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Induced pluripotent stem cells (iPSCs) harboring patient derived SAMD9 mutation offer a unique platform to study the multi-organ involvement observed in this rare disease, referred to as myelodysplasia, infections, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE) syndrome. The pluripotent nature of iPSCs allows in vitro differentiation into various somatic cell types representing multiple organ systems affected in SAMD9-mutated patients. Hence, in this paper, we present a CRISPR/Cas9-engineered iPSC model carrying SAMD9 c.2948T>G, p.I983S mutation previously reported in two patients with severe MIRAGE syndrome.

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder that typically presents in infancy as hypoplastic anemia and developmental abnormalities in approximately 50% of cases. DBAS is caused by haploinsufficiency in one of 24 ribosomal protein genes, with RPS19 mutations accounting for 25% of cases. We generated iPSC lines from two patients with different heterozygous RPS19 mutations (c.191T > C and c.184C > T) and isogenic lines in which the mutations were corrected by Cas9-mediated homology directed repair.

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IntroductionThe pupillary light reflex (PLR) is the constriction of the pupil in response to light. The PLR in response to a pulse of light follows a complex waveform that can be characterized by several parameters. It is a sensitive marker of acute neurological deterioration, but is also sensitive to the background illumination in the environment in which it is measured. To detect a pathological change in the PLR, it is therefore necessary to separate the contributions of neuro-ophthalmic factors from ambient illumination. Illumination varies over several orders of magnitude and is difficult to control due to diurnal, seasonal, and location variations.Methods and resultsWe assessed the sensitivity of seven PLR parameters to differences in ambient light, using a smartphone-based pupillometer (AI Pupillometer, Solvemed Inc.). Nine subjects underwent 345 measurements in ambient conditions ranging from complete darkness (

Introduction: Germline SAMD9 and SAMD9L mutations (SAMD9/9Lmut) cause a novel bone marrow failure and pediatric myelodysplastic syndrome. Despite >400 patients reported, evaluating variants remains challenging with >70% of germline SAMD9/9Lmut classified as variants of uncertain significance, mainly due to heterogenous phenotypes and lack of functional assays. Many patients acquire compensatory clones including secondary SAMD9/9Lmut and UPD7q with loss of the mutant allele, along with maladaptive, stress-induced monosomy 7. Monosomy 7 poses unique surveillance challenges as it may disappear spontaneously over time, precluding the need for HSCT. Methods: We utilized our prospective somatic surveillance database to identify genetic patterns and evolution in SAMD9/9Lmut patients (median age 8 years). Using high-sensitivity myeloid gene panel and SNP array, we evaluated hematopoietic specimens of 23 patients with SAMD9/9L syndromes. For comparison, we analyzed a cohort of 132 patients with other BMF/MDS conditions. Serial analysis was performed in 39% (61/155) of patients for a median duration of 15.7 (1.4-53.2) months. Results: We found 33 somatic SAMD9/9Lmut in 61% (14/23), UPD7q in 26% (6/23), and monosomy 7 in 48% (11/23) of patients with germline SAMD9/9Lmut. Somatic SAMD9/9Lmut and UPD7q were not identified in the comparative cohort, resulting in 100% specificity and positive predictive value to rule-in germline SAMD9/9L syndromes. Notably, no patient (including monosomy 7 cases) developed advanced MDS, leukemia, or cancer driver mutations with up to 4.4 years of follow-up. Conclusions: Somatic SAMD9/9Lmut and UPD7q act as a “natural functional assay” confirming pathogenicity of germline SAMD9/9Lmut. Despite high rates of monosomy 7, leukemic progression is rare in SAMD9/9L syndromes.

