hMAF, a Small Human Transcription Factor That Heterodimerizes Specifically with Nrf1 and Nrf2

A 1.6-kilobase pair full-length cDNA encoding a transcription factor homologous to the Maf family of proteins was isolated by screening a K562 cDNA library with the NFE2 tandem repeat probe derived from the globin locus control region. The protein, which was designated hMAF, contains a basic DNA binding domain and an extended leucine zipper but lacks any recognizable activation domain. Expressed in vitro, the hMAF protein is able to homodimerize in solution and band-shift the NFE2 tandem repeat probe. In addition to homodimers, hMAF can also form high affinity heterodimers with two members of the NFE2/CNC-bZip family (Nrf1 and Nrf2) but not with a third family member, p45-NFE2. Although hMAF/hMAF homodimers and hMAF/Nrf1 and hMAF/Nrf2 heterodimers bind to the same NFE2 site, they exert functionally opposite effects on the activity of a linked gamma-globin gene. In fact, whereas all hMAF/CNC-bZip heterodimers stimulate the activity of a gamma-promoter reporter construct in K562 cells, the association into homodimers that is induced by overexpressing hMAF inhibits the activity of the same construct. Thus variations in the expression of hMAF may account for the modulation in the activity of the genes that bear NFE2 recognition sites

A novel high-content immunofluorescence assay as a tool to identify at the single cell level γ-globin inducing compounds

The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ-and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathie

Cross-sectional analysis of the humoral response after SARS-CoV-2 vaccination in Sardinian multiple sclerosis patients, a follow-up study

Monitoring immune responses to SARS-CoV-2 vaccination and its clinical efficacy over time in Multiple Sclerosis (MS) patients treated with disease-modifying therapies (DMTs) help to establish the optimal strategies to ensure adequate COVID-19 protection without compromising disease control offered by DMTs. Following our previous observations on the humoral response one month after two doses of BNT162b2 vaccine (T1) in MS patients differently treated, here we present a cross-sectional and longitudinal follow-up analysis six months following vaccination (T2, n=662) and one month following the first booster (T3, n=185). Consistent with results at T1, humoral responses were decreased in MS patients treated with fingolimod and anti-CD20 therapies compared with untreated patients also at the time points considered here (T2 and T3). Interestingly, a strong upregulation one month after the booster was observed in patients under every DMTs analyzed, including those treated with fingolimod and anti-CD20 therapies. Although patients taking these latter therapies had a higher rate of COVID-19 infection five months after the first booster, only mild symptoms that did not require hospitalization were reported for all the DMTs analyzed here. Based on these findings we anticipate that additional vaccine booster shots will likely further improve immune responses and COVID-19 protection in MS patients treated with any DMT

hMAF, a small human transcription factor that heterodimerizes specifically with Nrf1 and Nrf2

A 1.6-kilobase pair full-length cDNA encoding a transcription factor homologous to the Maf family of proteins was isolated by screening a K562 cDNA library with the NFE2 tandem repeat probe derived from the globin locus control region. The protein, which was designated hMAF, contains a basic DNA binding domain and an extended leucine zipper but lacks any recognizable activation domain. Expressed in vitro, the hMAF protein is able to homodimerize in solution and bandshift the NFE2 tandem repeat probe. In addition to homodimers, hMAF can also form high affinity heterodimers with two members of the NFE2/CNC-bZip family (Nrf1 and Nrf2) but not with a third family member, p45-NFE2. Although hMAF/hMAF homodimers and hMAF/Nrf1 and hMAF/Nrf2 heterodimers bind to the same NFE2 site, they exert functionally opposite effects on the activity of a linked gamma-globin gene. In fact, whereas all hMAF/CNC-bZip heterodimers stimulate the activity of a gamma-promoter reporter construct in K562 cells, the association into homodimers that is induced by overexpressing hMAF inhibits the activity of the same construct. Thus variations in the expression of hMAF may account for the modulation in the activity of the genes that bear NFE2 recognition sites

A novel silent beta-thalassemia mutation in the distal CACCC box affects the binding and responsiveness to EKLF

