RESUMEN

Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology.

Asunto(s)

RESUMEN

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

Asunto(s)

RESUMEN

BACKGROUND: Lebanon endured its worst economic and financial crisis in 2020-2021. To minimize the impact of COVID-19 pandemic, it is important to improve the overall COVID-19 vaccination rate. Given that vaccine hesitancy among health care workers (HCWs) affects the general population's decision to be vaccinated, our study assessed COVID-19 vaccine acceptance among Lebanon HCWs and identified barriers, demographic differences, and the most trusted sources of COVID-19 information. METHODS: A cross-sectional study was conducted between January and May 2021 among HCWs across nine hospitals, the Orders of Physicians, Nurses, and Pharmacists in Lebanon. Descriptive statistics were performed to evaluate the COVID-19 vaccine acceptance, and univariate and multivariable to identify their predictors. RESULTS: Among 879 participants, 762 (86.8%) were willing to receive the COVID-19 vaccine, 52 (5.9%) refused, and 64 (7.3%) were undecided. Males (226/254; 88.9%) and those ≥ 55 years (95/100; 95%) had the highest rates of acceptance. Of the 113 who were not willing to receive the vaccine, 54.9% reported that the vaccine was not studied well enough. Participants with a previous SARS-CoV-2 infection and those who did not know if they had a previous infection (p = 0.002) were less likely to accept the vaccine compared to those with no previous infection. The most trusted COVID-19 sources of information were WHO (69.3%) and healthcare providers (68%). CONCLUSION: Lebanese HCWs had a relatively high acceptance rate for COVID-19 vaccination compared to other countries. Our findings are important in informing the Lebanese health care authorities to establish programs and interventions to improve vaccine uptake among HCWs and the general population.

Asunto(s)

RESUMEN

Two recent papers, Rao et al. (2021), in this issue, and Sönmezer et al. (2021), investigate transcription factor cooperativity at cis-regulatory elements. Using data from nuclease- and methyltransferase-footprinting experiments, they demonstrate that factor co-occupancy at regulatory elements commonly occurs in vivo and is conserved from fly to mouse.

Asunto(s)

RESUMEN

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.

Asunto(s)

RESUMEN

Camurati-Engelmann Disease (CED), also known as progressive diaphyseal dysplasia, is a rare congenital disorder inherited in an autosomal-dominant pattern, most commonly affecting the skull and diaphysis of long tubular bones. Clinical symptoms start in early age and include ostealgia, muscle atrophy and weakness in the lower limbs, generalized fatigue in addition to gait disturbances (Garcia Armario and Lebron, 2011, Andreu-Arasa et al., 2019; Fyrgiola et al., 2017; Damiá and García Gómez, 2017; Mwasamwaja et al., 2018). CED is believed to be caused by mutation in the gene coding for Transforming Growth Factor ß-1 (TGFß-1) (Fyrgiola et al. 2017). This article presents a rare clinical case of CED, with bilaterally hypertrophic articular apparatus and subsequent ankylosis. A 33-year-old male is reported with temporomandibular joint (TMJ) ankylosis, bone pain, generalized muscle weakness, abnormal gait and bulging eyes. Diagnosis of CED was based on genetic mapping performed by genetist. Upon clinical and radiological examination, a massive bony mass in the condyloid and coronoid was discovered and treatment of choice was surgical resection and installation of bilateral stock articular prostheses.

RESUMEN

Studies of genetic blood disorders have advanced our understanding of the intrinsic regulation of hematopoiesis. However, such genetic studies have only yielded limited insights into how interactions between hematopoietic cells and their microenvironment are regulated. Here, we describe two affected siblings with infantile myelofibrosis and myeloproliferation that share a common de novo mutation in the Rho GTPase CDC42 (Chr1:22417990:C>T, p.R186C) due to paternal germline mosaicism. Functional studies using human cells and flies demonstrate that this CDC42 mutant has altered activity and thereby disrupts interactions between hematopoietic progenitors and key tissue microenvironmental factors. These findings suggest that further investigation of this and other related disorders may provide insights into how hematopoietic cell-microenvironment interactions play a role in human health and can be disrupted in disease. In addition, we suggest that deregulation of CDC42 may underlie more common blood disorders, such as primary myelofibrosis.

