Search Results (34)

Background/Objectives: Chronic myeloid leukemia (CML) is characterized by the presence of the BCR::ABL1 fusion gene, most commonly in the e14a2 or e13a2 variants. Studies show that the transcript type in CML may be important for achieving treatment-free remission (TFR). This study aimed to immunologically characterize CML patients with e13a2 and e14a2 transcripts to search for differences that may contribute to achieving remission in patients after therapy withdrawal. Methods: Using multicolor flow cytometry, we analyzed the differences in the immune system at the time of imatinib discontinuation and the early stage of TFR in fifty-one CML patients with different transcripts. RQ-PCR and ddPCR were used to monitor the dynamics of BCR::ABL1 transcript changes. The patients were grouped using principal component analysis (PCA) based on the percentage of detected immune cells that were classified as populations consistently selected by the MCFS-ID algorithm from randomly selected data. Results: PCA separated CML patients into two groups defined by k-means clustering, indicating significant heterogeneity within the studied population. We found a significant association between Cluster metrics (Cluster 1 and 2) and BCR::ABL1 transcript types (e13a2 or e14a2) (p = 0.003, 95% CI: 0.026–0.595, OR = 0.14, Fisher test). The e13a2 transcript was less frequent in Cluster 2 than in Cluster 1, while e14a2 was more common in Cluster 2. Additionally, patients grouped into Cluster 1 had significantly higher percentages of the PD1 expressing populations cDC PD1⁺, CD56^dimCD16⁺PD1⁺, CD8⁺PD1⁺, CD4⁺PD1⁺, and CD19⁺PD1⁺, as identified by the MCFS-ID algorithm, compared to patients in Cluster 2. Conclusions: Our results suggest that immunological differences may be related to the BCR::ABL1 transcript type, which could affect the number of active CML cells represented by the BCR::ABL1 transcript amount and thus may determine molecular recurrence. Full article

Background: Hodgkin lymphoma (HL) is a B-cell-derived malignancy and one of the most frequent types of lymphoma. The tumour cells typically exhibit multiple genomic alterations together with aberrantly activated signalling pathways, driven by paracrine and/or autocrine modes. SPP1 (alias osteopontin) is a cytokine acting as a signalling activator and has been connected with relapse in HL patients. To understand its pathogenic role, here, we investigated the mechanisms and function of deregulated SPP1 in HL. Methods: We screened public patient datasets and cell lines for aberrant SPP1 expression. HL cell lines were stimulated with SPP1 and subjected to siRNA-mediated knockdown. Gene and protein activities were analyzed by RQ-PCR, ELISA, Western blot, and immuno-cytology. Results: SPP1 expression was detected in 8.3% of classic HL patients and in HL cell line SUP-HD1, chosen to serve as an experimental model. The gene encoding SPP1 is located at chromosomal position 4q22 and is genomically amplified in SUP-HD1. Transcription factor binding site analysis revealed TALE and HOX factors as potential regulators. Consistent with this finding, we showed that aberrantly expressed PBX1 and HOXB9 mediate the transcriptional activation of SPP1. RNA-seq data and knockdown experiments indicated that SPP1 signals via integrin ITGB1 in SUP-HD1. Accordingly, SPP1 activated NFkB in addition to MAPK/ERK which in turn mediated the nuclear import of ETS2, activating oncogenic JUNB expression. Conclusions: SPP1 is aberrantly activated in HL cell line SUP-HD1 via genomic copy number gain and by homeodomain transcription factors PBX1 and HOXB9. SPP1-activated NFkB and MAPK merit further investigation as potential therapeutic targets in affected HL patients. Full article

Aging and age-related neurodegenerative disorders are characterized by the dysfunction or loss of brain nicotinic acetylcholine receptors (nAChRs), and these changes may be related to other senescence markers, such as oxidative stress and DNA repair dysfunction. However, the mechanism of nAChR loss in the aging brain and the modification of this process by drugs (e.g., memantine, Mem) are not yet fully understood. To study whether the differences in nAChR expression in the rat brain occur due to aging or oxidative stress and are modulated by Mem, we analyzed nAChR subunits (at RNA and protein levels) and other biomarkers by real-time quantitative polymerase chain reaction (RQ-PCR) and Western blot validation. Twenty-one female Wistar rats were divided into four groups, depending on age, and the oldest group received injections of Mem or water with the use of intragastric catheters. We studied the cerebral grey matter (CGM), subcortical white matter (SCWM), and cerebellum (Ce). Results showed an age-related decrease of α7 nAChR mRNA level in SCWM. The α7 nAChR mRNA loss was accompanied by reduced expression of 8-oxoguanine DNA glycosylase 1 (OGG1) and an increased tumor necrosis factor alpha (TNFα) level. In the water group, we observed a higher level of α7 nAChR protein in the SCWM and Ce. Biomarker levels changed, but to a different extent depending on the brain area. Importantly, the dysfunction in antioxidative status was stopped and even regressed under Mem treatment. After two weeks of treatment, an increase in TP53 protein level and a decrease in 8-oxo-2′deoxyguanosine (8-oxo-2′dG) level were observed. We conclude that Mem administration may be protective against the senescence process by antioxidative mechanisms. Full article

