 and p = 0.011, respectively). When studying the co-expression of pSTAT3 and pSyk, the lowest OS and PFS were recorded in the subjects with a simultaneous high expression of these proteins. With this combination of markers, the probability of an adverse event in patients when calculating OS increased by 2.9 times (p = 0.003; hazard ratio 2.9; 95 % confidence interval 1.43–5.85), and when analyzing PFS – by 2.3 times (p = 0.021; hazard ratio 2.3; 95 % confidence interval 1.14–4.87) compared with other variants of their simultaneous expression. Conclusion . Overexpression of pSTAT3 is associated with unfavorable biological tumor characteristics and poor patient survival. The combined suprathreshold expression of the pSTAT3 and pSyk proteins is associated with lower OS and PFS values compared to their isolated expression.

Aldh1a1 and Scl25a30 in diaphragmatic dysfunction.

New methods to prevent ventilator-induced diaphragmatic dysfunction (VIDD) are urgently needed, and the cellular basis of VIDD is poorly understood. This study evaluated whether transvenous phrenic nerve stimulation (PNS) could prevent VIDD in rabbits undergoing mechanical ventilation (MV) and explored whether oxidative stress-related genes might be candidate molecular markers for VIDD. Twenty-four adult male New Zealand white rabbits were allocated to control, MV, and PNS groups (n = 8 in each group). Rabbits in the MV and PNS groups underwent MV for 24 h. Intermittent bilateral transvenous PNS was performed in rabbits in the PNS group. Transdiaphragmatic pressure was recorded using balloon catheters. The diameters and cross-sectional areas (CSAs) of types I and II diaphragmatic fibers were measured using immunohistochemistry (IHC) techniques. Genes associated with VIDD were identified by RNA sequencing (RNA-seq), differentially expressed gene (DEG) analysis, and weighted gene co-expression network analysis (WGCNA). Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and IHC analyses were carried out to verify the transcriptome profile. Pdi60Hz, Pdi80Hz, and Pdi100Hz were significantly higher in the PNS group than in the MV group at 12 and 24 h (P < 0.05 at both time points). The diameters and CSAs of types I (slow-twitch) and II (fast-twitch) fibers were significantly larger in the PNS group than in the MV group (P < 0.05). RNA-seq, RT-PCR, Western blotting, and IHC experiments identified two candidate genes associated with VIDD: Aldh1a1 and Scl25a30. The MV group had significantly higher mRNA and protein expressions of Aldh1a1/ALDH1A1 and significantly lower mRNA and protein expressions of Scl25a30/SCL25A30 than the control or PNS groups (P < 0.05). We have identified two candidate genes involved in the prevention of VIDD by transvenous PNS. These two key genes may provide a theoretical basis for targeted therapy against VIDD.

Clinical outcomes associated with expression of aurora kinase and p53 family members in muscle-invasive bladder cancer.

Biomarkers are needed in muscle-invasive bladder cancer (MIBC). We previously reported that high tumor aurora kinase (AURK) A expression identifies patients with MIBC with poor prognosis. Aberrant p53 expression has also been associated with poor outcomes in MIBC, though to the best of our knowledge, co-expression rates of p53 and aurora kinases have not been previously described in MIBC. As aurora kinase and p53 family members may co-regulate each other, the present study investigated whether tumor p53 or p63 protein expression influenced the prognostic value of AURKA in a pilot study of 50 patients with MIBC treated with curative intent. Immunohistochemistry for AURKA, AURKB, p53 and p63 were performed on archival pre-treatment tumor specimens and correlated with clinical outcomes in patients with MIBC who received neoadjuvant chemotherapy (NAC) prior to cystectomy. Baseline p53 [hazard ratio (HR) 1.46; 95% confidence interval (CI)=0.55-3.9; P=0.448) and p63 (HR 2.02; 95% CI=0.51-8.1; P=0.313) protein expression did not predict for overall survival (OS). Low p53 protein expression did not correlate with high AURKA (φ=0.190) or AURKB (φ=0.075) expression. However, in tumors with low p53 expression (n=17), the presence of either high AURKA or AURKB expression levels predicted an increased risk for relapse (HR 27.1; 95% CI=2.7-270.1; P=0.005) and mortality (HR 14.9; 95% CI=2.3-95.6; P=0.004) compared to tumors with both low AURKA and AURKB levels. The relationship between p63 and AURKA/B expression levels was not tested due to the prevalence (80%) of high p63 expression in the present cohort. In tumors with low AURKA expression, p53 status did not predict for OS (HR 0.62; 95% CI 0.2-3.2; P=0.572). In multivariable analysis, only high baseline AURKA expression predicted for inferior OS (HR 4.9; 95% CI 1.7-14.1; P=0.003). To the best of our knowledge, the present study was the first to report co-expression of p53 and aurora kinase family members in MIBC, and although wild-type p53 may regulate the aurora kinases in preclinical models, the adverse prognostic value of tumor AURKA overexpression was independent from baseline tumor p53 protein expression in the present cohort. AURKA remains an important prognostic biomarker in patients with MIBC and warrants further evaluation in prospective studies to validate whether baseline AURKA can identify patients that are unlikely to benefit from standard of care with NAC.