Replication Protein A (RPA) is single-strand DNA binding protein that plays a key role in the replication and repair of DNA. RPA is a heterotrimer made of 3 subunits – RPA1, RPA2, and RPA3. Germline pathogenic variants affecting RPA1 were recently described in patients with Telomere Biology Disorders (TBD), also known as dyskeratosis congenita or short telomere syndrome. Premature telomere shortening is a hallmark of TBD and results in bone marrow failure and predisposition to hematologic malignancies. Building on the finding that somatic mutations in RPA subunit genes occur in ~1% of cancers, we hypothesized that germline RPA alterations might be enriched in human cancers. Because germline RPA1 mutations are linked to early onset TBD with predisposition to myelodysplastic syndromes, we interrogated pediatric cancer cohorts to define the prevalence and spectrum of rare/novel and putative damaging germline RPA1, RPA2, and RPA3 variants. In this study of 5,993 children with cancer, 75 (1.25%) harbored heterozygous rare (non-cancer population allele frequency (AF) < 0.1%) variants in the RPA heterotrimer genes, of which 51 cases (0.85%) had ultra-rare (AF < 0.005%) or novel variants. Compared with Genome Aggregation Database (gnomAD) non-cancer controls, there was significant enrichment of ultra-rare and novel RPA1, but not RPA2 or RPA3, germline variants in our cohort (adjusted p-value < 0.05). Taken together, these findings suggest that germline putative damaging variants affecting RPA1 are found in excess in children with cancer, warranting further investigation into the functional role of these variants in oncogenesis.

Diamond-Blackfan anemia (DBA) is one of the most common inherited causes of bone marrow failure in children. DBA typically presents with isolated erythroid hypoplasia and anemia in infants. Congenital anomalies are seen in 50% of the patients. Over time, many patients experience panhematopoietic defects resulting in immunodeficiency and multilineage hematopoietic cytopenias. Additionally, DBA is associated with increased risk of myelodysplastic syndrome, acute myeloid leukemia and solid organ cancers. As a prototypical ribosomopathy, DBA is caused by heterozygous loss-of-function mutations or deletions in over 20 ribosomal protein genes, with RPS19 being involved in 25% of patients. Corticosteroids are the only effective initial pharmacotherapy offered to transfusion-dependent patients aged 1 year or older. However, despite good initial response, only ~20-30% remain steroid-responsive while the majority of the remaining patients will require life-long red blood cell transfusions. Despite continuous chelation, iron overload and related toxicities pose a significant morbidity problem. Allogeneic hematopoietic cell transplantation (HCT) performed to completely replace the dysfunctional hematopoietic stem and progenitor cells is a curative option associated with potentially uncontrollable risks. Advances in HLA-typing, conditioning regimens, infection management, and graft-versus-host-disease prophylaxis have led to improved transplant outcomes in DBA patients, though survival is suboptimal for adolescents and adults with long transfusion-history and patients lacking well-matched donors. Additionally, many patients lack a suitable donor. To address this gap and to mitigate the risk of graft-versus-host disease, several groups are working towards developing autologous genetic therapies to provide another curative option for DBA patients across the whole age spectrum. In this review, we summarize the results of HCT studies and review advances and potential future directions in hematopoietic stem cell-based therapies for DBA.

Monosomy 7 is the most common cytogenetic abnormality in pediatric myelodysplastic syndrome (MDS) and associated with a high risk of disease progression. However, in young children, spontaneous loss of monosomy 7 with concomitant hematologic recovery has been described, especially in the presence of germline mutations in SAMD9 and SAMD9L genes. Here, we report on our experience of close surveillance instead of upfront hematopoietic stem cell transplantation (HSCT) in seven patients diagnosed with SAMD9L syndrome and monosomy 7 at a median age of 0.6 years (range, 0.4-2.9). Within 14 months from diagnosis, three children experienced spontaneous hematological remission accompanied by a decrease in monosomy 7 clone size. Subclones with somatic SAMD9L mutations in cis were identified in five patients, three of whom attained hematological remission. Two patients acquired RUNX1 and EZH2 mutations during the observation period, of whom one progressed to myelodysplastic syndrome with excess of blasts (MDS-EB). Four patients underwent allogeneic HSCT at a median time of 26 months (range, 14-40) from diagnosis for MDSEB, necrotizing granulomatous lymphadenitis, persistent monosomy 7, and severe neutropenia. At last follow-up, six patients were alive, while one passed away due to transplant-related causes. These data confirm previous observations that monosomy 7 can be transient in young children with SAMD9L syndrome. However, they also indicate that delaying HSCT poses a substantial risk of severe infection and disease progression. Finally, surveillance of patients with SAMD9L syndrome and monosomy 7 is critical to define the evolving genetic landscape and to determine the appropriate timing of HSCT (clinicaltrials gov. Identifier: NCT00662090).