Background/Objectives: Osteoarthritis (OA) is a very common degenerative joint disease that has a significant negative impact on patients’ lives and which can lead to functional limitations and disability. Matrix metalloproteinase 13 (MMP-13) is a key enzyme responsible for the degenerative changes in cartilage occurring during the pathogenesis of OA. This cohort study analyzed the differences in the expression level of MMP13 mRNA in articular cartilage with subchondral bone and in the synovium of patients with OA, according to the disease stage, in order to develop potential markers for OA progression, as well as for the degree of pain perception, in order to discover a molecular biomarker related to pain. Methods: In thirty-one patients (n = 31), the expression level of the studied gene was assessed in the affected and unaffected areas of the knee joint using the qPCR method. Statistical analysis was performed using the Mann–Whitney U test, the Kruskal–Wallis test, and Spearman’s rank correlation coefficient. Results: A significantly higher expression level of MMP13 mRNA was noticed in the OA-affected articular cartilage with subchondral bone compared to the control tissue (p = 0.027, Mann–Whitney U test). The expression level of MMP13 mRNA was higher in patients with stage 4 knee OA than in those with stage 3, but the difference in MMP13 mRNA expression level was statistically insignificant (p > 0.05, Mann–Whitney U test). A higher MMP13 mRNA expression level was noticed in the OA-affected synovium compared to the control tissue (median RQ: 0.068 and 0.037, respectively), but these differences were not significant (p > 0.05, Mann–Whitney U test). A significantly higher MMP13 mRNA expression level was observed in the synovium of stage 4 knee OA patients compared to stage 3 patients (p = 0.015, Mann–Whitney U test). There was no significant difference in the expression level of MMP13 mRNA between both tissues, i.e., the articular cartilage with subchondral bone and the synovium from the stage 3 group and the control tissue (p > 0.05, Mann–Whitney U test); however, a significant difference was found between these tissues in stage 4 and in the control tissue (p = 0.014, Mann–Whitney U test). Conclusions: The results of our pilot study indicated the diagnostic potential of MMP13 mRNA and proved its role in the development and progression of OA. Further studies are needed to verify the potential utility of MMP13 mRNA in the development of molecularly targeted therapy for patients with OA. Full article

Background: Among numerous genes that have been a focus of equine genetic research, the KL (Klotho) and ACTN3 (Alpha-actinin-3) genes stand out due to their significant roles in muscle function and overall health, as well as performance ability. Previous studies on Arabian horses and other mammalians have shown that both KL and ACTN3 occur in different isoforms that seem to have different roles in metabolism. The main purpose of this present study was to describe different isoforms (ACTN3, ACTN3-201, ACTN3-202, KL, KL-202, KL-203) expression levels affected by the endurance effort in Arabian horses. Methods: Blood samples were taken from a group of n = 10 Arabian horses taking part in a long-distance 120 km endurance ride. After RNA isolation and reverse transcription, real-time PCR was performed. The expression levels (Relative Quantity, RQ) were calculated using the delta-delta CT method. The results showed surprisingly large differences between different isoforms expression levels which brought us to the conclusion that both KL and ACTN3 genes are suitable genetic markers to measure endurance performance. Moreover, the correlation network analyses showed that the MIOX (myo-inositol oxygenase), SH3RH2 (SH3 domain-containing ring finger 2) and TNNI2 (Troponin I2, fast skeletal type) genes are significantly involved in the endurance effort metabolism. Full article

Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8⁺PD-1⁺ cells in patients losing TFR. The level of CD8⁺PD-1⁺ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8⁺PD-1⁺ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8⁺PD-1⁺ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib. Full article