Asunto(s)

RESUMEN

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five-amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

Asunto(s)

RESUMEN

Nitrite and Nitrate have been used extensively as additives in various meat products to enhance flavor, color, and to preserve the meat from the bacterial growth. High concentrations of nitrite can threat human health since several studies in the literature claim that nitrite is associated with cancer incidences, leukemia, and brain tumors. Therefore, it is vital to measure the nitrite concentrations in processed meat products. In this study, an in-lab miniaturized photometric detection system is fabricated to inspect the nitrite concentration in processed meat products in Jordan. The analytical performance of nitrite detection is evaluated based on three key statistical parameters; linearity, limit of detection, and limit of quantitation. Respectively, for the fabricated system, the three values are found to be equal to 0.995, 1.24 × 10-2 ppm, and 4.12 × 10-2 ppm. Adherence to Beer's law is found over the investigated range from 2.63 ppm to 96.0 ppm. The developed system is utilized for photometric detection of nitrite in processed meat products available in the Jordanian market like pastrami, salami, and corned beef. In all of the analyzed samples, the nitrite content is found to be lower than 150 ppm, which represents the maximum allowable nitrite limit.

RESUMEN

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

Asunto(s)

RESUMEN

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.

Asunto(s)

RESUMEN

BACKGROUND The 800 nm long-pulsed diode laser machine is safe and effective for permanent hair reduction. Traditionally, most long-pulsed diode lasers used for hair removal had a relatively small spot size. Recently, a long-pulsed diode laser with a large spot size and vacuum assisted suction handpiece was introduced. The treatment parameters of each type of handpiece differ. Short and long-term clinical efficacy, treatment associated pain, and patient satisfaction are important factors to be considered. OBJECTIVE: This study aims to conduct a direct head to head comparison of both handpieces of the 800nm long-pulsed diode laser by evaluating long term hair reduction, treatment associated pain and patient satisfaction. METHODS: Thirteen subjects were enrolled in this prospective, self-controlled, single-center study of axillary laser hair removal. The study involved 4 treatments using a long pulsed diode laser with a large spot size HS handpiece (single pass), HS handpiece (double pass), and a small spot size ET handpiece according to a randomized choice. The treatment sessions were done at 4-8 week intervals with follow up visits taken at 6 and 12 months after the last treatment session. Hair clearance and thickness analysis were assessed using macro hair count photographs taken at baseline visit, at each treatment session visit and at follow up visits. Other factors including pain, treatment duration, and patients' preference were secondary study endpoints. RESULTS: At 6 months follow up visits after receiving four laser treatments, there was statistically significant hair clearance in the three treatment arms with 66.1 % mean percentage hair reduction with the ET handpiece, 43.6% with the HSS (single pass) and 64.1 % with the HSD (double). However, at one year follow up, the results significantly varied from the 6 months follow up. The mean percentage hair reduction was 57.8% with the ET handpiece treated axillas (n=9), 16.5% with the HSS (single pass) handpiece treated axillas (n=7), and 46.9% with the HSD (double pass) handpiece treated axillas (n=6). Thus, at one year follow up, there was a significant hair reduction that was similar in both the ET and HSD treated axillae (57.8% and 46.9 %), but only minimal hair reduction (16.5%)was observed in the HSS treated axillae. CONCLUSIONS: This is the first study that compared the long-term efficacy of the ET and HS handpieces after four treatment sessions with up to 12 months follow up after the last treatment session. It is also the first study that provided head to head comparison between HS (double pass), HS (single pass), and ET handpiece taking into consideration the end hair reduction result, the time consumed, the pain score experienced, and the overall patient satisfaction. HSD had better hair clearance and patient satisfaction when compared to ET and HSS. The long term follow up results showed that ET was superior to HSS (P less than .05), but was not superior to HSD (P greater than 0.05). However, HSD treated patients had lower pain scores with HSD than with ET. We conclude that ET handpiece is almost as efficacious as HSD handpiece, and the desired end results could be achieved with HDD with better patient satisfaction, less treatment duration and less pain.

J Drugs Dermatol. 2017;16(9):893-898.