The silent beta-thalassemia mutation, beta(+)-101C --> T, is the only mutation currently described in the distal beta-globin CACCC box. We present a novel mutation, a C --> G transversion, in the same position. Expression analysis in heterozygous subjects demonstrated that the mutation determines a 20% reduction in the output of the P-globin gene. DNA-protein interaction and transactivation analysis correlated the decrease in the beta-globin synthesis with the reduced binding and transactivation of EKLF to the mutant promoter. These data predict that the beta-101C --> G mutation will display a silent thalassemia phenotype similar to that of the beta-101 C --> T mutation

A novel splicing defect (IVS6+1G > T) in a patient with pseudovitamin D deficiency rickets

A 15-month-old boy with severe rickets, that by clinical analysis was diagnosed as affected by hereditary pseudovitamin D deficiency rickets (PDDR), was evaluated for mutations in the 25OHD(3) 1alpha-hydroxylase gene. Molecular analysis showed a double heterozygous state for a novel splicing mutation in the invariant dinucleotide of the donor site of IVS6 and a 7 nucleotide insertion in the exon 8, which is common in different ethnical backgrounds. (C) 2002, Editrice Kurtis

Papillary thyroid cancer in a young woman affected by giant congenital melanocytic nevus, ultrasound diagnosis

Data literatures report numerous association between giant congenital nevus and development alteration; only two cases describe its coexistence with thyroid disorders. However, we report the association of papillary thyroid cancer and giant congenital nevus. Papillary thyroid cancer is the most common differentiated thyroid cancer and has high prevalence in young women. In this paper we report: the case of a 18 years-old woman, affected by giant congenital melanocytic nevus on her back, who came to our observation because of one month of fever and increased volume of latero-cervical lymph nodes. Negative serologic tests allowed us to exclude lymphoma and mononucleosis. Because of the high risk (6%) that giant congenital melanocytic nevi could transform into malignant melanoma, we performed an ultrasound examination (US) of the cervical lymph nodes. The examination extended to the thyroid gland enabled us to visualize the same parenchyma alteration in both thyroid gland and lymph nodes. At last, fine-needle percoutaneus aspiration on thyroid lesion confirmed the presence of papillary carcinoma. In our case, thank to the optimal visualization of the parenchyma structure, US was diriment allowing a diagnosis of primitive thyroid lesion with an involvement of all lymph nodes in the neck. This findings legitimate the role of US as an accurate, noninvasive, radiation free and low-cost imaging technique in detecting differential diagnosis in the cervical lymphadenopathy, as well in preoperative staging thyroid carcinoma

Papillary thyroid cancer in a young woman affected by giant congenital melanocytic nevus, ultrasound diagnosis

Data literatures report numerous association between giant congenital nevus and development alteration; only two cases describe its coexistence with thyroid disorders. However, we report the association of papillary thyroid cancer and giant congenital nevus. Papillary thyroid cancer is the most common differentiated thyroid cancer and has high prevalence in young women. In this paper we report: the case of a 18 years-old woman, affected by giant congenital melanocytic nevus on her back, who came to our observation because of one month of fever and increased volume of latero-cervical lymph nodes. Negative serologic tests allowed us to exclude lymphoma and mononucleosis. Because of the high risk (6%) that giant congenital melanocytic nevi could transform into malignant melanoma, we performed an ultrasound examination (US) of the cervical lymph nodes. The examination extended to the thyroid gland enabled us to visualize the same parenchyma alteration in both thyroid gland and lymph nodes. At last, fine-needle percoutaneus aspiration on thyroid lesion confirmed the presence of papillary carcinoma. In our case, thank to the optimal visualization of the parenchyma structure, US was diriment allowing a diagnosis of primitive thyroid lesion with an involvement of all lymph nodes in the neck. This findings legitimate the role of US as an accurate, noninvasive, radiation free and low-cost imaging technique in detecting differential diagnosis in the cervical lymphadenopathy, as well in preoperative staging thyroid carcinoma