Linagliptin is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor that indirectly elevates the glucagon-like peptide-1 (GLP-1) level. The aim of the present study was to check whether linagliptin has an influence on neurotransmission in rat brain. Rats were acutely and chronically exposed to linagliptin (10 and 20 mg/kg, intraperitoneally (i.p.)). Twenty-four hours later, the striatum and hippocampus were selected for further studies. In neurochemical experiments, using high-performance liquid chromatography with electrochemical detection (HPLC-ED), the concentrations of three major neurotransmitters—dopamine, serotonin and noradrenaline—and their metabolites were measured. The analysis of mRNA expression of dopamine (D1 and D2), serotonin (5-HT-1 and 5-HT-2) and noradrenaline (α1 and α2a) receptors was also investigated using real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) in the same brain areas. Linagliptin has the ability to influence the dopaminergic system. In the striatum, the elevation of dopamine and its metabolites was observed after repeated administration of that linagliptin, and in the hippocampus, a reduction in dopamine metabolism was demonstrated. Acute linagliptin exposure increases the serotonin level in both areas, while after chronic linagliptin administration a tendency for the mRNA expression of serotoninergic receptors (5-HT1A and 5-HT2A) to increase was observed. A single instance of exposure to linagliptin significantly modified the noradrenaline level in the striatum and intensified noradrenaline turnover in the hippocampus. The recognition of the interactions in the brain between DPP-4 inhibitors and neurotransmitters and/or receptors is a crucial step for finding novel discoveries in the pharmacology of DPP-4 inhibitors and raises hope for further applications of DPP-4 inhibitors in clinical practices. Full article

Homeobox genes encode developmental transcription factors regulating tissue-specific differentiation processes and drive cancerogenesis when deregulated. Dendritic cells (DCs) are myeloid immune cells occurring as two types, either conventional or plasmacytoid DCs. Recently, we showed that the expression of NKL-subclass homeobox gene VENTX is restricted to conventional DCs, regulating developmental genes. Here, we identified and investigated homeobox genes specifically expressed in plasmacytoid DCs (pDCs) and derived blastic plasmacytoid dendritic cell neoplasm (BPDCN). We analyzed gene expression data, performed RQ-PCR, protein analyses by Western blot and immuno-cytology, siRNA-mediated knockdown assays and subsequent RNA-sequencing and live-cell imaging. Screening of public gene expression data revealed restricted activity of the CUT-class homeobox gene CUX2 in pDCs. An extended analysis of this homeobox gene class in myelopoiesis showed that additional CUX2 activity was restricted to myeloid progenitors, while BPDCN patients aberrantly expressed ONECUT2, which remained silent in the complete myeloid compartment. ONECUT2 expressing BPDCN cell line CAL-1 served as a model to investigate its regulation and oncogenic activity. The ONECUT2 locus at 18q21 was duplicated and activated by IRF4, AUTS2 and TNF-signaling and repressed by BMP4-, TGFb- and IL13-signalling. Functional analyses of ONECUT2 revealed the inhibition of pDC differentiation and of CDKN1C and CASP1 expression, while SMAD3 and EPAS1 were activated. EPAS1 in turn enhanced survival under hypoxic conditions which thus may support dendritic tumor cells residing in hypoxic skin lesions. Collectively, we revealed physiological and aberrant activities of CUT-class homeobox genes in myelopoiesis including pDCs and in BPDCN, respectively. Our data may aid in the diagnosis of BPDCN patients and reveal novel therapeutic targets for this fatal malignancy. Full article

T-box genes encode transcription factors, which control developmental processes and promote cancer if deregulated. Recently, we described the lymphoid TBX-code, which collates T-box gene activities in normal lymphopoiesis, enabling identification of members deregulated in lymphoid malignancies. Here, we have extended this analysis to cover myelopoiesis, compiling the myeloid TBX-code and, thus, highlighting which of these genes might be deregulated in myeloid tumor types. We analyzed public T-box gene expression datasets bioinformatically for normal and malignant cells. Candidate T-box-gene-expressing model cell lines were identified and examined by RQ-PCR, Western Blotting, genomic profiling, and siRNA-mediated knockdown combined with RNA-seq analysis and live-cell imaging. The established myeloid TBX-code comprised 10 T-box genes, including progenitor-cell-restricted TBX1. Accordingly, we detected aberrant expression of TBX1 in 10% of stem/progenitor-cell-derived chronic myeloid leukemia (CML) patients. The classic CML cell line K-562 expressed TBX1 at high levels and served as a model to identify TBX1 activators, including transcription factor GATA1 and genomic amplification of the TBX1 locus at 22q11; inhibitors, including BCR::ABL1 fusion and downregulated GNAI2, as well as BMP, FGF2, and WNT signaling; and the target genes CDKN1A, MIR17HG, NAV1, and TMEM38A. The establishment of the myeloid TBX-code permitted identification of aberrant TBX1 expression in subsets of CML patients and cell lines. TBX1 forms an integral part of an oncogenic regulatory network impacting proliferation, survival, and differentiation. Thus, the data spotlight novel diagnostic markers and potential therapeutic targets for this malignancy. Full article