Status of higher TGF-\u03b21 and TGF-\u03b22 levels in the aqueous humour of patients with diabetes and cataracts

BackgroundTransforming growth factor (TGF) is a cytokine that acts on the proliferation, migration, differentiation, and apoptosis of cells and the accumulation of extracellular matrix components. Very few studies have precisely evaluated the concentration of TGF-β in the aqueous humour (AH) of diabetic and cataract (DMC) eyes due to the low expression of proteins in the AH or other reasons. The concentrations of TGF-β1, -β2, and -β3 in the AH of the DMC group were compared with those of the age-related cataract (ARC) group.MethodsWe collected AH and lens epithelium samples from 33 DMC patients and 36 ARC patients. Luminex liquid suspension chip detection was applied to detect the concentration of TGF-β1, -β2, and -β3 in the AH samples. The expression of TGFB1/2/3 in lens epithelium samples was determined by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsThe concentrations of TGF-β1 and TGF-β2 in AH samples of DMC eyes were higher than those of ARC eyes. The differences in TGF-β1 and TGF-β2 between the two groups were statistically significant (P value = 0.001 for TGF-β1, P value = 0.023 for TGF-β2). The difference of the correlation between TGF-β1 and glycosylated haemoglobin was significant (P value = 0.011, and Pearson correlation coefficient = 0.306). The difference of the correlation between TGF-β2 and glycosylated haemoglobin was significant (P value = 0.026, and Pearson correlation coefficient = 0.269). The mRNA expression levels of TGFB1 and TGFB2 were upregulated in DMC epithelium samples compared with ARC epithelium samples. The differences in TGFB1 and TGFB2 between the two groups were statistically significant (P value for TGFB1 = 0.041, P value for TGFB2 = 0.021).ConclusionsThe concentrations of TGF-β1 and TGF-β2 in AH samples were significantly higher in DMC eyes than in ARC eyes. The higher the glycosylated haemoglobin was, the higher the concentrations of TGF-β1 and -β2 were. The mRNA expression of TGFB1 and TGFB2 was significantly upregulated in DMC epithelial samples compared with ARC epithelial samples, suggesting the proinflammatory status of the anterior chamber of DMC eyes.

IDF21-0381 Expression Levels and Genetic Polymorphism of Scavenger Receptor Class B Type 1 as a Biomarker of Type 2 Diabetes