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multilineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/− HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

In situ Raman spectroscopy of flexible films of bare graphene oxide (GO) and GO decorated with Pt and Au nanoparticles (NPs) were examined upon exposure to synthetic air and 1% hydrogen in synthetic air. Initially, GO possesses an sp2 rich-structure at room temperature; however, it suffers from a drastic sp2 loss at higher temperatures under argon laser exposure leading to structural damage above 65 °C. Conversely, metal nano particle (MNP) decorated GO undergoes a drastic reduction of sp2 content mainly due to an increased defect density. In MNP composites, the formation rate of reduced graphene oxide (rGO) elevates at higher temperatures leading to an improvement in the sp2 fraction to some extent under argon laser exposure. In fact, the optical properties of MNP decorated GO drastically change against those of bare GO. Despite Pt NPs demonstrating a large catalytic effect with temperature, Au NPs do not. Similarly, the catalytic effect of Pt-GO gives rise to a slight excess in rGO formation in favor of the sp2 domain recovery. Here, there is a dominant temperature effect and a minor catalytic effect during laser exposure to GO.

© 2015 The Authors. Published by Elsevier Ltd. In this work for the first time, we report the use of molybdenum oxide (MoO3) nanoflakes on gold as active material for plasmonic optical gas sensor. The M0O3 flakes were deposited over a monolayer of gold nanoparticles, chemically attached to a functionalized fused silica substrate. The coupling between MoO3and gold nanoparticles led to reversible optical changes of the localized surface plasmon resonance of gold nanoparticles upon exposure to H2.

Summary: In contrast to their molecular mode of action, the system-level effect of antibiotics on cells is only beginning to be quantified. Molecular crowding is expected to be a relevant global regulator, which we explore here through the dynamic response phenotypes in Escherichia coli, at single-cell resolution, under sub-lethal regimes of different classes of clinically relevant antibiotics, acting at very different levels in the cell. We measure chromosomal mobility through tracking of fast (

DNA damage response is essential to human physiology. A broad spectrum of pathologies are displayed by individuals carrying monoallelic or biallelic loss-of-function mutations in DNA damage repair genes. DNA repair syndromes with biallelic disturbance of essential DNA damage response pathways manifest early in life with multi-systemic involvement and a high propensity for hematologic and solid cancers, as well as bone marrow failure. In this review, we describe classic biallelic DNA repair cancer syndromes arising from faulty single- and double-strand DNA break repair, as well as dysfunctional DNA helicases. These clinical entities include xeroderma pigmentosum, constitutional mismatch repair deficiency, ataxia telangiectasia, Nijmegen breakage syndrome, deficiencies of DNA ligase IV, NHEJ/Cernunnos, and ERCC6L2, as well as Bloom, Werner, and Rothmund-Thompson syndromes. To give an in-depth understanding of these disorders, we provide historical overview and discuss the interplay between complex biology and heterogeneous clinical manifestations.