Asunto(s)

RESUMEN

Immunodeficient mouse models have been valuable for studies of human hematopoiesis, but high-fidelity recapitulation of erythropoiesis in most xenograft recipients remains elusive. Recently developed immunodeficient and Kit mutant mice, however, have provided a suitable background to achieve higher-level human erythropoiesis after long-term hematopoietic engraftment. While there has been some characterization of human erythropoiesis in these models, a comprehensive analysis from various human developmental stages has not yet been reported. Here, we have utilized cell surface phenotypes, morphologic analyses, and molecular studies to fully characterize human erythropoiesis from multiple developmental stages in immunodeficient and Kit mutant mouse models following long-term hematopoietic stem and progenitor cell engraftment. We show that human erythropoiesis in such models demonstrates complete maturation and enucleation, as well as developmentally appropriate globin gene expression. These results provide a framework for future studies to utilize this model system for interrogating disorders affecting human erythropoiesis and for developing improved therapeutic approaches.

Asunto(s)

RESUMEN

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

Asunto(s)

RESUMEN

Cheilitis glandularis (CG) is a rare inflammatory salivary gland disease that usually affects the lips. Although the etiology of CG is still unknown, it is believed to be a hereditary disease with an autosomal dominant pattern of inheritance. Three clinical presentations of CG are described in the literature: simple, superficial suppurative, and deep suppurative. A case of deep suppurative CG that extended to the buccal mucosa has been previously reported as suppurative stomatitis glandularis (SSG). Here we report a case of SSG in a 64-year-old white female with a history of bilateral renal transplants for adult polycystic kidney disease, who presented with painful swollen lips and bilateral buccal mucosal lesions. The diagnosis and management of the case is discussed. To the best of our knowledge, this is the second report of SSG, a rare condition affecting the minor salivary glands in the oral cavity.

Asunto(s)

RESUMEN

BACKGROUND: Cicatricial pemphigoid (benign mucous membrane pemphigoid) is an autoimmune vesiculobullous disease that affects mucosal tissues of adults and rarely presents in children. Only 9 cases in the English literature have reported cicatricial pemphigoid in children, primarily as oral mucosal lesions. This paper presents a case of childhood cicatricial pemphigoid that clinically manifested as necrotizing ulcerative gingivitis (NUG). METHODS: A 9-year-old girl presented with gingival bleeding and discomfort for 2 weeks. NUG was suspected and the patient was treated with antibiotics and an oral hygiene regimen. When the condition did not improve after repeated treatment trials, routine hematoxylin and eosin (H&E) and direct immunofluorescence examinations were performed. RESULTS: Microscopic examination of H&E stained sections showed a non-specific ulceration with chronic inflammation. Direct immunofluorescence studies of peri-lesional tissue showed linear deposition of C3 at the basement membrane zone that was consistent with a diagnosis of cicatricial pemphigoid. CONCLUSION: Cicatricial pemphigoid is an autoimmune ulcerative condition that is rarely seen in children. Immunofluorescence studies are essential to differentiate this condition from other ulcerative oral lesions.

Asunto(s)

RESUMEN

The one-carbon metabolism enzymes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) can be found on a single trifunctional protein in the eukaryotes examined. The one exception is in spinach leaves where 10-formyltetrahydrofolate synthetase is monofunctional (Nour, J. M., and Rabinowitz, J. C. (1991) J. Biol. Chem. 266, 18363-18369). In the prokaryotes examined, 10-formyltetrahydrofolate synthetase is either absent or is monofunctional. A cDNA clone encoding spinach leaf 10-formyltetrahydrofolate synthetase was isolated through the use of antibodies to the purified enzyme. This clone had an open reading frame of 1914 base pairs and encoded for a protein containing 636 amino acids with a calculated M(r) of 67,727. The percentage identity between spinach 10-formyltetrahydrofolate synthetase and the synthetase domains in the four trifunctional eukaryotic enzymes and the two monofunctional prokaryotic enzymes that have been cloned and sequenced was: 64.9% human, 63.8% rat, 55.6% yeast cytoplasm, 53.8% yeast mitochondria, 47.8% Clostridium acidi-urici, and 47.9% Clostridium thermoaceticum. Clearly the spinach monofunctional protein had greatest homology with the mammalian proteins. The spinach protein is longer than the two other monofunctional prokaryotic proteins. Possible reasons for this are presented. The codon usage and the putative translation initiation sites are examined and compared with other spinach proteins.

Asunto(s)

RESUMEN

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.

Asunto(s)

RESUMEN

One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.

Asunto(s)