Background: Type 2 Diabetes Mellitus (T2DM) persists to be a major health problem worldwide and it is well accepted that T2DM is a metabolic disorder caused by hyperglycemia, which arises from insufficient pancreatic insulin secretion to peripheral tissues. Moreover, scavenger receptor class B type 1 (SRB1) protein plays a major role in the uptake of cholesteryl ester from high-density lipoprotein (HDL) and eliminates extra cholesterol from peripheral tissues as bile by the process of reverse cholesterol transport (RCT) Lack of SRB1 protein can result in an imbalance of cholesterol, dyslipidemia and eventually lead to insulin resistance. Aim: The present study aimed to determine whether the expression level and genetic polymorphism scavenger receptor class B type 1 (SRB1) rs5888 may be used as biological markers in type 2 diabetes mellitus (T2DM). Method: A total of 600 individuals, including 300 T2DM and 300 healthy individuals, were enrolled in the study from April 2016 to April 2017. Blood samples were collected from each T2DM and healthy individual. Total proteins were determined using western blot analysis. Also, restriction fragment length polymorphism (RFLP) analysis was achieved to detect the incidence of genetic polymorphisms. Results: Western blot analysis results revealed that the protein expression of SRB1 was significantly decreased in T2DM of SRB1 CC variant as compared with controls. The genotype distribution and the allelic frequencies for the SRB1 polymorphism were significantly different than T2DM and controls. CC genotype of the SRB1 polymorphism showed a potential association with the incidence of T2DM (OR = 1.19, 95% CI 0.63 - 2.25, P = 0.577). Discussion: In the present study, SRB1 CC variant had decreased SRB1 protein expression which might be due to variation in the regulatory region of the SRB1 gene that could involve in differential expression of SRB1 as the variant was located in the promoter region of the gene and could hinder the binding of transcriptional factor. Additionally, previous reports also found that the rs5888 SNP affected SRBI RNA secondary structure, which changed its ability to undergo productive protein translation leading ultimately to significantly lower SRB1 protein expression.

U2 small nuclear RNA auxiliary factor 2, transcriptionally activated by the transcription factor Dp-1/E2F transcription factor 1 complex, enhances the growth and aerobic glycolysis of leiomyosarcoma cells

ABSTRACT The dysregulation of U2 Small Nuclear RNA Auxiliary Factor 2 (U2AF2) is associated with malignant behaviors of multiple types of tumors. In this study, we explored the association between U2AF2 dysregulation and the survival of patients with primary leiomyosarcoma, the regulatory effect of U2AF2 on cell growth/aerobic glycolysis, and the mechanisms of U2AF2 dysregulation at the transcriptional level. Gene expression and survival time of patients with primary leiomyosarcoma were extracted from TCGA-Sarcoma (SARC). Leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1 were utilized to construct in vitro and in vivo models. Results showed that the higher U2AF2 expression group had significantly shorter progression-free survival (HR: 2.049, 95%CI: 1.136–3.697, p = 0.011) and disease-specific survival (4.656, 95%CI: 2.141–10.13, p < 0.001) compared to the lower U2AF2 expression group. U2AF2 knockdown suppressed leiomyosarcoma cell growth and aerobic glycolysis (decreased glucose uptake, lactate production, and extracellular acidification rate) in vitro. Tumors derived from SK-LMS-1 cells with U2AF2 knockdown grew significantly slower, with lower GLUT1, PGK1, and PGAM1 protein expression than the control groups. TFDP1 and E2F1 could interact with each other in leiomyosarcoma cells. Both TFDP1 and E2F1 could bind to the promoter of U2AF2 and exert a synergistic activating effect on U2AF2 transcription. In conclusion, this study revealed that U2AF2 upregulation is associated with poor survival of leiomyosarcoma. Its upregulation enhances proliferation and aerobic glycolysis of leiomyosarcoma cells in vitro and in vivo. TFDP1 and E2F1 can form a complex, which binds to the U2AF2 gene promoter and synergistically activates its transcription.

Targeting GLI1 Transcription Factor for Restoring Iodine Avidity with Redifferentiation in Radioactive-Iodine Refractory Thyroid Cancers.