Diamond Blackfan anemia (DBA) is predominantly an autosomal dominant inherited red cell aplasia primarily caused by pathogenic germline variants in ribosomal protein genes. DBA due to pathogenic RPL35A variants has been associated with large 3q29 deletions and phenotypes not common in DBA. We conducted a multi-institutional genotype-phenotype study of 45 patients with DBA associated with pathogenic RPL35A germline variants and curated the variant data on 21 additional cases from the literature. Genotype-phenotype analyses were conducted comparing patients with large deletions versus all other pathogenic variants in RPL35A. Twenty-two of the 45 cases had large deletions in RPL35A. After adjusting for multiple tests, a statistically significant association was observed between patients with a large deletion and steroid-resistant anemia, neutropenia, craniofacial abnormalities, chronic gastrointestinal problems, and intellectual disabilities (p

HLX transcription factor regulates haematopoietic stem and progenitor cell (HSPC) differentiation and is overexpressed in acute myeloid leukemia. Here the authors show that HLX overexpression leads to myeloid differentiation block in zebrafish and human HSPCs by direct regulation of metabolic pathways.

A template-free rapidly mixed reaction was employed to synthesize polyaniline nanofibers using chemical oxidative polymerization of aniline. Hydrochloric acid (HCl) and camphor sulfonic acid (CSA) were used in the synthesis to obtain 30- and 50-nm average diameter polyaniline nanofibers. The nanofibers were deposited onto layered ZnO/64 degrees YX LiNbO3 surface-acoustic-wave transducers. The sensors were tested toward hydrogen (H-2) gas while operating at room temperature. The dopant for the polyaniline nanofiber synthesis was found to have a significant effect on the device sensitivity. The sensor response was found to be larger for the 50-nmdiameter CSA-doped nanofiber based sensors, while the response and recovery times were faster for the 30-nm diameter HCl-doped nanofibers.

A polyaniline/In2O3 nanofibre composite based layered surface acoustic wave ( SAW) sensor has been developed and investigated for different gases. Chemical oxidative polymerization of aniline in the presence of finely divided In2O3 was employed to synthesize a polyaniline nanofibre/In2O3 nanoparticle composite. The nanocomposite was deposited onto a layered ZnO/64 degrees YX LiNbO3 SAW transducer. The novel sensor was exposed to H-2, NO2 and CO gases. Fast response and recovery times with good repeatability were observed at room temperature.

Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors analyse the DNA methylome and mutational profile of JMML to define three subgroups with unique molecular and clinical characteristics.

O objetivo com o trabalho foi avaliar as características morfogênicas e a densidade de perfilhos do capim-Aruana manejado em diferentes alturas sob pastejo de ovinos. Em 12 piquetes de 250m² cada, o capim-Aruana foi manejado em quatro alturas médias (12, 15, 20 e 25 cm), distribuídas em um delineamento experimental de blocos casualizados, com três repetições para cada tratamento. Utilizou-se vinte e quatro borregas mestiças das raças Dorper x Santa Inês com idade e peso médio de 60 (± 15) dias e 16,69 (± 2,70) kg, respectivamente, distribuídas aleatoriamente nos tratamentos. Para mensuração das variáveis morfogênicas, foram demarcados cinco perfilhos por piquete. Para contagem do número de perfilhos, foi escolhida uma área representativa do piquete. Não foram observadas diferenças (P > 0,05) para a taxa de aparecimento foliar, taxa de alongamento foliar, taxa de alongamento do colmo, taxa de senescência foliar, comprimento final de folha inteira e duração de vida das folhas entre as alturas avaliadas. O filocrono foi maior na altura de 25 cm quando comparado às alturas de 15 e 20 cm (19,35, 12,11 e 12,75 dias, respectivamente). O número de folhas vivas foi superior nos tratamentos de 12, 15 e 20, quando comparados ao de 25 cm (3,99, 4,35, 4,15 e 2,86, respectivamente). O número de perfilhos basais e aéreos não variaram (P > 0,05) entre as alturas de dossel. Alturas de manejo entre 15 e 20 cm permitem maior número de folhas vivas e menor filocrono em pastagens de capim-Aruana manejada para ovinos. A altura de manejo do dossel não influenciou nas demais características morfogênicas e densidade de perfilhos.