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100–200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500–3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern. Full article

Alterations in the expression of numerous genes and the miRNAs that are recognized as their regulators in the endometrial cells of women with endometriosis may disrupt the intracellular signaling pathways associated with epithelial–mesenchymal transition (EMT). So far, the functional role of BMP7 in endometrial physiology has been confirmed, especially in the context of fertility, but the role of the activation of a specific mechanism operating through the BMP–SMAD–CDH1 axis in the formation of endometrial lesions remains unexplored. The aim of this study was to evaluate the expression profile of miR-542-3p and the EMT markers (BMP7, SMAD4, CDH1) in matched eutopic endometrium (EUE) and ectopic endometrium (ECE) samples from women with endometriosis in relation to healthy women. The levels of expression of the studied genes and miRNA in peripheral blood mononuclear cells (PBMCs) obtained from women diagnosed with endometriosis and those without the disease were also evaluated. Fifty-four patients (n = 54: with endometriosis—n = 29 and without endometriosis—n = 25) were included in the study. A comparative analysis of the relative mean expression values (RQ) of the studied mRNA and miRNA assessed by RT-qPCR demonstrated downregulation of BMP7, SMAD4, and CDH1 expression in ectopic lesions and upregulation in the eutopic endometrium compared with the control group. In the eutopic tissue of women with endometriosis, miR-542-3p expression was similar to that of the control but significantly lower than in endometrial lesions. We also confirmed a trend towards a negative correlation between miR-542-3p and BMP7 in ectopic tissue, and in PBMC, a significant negative correlation of miR-542-3p with further BMP signaling genes, i.e., SMAD4 and CDH1, was observed. These results indicate that the miRNA selected by us may be a potential negative regulator of BMP7-SMAD4-CDH1 signaling associated with EMT. The different patterns of BMP7, SMAD4, and CDH1 gene expression in ECE, EUE, and the control endometrium observed by us suggests the loss of the endometrial epithelium phenotype in women with endometriosis and demonstrates their involvement in the pathogenesis and pathomechanism of this disease. Full article

Background: CrossFit is known as a functional fitness training high-intensity exercise to improve physical performance. The most studied polymorphisms are the ACTN3 R577X gene, known for speed, power, and strength, and ACE I/D, related to endurance and strength. The present investigation analyzed the effects of training on ACTN3 and ACE gene expression in CrossFit athletes for 12 weeks. Methods: the studies included 18 athletes from the Rx category, where ACTN3 (RR, RX, XX) and ACE (II, ID, DD) characterization of genotypes and tests of maximum strength (NSCA), power (T-Force), and aerobic endurance (Course Navette) were performed. The technique used was the reverse transcription-quantitative PCR real-time polymerase chain reaction (RT-qPCR) for the relative expression analysis. Results: the relative quantification (RQ) values for the ACTN3 gene increased their levels 2.3 times (p = 0.035), and for ACE, they increased 3.0 times (p = 0.049). Conclusions: there is an overexpression of the ACTN3 and ACE genes due to the effect of training for 12 weeks. Additionally, the correlation of the expression of the ACTN3 (p = 0.040) and ACE (p = 0.030) genes with power was verified. Full article

The energy homeostasis-associated (Enho) gene, the transcript for the Adropin peptide, is usually linked to energy homeostasis, adiposity, glycemia, and insulin resistance. Studies on Enho expression in stressful conditions are lacking. This work aimed to investigate Enho mRNA expression and energy homeostasis in acute stress (AS) versus chronic unpredictable mild stress (CUMS) rat models. A total of thirty male Wistar rats (180–220 g) were fed a balanced diet with free access to water. Rats were divided into three equal groups (n = 10): (a) the normal control (NC) group; (b) the AS group, where one episode of stress for 2 h was applied; and (c) the CUMS group, in which rats were exposed to a variable program of mild stressors for 4 weeks. Energy homeostasis was analyzed by the PhenoMaster system for the automatic measuring of food intake (FI), respiratory O₂ volume (VO₂), CO₂ volume (VCO₂), respiratory quotient (RQ), and total energy expenditure (TEE). Finally, liver, whole brain, and adipose (WAT) tissue samples were collected, total RNA was prepared, and RT-PCR analysis of the Enho gene was performed. The CUMS group showed higher VO₂ consumption and VCO₂ production, and a higher RQ than the AS group. Furthermore, the TEE and FI were higher in the CUMS group compared to the AS group. Enho gene expression in the liver, brain, and WAT was significantly higher in the CUMS group than in the AS and NC groups. We can conclude that in the chew-fed AS rats, hypophagia was evident, with a shift in the RQ toward fat utilization, with no changes in body weight despite the increase in Enho mRNA expression in all studied tissues. In the CUMS group, the marked rise in Enho mRNA expression may have contributed to weight loss despite increased FI and TEE. Full article