Simple SummaryThyroid cancers have an excellent prognosis by standard therapy of surgery followed by radioactive-iodine therapy. However, metastatic thyroid cancers do not response to radioactive-iodine therapy by losing iodine avidity. Therefore, reversing iodine avidity to metastatic thyroid cancers gives a new chance of applying radioactive-iodine therapy to the cancers. In the current study, we found that GLI1 knockdown can revert iodine non-avid thyroid cancers to iodine avid cancers by increasing expression of thyroid-specific proteins. Restoration of iodine avidity in thyroid cancers makes the cancers sensitive to radioactive-iodine therapy again. Therefore, the GLI1 can be a potential therapeutic target of radioactive-iodine resistant thyroid cancers.Radioactive-iodine (RAI) therapy is the mainstay for patients with recurrent and metastatic thyroid cancer. However, many patients exhibit dedifferentiation characteristics along with lack of sodium iodide symporter (NIS) functionality, low expression of thyroid-specific proteins, and poor RAI uptake, leading to poor prognosis. Previous studies have demonstrated the effect of GLI family zinc finger 1 (GLI1) inhibition on tumor growth and apoptosis. In this study, we investigated the role of GLI1 in the context of redifferentiation and improvement in the efficacy of RAI therapy for thyroid cancer. We evaluated GLI1 expression in several thyroid cancer cell lines and selected TPC-1 and SW1736 cell lines showing the high expression of GLI. We performed GLI1 knockdown and evaluated the changes of thyroid-specific proteins expression, RAI uptake and I-131-mediated cytotoxicity. The effect of GANT61 (GLI1 inhibitor) on endogenous NIS expression was also assessed. Endogenous NIS expression upregulated by inhibiting GLI1, in addition, increased expression level in plasma membrane. Also, GLI1 knockdown increased expression of thyroid-specific proteins. Restoration of thyroid-specific proteins increased RAI uptake and I-131-mediated cytotoxic effect. Treatment with GANT61 also increased expression of endogenous NIS. Targeting GLI1 can be a potential strategy with redifferentiation for restoring RAI avidity in dedifferentiated thyroid cancers.

Validation of CD98hc as a Therapeutic Target for a Combination of Radiation and Immunotherapies in Head and Neck Squamous Cell Carcinoma.

Simple SummaryThe outcome of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) has not changed for the past decade despite advances in treatment strategies. SLC3A2, which encodes CD98hc, was identified as a putative biomarker for HNSCC radioresistance. Chimeric antigen receptor (CAR) T cell therapy presents a promising immunotherapy approach for the treatment of different cancer types. Due to the limitations of this system, including a lack of self-control mechanisms, the adapter UniCAR system with a switchable mechanism was developed. This study demonstrated a synergistic effect of the combination of fractionated irradiation and immune targeting of CD98hc. The sequential combination of fractionated radiotherapy and UniCAR-based immunotherapy revealed itself to be a promising approach for the treatment of high-risk HNSCC patients.Most patients with head and neck squamous cell carcinomas (HNSCC) are diagnosed at a locally advanced stage and show heterogeneous treatment responses. Low SLC3A2 (solute carrier family 3 member 2) mRNA and protein (CD98hc) expression levels are associated with higher locoregional control in HNSCC patients treated with primary radiochemotherapy or postoperative radiochemotherapy, suggesting that CD98hc could be a target for HNSCC radiosensitization. One of the targeted strategies for tumor radiosensitization is precision immunotherapy, e.g., the use of chimeric antigen receptor (CAR) T cells. This study aimed to define the potential clinical value of new treatment approaches combining conventional radiotherapy with CD98hc-targeted immunotherapy. To address this question, we analyzed the antitumor activity of the combination of fractionated irradiation and switchable universal CAR (UniCAR) system against radioresistant HNSCC cells in 3D culture. CD98hc-redirected UniCAR T cells showed the ability to destroy radioresistant HNSCC spheroids. Also, the infiltration rate of the UniCAR T cells was enhanced in the presence of the CD98hc target module. Furthermore, sequential treatment with fractionated irradiation followed by CD98hc-redirected UniCAR T treatment showed a synergistic effect. Taken together, our obtained data underline the improved antitumor effect of the combination of radiotherapy with CD98hc-targeted immunotherapy. Such a combination presents an attractive approach for the treatment of high-risk HNSCC patients.

Genetic association study of intron variants in the forkhead box protein P3 gene in Chinese patients diagnosed with cervical cancer.