The molecular pathogenesis of endometriosis has been associated with pathological alterations of protein expression via disturbances in homeostatic genes, miRNA expression profiles, and signaling pathways that play an essential role in the epithelial-mesenchymal transition (EMT) process. TGF-β1 has been hypothesized to play a key role in the development and progression of endometriosis, but the activation of a specific mechanism via the TGF-β-SMAD-ILK axis in the formation of endometriotic lesions is poorly understood. The aim of this study was to assess the expression of EMT markers (TGF-β1, SMAD3, ILK) and miR-21 in ectopic endometrium (ECE), in its eutopic (EUE) counterpart, and in the endometrium of healthy women. The expression level of the tested genes and miRNA was also evaluated in peripheral blood mononuclear cells (PBMC) in women with and without endometriosis. Fifty-four patients (n = 54; with endometriosis, n = 29, and without endometriosis, n = 25) were enrolled in the study. The expression levels (RQ) of the studied genes and miRNA were evaluated using qPCR. Endometriosis patients manifested higher TGF-β1, SMAD3, and ILK expression levels in the eutopic endometrium and a decreased expression level in the ectopic lesions in relation to control tissue. Compared to the endometrium of healthy participants, miR-21 expression levels did not change in the eutopic endometrium of women with endometriosis, but the RQ was higher in their endometrial implants. In PBMC, negative correlations were found between the expression level of miR-21 and the studied genes, with the strongest statistically significant correlation observed between miR-21 and TGF-β1. Our results suggest the loss of the endometrial epithelial phenotype defined by the differential expression of the TGF-β1, SMAD3 and ILK genes in the eutopic and ectopic endometrium. We concluded that the TGF-β1-SMAD3-ILK signaling pathway, probably via a mechanism related to the EMT, may be important in the pathogenesis of endometriosis. We also identified miR-21 as a possible inhibitor of this TGF-β1-SMAD3-ILK axis. Full article

Female sex, high estrogen levels, aging, obesity, and dyslipidemia are some of the risk factors associated with gallstone formation. HIV-infected patients on combination antiretroviral therapy (cART) are more prone to hypercholesterolemia. Bile acid synthesis is initiated by cholesterol 7-alpha hydroxylase (CYP7A1) and regulated by hepatocyte nuclear factors (HNF1α, HNF4α, and LXRb). The aim of this study was to evaluate the expression of HNF1α, HNF4α, LXRb, and miRNAs (HNF4α specific: miR-194-5p and miR-122^*_1) that regulate CYP7A1 transcription in HIV-infected Black South African women on cART and presenting with gallstones relative to HIV-negative patients with gallstone disease. Females (n = 96) presenting with gallstone disease were stratified based on HIV status. The gene expression of CYP7A1, HNF1α, HNF4α, LXRb, miR-194-5p, and miR-122^*_1 was determined using RT-qPCR. Messenger RNA and miRNA levels were reported as fold change expressed as 2^−ΔΔCt (RQ min; RQ max). Fold changes >2 and <0.5 were considered significant. HIV-infected females were older in age (p = 0.0267) and displayed higher low-density lipoprotein cholesterol (LDL-c) (p = 0.0419), CYP7A1 [2.078-fold (RQ min: 1.278; RQ max: 3.381)], LXRb [2.595-fold (RQ min: 2.001; RQ max: 3.000)], and HNF1α [3.428 (RQ min: 1.806; RQ max: 6.507] levels. HNF4α [0.642-fold (RQ min: 0.266; RQ max: 1.55)], miR-194-5p [0.527-fold (RQ min: 0.37; RQ max: 0.752)], and miR-122*_1 [0.595-fold (RQ min: 0.332; RQ max: 1.066)] levels were lower in HIV-infected females. In conclusion, HIV-infected women with gallstone disease displayed higher LDL-c levels and increased bile acid synthesis, which was evidenced by the elevated expression of CYP7A1, HNF1α, and LXRb. This could have been further influenced by cART and aging. Full article