The aim of this study was to investigate the effects of forkhead box protein P3 (FOXP3) intron single nucleotide variants (SNVs) in high‐risk human papilloma virus (HR‐HPV) infection and cervical cancer (CC) malignant lesions. We performed FOXP3 genotyping in 350 patients with CC and 350 healthy controls using the ImLDR multiple single nucleotide polymorphism genotyping technology. The heterozygous mutation TC in rs2294021 decreased the risk of HR‐HPV infection and CC malignant lesions (TC vs. TT: OR = 0.71, 95% CI = 0.51–0.99); the dominant model TC+CC and allele C in rs2294021 decreased the risk of CC malignant lesions (TC+CC vs. TT: OR = 0.69, 95% CI = 0.50–0.95; C vs. T: OR = 0.78, 95% CI = 0.63–0.97). The heterozygous mutation GA, dominant model GA+AA and allele A in rs3761549 also decreased the risk of HR‐HPV infection and CC malignant lesions (GA vs. GG: OR = 0.70, 95% CI = 0.51–0.96; GA+AA vs. GG: OR = 0.69, 95% CI = 0.51–0.94; A vs. G: OR = 0.75, 95% CI = 0.58–0.96). Patients with CC and HR‐HPV infection carrying rs2294021 TC and rs3761549 GA had lower expression of FOXP3 protein. Haplotype analysis revealed that T‐C‐A decreased the risk of HR‐HPV infection. Furthermore, we found a significant association between immune cells infiltration and prognosis in patients with CC. Our findings demonstrated that rs2294021 and rs3761549 variants may protect against HR‐HPV and CC malignant lesions by downregulating FOXP3 and that FOXP3 was associated with immune cells infiltration, which affected the prognosis of CC.

CD55-deficiency in Jews of Bukharan descent is caused by the Cromer blood type Dr(a-) variant.

The complement system regulator CD55 was initially found to carry the Cromer blood group system antigens, and its complete loss of function was subsequently revealed to cause a severe monogenic gastrointestinal syndrome characterized by protein-losing enteropathy and susceptibility to venous thrombosis. Here we present homozygosity to the CD55 c.596C>T; p.Ser199Leu variant, which was previously described as the Cromer Dr(a−) genotype, in two Bukharan Jewish CD55-deficiency patients with variable disease severity. We confirm that this missense variant causes aberrant splicing and deletion of 44 bp in exon 5, leading to premature termination and low expression of the CD55 protein. Furthermore, Patient 1 exhibited a mildly abnormal B cell immunophenotyping profile. By population screening we established that this variant is highly prevalent in the Bukharan Jewish population, with a carrier frequency of 1:17, suggesting that many similar patients are un- or mis-diagnosed. The phenotypic variability, ranging from abdominal pain when eating a high-fat diet to the full CD55-deficiency phenotype, is likely related to modifiers affecting the proportion of the variant that is able to escape aberrant splicing. Establishing the diagnosis of CD55-deficiency in a timely manner, even in patients with milder symptoms, may have a critical effect on their management and quality-of-life since treatment with the complement inhibitor eculizumab is highly effective in ameliorating disease manifestations. Awareness of founder mutations within certain populations can further guide genetic testing and prevent a diagnostic odyssey, by placing this CD55 variant high on the differential diagnosis.

Controlled microwave-assisted reactions: A facile synthesis of polyfunctionally substituted phthalazines as dual EGFR and PI3K inhibitors in CNS SNB-75 cell line

Brain tumors are stubborn cancers with poor prognosis and disappointing survival rates. Targeted cancer therapeutics with higher efficacy and lower resistance are highly demanded. An efficient one-pot synthesis of polyfunctionalized phthalazines derivatives was developed by reacting ethyl 1-aryl-5-cyano-1,6-dihydro-4-methyl-6-oxo-3-pyridazine-carboxylates with cinnamonitrile derivatives and the cycloaddition reaction of thieno[3,4-d]pyridazines with activated double or triple bond systems under controlled microwave heating with high yields. The resultant synthesized phthalazines (5a-e, 9 and 13) were tested for their in vitro anti-cancer activities by using in vitro one dose assay at National Cancer institute, USA. Only phthalazine (5b) showed broad spectrum anti-tumor activity against most tested cancer cell lines from all subpanels with mean % GI = 22.61. Interestingly, all tested compounds showed varying growth inhibitory activity against a particular cell line, CNS SNB-75 cell line, but (5b) exhibited the highest growth inhibitory activity against CNS-SNB-75 cell line with (GI% = 108.81) and (IC50 = 3.703 ± 0.2) compared to erlotinib; (IC50 = 12.5 ± 0.68). It caused Pre-G1 apoptosis and growth arrest at S phase. It also increased percentage of the total apoptotic cells in CNS-SNB-75 cell line (39.26%) compared to control cells (2.17%) in the annexin V-FITC experiment. It revealed pronounced EGFR inhibitory activity (IC50 = 47.27 ± 2.41 ng/mL) compared to erlotinib (IC50 = 30.7 ± 1.56 ng/mL). It also inhibited the different PI3K isoforms α, β, γ and δ (with IC50 of 4.39, 13.6, 12.5 and 3.11 μg/mL, respectively compared to LY294002 (with IC50 of 12.7, 8.57, 6.89 and 5.7 μg/mL, respectively). It also caused significant lower protein expression levels of pPI3K, AKT, pAKT and Bcl2 and higher protein expression levels of BAX, Casp3 and Casp9 when compared to untreated cells. Conclusion: Phthalazine (5b) may be an effective, convenient and safe anti-cancer agent acting via proapoptotic and dual EGFR and PI3K kinase inhibitory actions in CNS SNB-75 cell line.

Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy.

BackgroundOxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity.MethodsWe assessed iron distribution and total elemental iron using LA‐ICP‐MS on skeletal muscle cross‐sections of 8‐week‐old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin‐null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron‐enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron‐ and mitochondria‐related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed.ResultsWe observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS‐1, the rate‐limiting enzyme for haem synthesis (dko −44%, P < 0.05), and the haem‐containing protein myoglobin (dko −54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (−30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem‐containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology.ConclusionsMuscles from dystrophic mice have increased iron levels and dysregulated iron‐related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem‐containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem‐containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.

FOXO1 Gene Downregulation and Promoter Methylation Exhibits Significant Correlation With Clinical Parameters in Indian Breast Cancer Patients.

Background: Forkhead box “O” one which is member of Forkhead box family of transcription factors is known to play key role in different physiological processes including cell cycle arrest, autophagy, and apoptosis. FOXO1 is defined to play tumor suppressive role in various malignancies including breast cancer and its Dysregulation is frequently reported. However, the evaluation of FOXO1 promoter methylation and its expression at mRNA and protein level in different stages of breast cancer and its association with different clinical parameters is still not studied. Therefore, for better understanding the role of FOXO1 in breast cancer, in our study we examined the FOXO1 mRNA and protein expression in Breast cancer samples of Indian breast cancer patients. Results: Total 127 breast cancer samples along with adjacent normal tissue (n = 127) were analyzed through methylation specific PCR (MS-PCR), mRNA expression (Real-time PCR) and Immunohistochemistry (IHC). We detected 69.29% cases to be downregulated at the mRNA level, and 77.95% of cases exhibited no or low protein expression. In our data we report a significant association (p = 0.0001) between the downregulated protein expression and promoter hypermethylation of FOXO1 gene. We also found a significant correlation of FOXO1 mRNA level with Age (p = 0.008), age at first live birth (p = 0,003), tumor size (p = 0.05) and lymph node status (p = 0.01). Conclusion: we in our study report the tumor suppressive role of FOXO1 in case of Indian breast cancer patients and our data suggest it to exhibit prognostic importance. However, further research is needed to evaluate FOXO1 significance in diagnostic and therapeutic targeting in breast cancer cases.

A comparative study on regulation of HLA-G expression in bad obstetric history and in head and neck squamous cell carcinoma from Northeast India

The expression of immunomodulatory molecule HLA-G is tissue restricted with abundant expression in placenta, mediating immune tolerance to fetus. Tumors hijack HLA-G to establish nutrient supply and evade host immune response. 14 base pair Insertion/Deletion polymorphism (rs371194629) and + 3142 G/C SNP (rs1063320) of 3′UTR of HLA-G were investigated in conjunction with miR-148A, miR-152 and HLA-G expression in SAB (Spontaneous abortion) history placenta and HNSCC tumor in a hospital-based case control study. Higher frequency of G allele of rs1063320 was seen in study participants as reported in other global populations. Both miR-148A and miR-152 were downregulated in tumor tissue. Predominance of 14 base pair “IN” allele of rs371194629 was noted in SAB placental tissue (p =<0.0001) with lower expression of HLA-G levels. In conclusion, 14 base pair Insertion/Deletion in linkage with + 3142 G/C SNP was related to lower HLA-G protein expression in SAB tissue, contradictorily HLA-G protein level was manipulated by tumors by suppressing microRNAs.