Lack Of Insertion Research Articles

Cancer-Associated Gangliosides as a Therapeutic Target for Host Defense Peptide Mimics.

Aberrant levels of glycolipids expressed on cellular surfaces are characteristic of different types of cancers. The oligomer of acylated lysine (OAK) mimicking antimicrobial peptides displays in vitro activity against human and murine melanoma cell lines with upregulated GD3 and GM3 gangliosides. Herein, we demonstrate the capability of OAK to intercalate into the sialo-oligosaccharides of DPPC/GD3 and DPPC/GM3 lipid monolayers using X-ray scattering. The lack of insertion into monolayers containing phosphatidylserine suggests that the mechanism of action by OAKs against glycosylated lipid membranes is not merely driven by charge effects. The fluorescence microscopy data demonstrates the membrane-lytic activity of OAK. Understanding the molecular basis for selectivity toward GD3 and GM3 gangliosides by antimicrobial lipopeptides will contribute to the development of novel therapies to cure melanoma and other malignancies.

VULNERABILIDADE DE GESTANTES USUÁRIAS DE ÁLCOOL E OUTRAS DROGAS EM PRÉ-NATAL DE BAIXO RISCO

ABSTRACT Objective: to verify the contexts that enhance the dimensions of individual, social, and programmatic vulnerability associated with the use of alcohol and other drugs during pregnancy. Method: qualitative, descriptive, and exploratory, cross-sectional study. Participants were 38 pregnant women who used alcohol and other drugs, at a moderate and severe level, in low-risk prenatal care in the Primary Health Care of two cities in the Metropolitan Region of Maringá - Paraná. Data was collected from, December 2019 to March 2020. The Vulnerability analytical framework guided the discussion. Results: at the individual level, the vulnerability contexts were issues of gender, brown and black ethnicity/color, low education, reproductive period, and high parity. At the social level, the lack of insertion in the job market, family income below the poverty line, abusive intra-family relationships, addictive behavior in the family, and violence in the living community. In the programmatic plan, there was a low demand for health services, lack of welcoming for the treatment of drug use, screening for deficient drug use, low bond with family health teams, absence of dental, psychological, and social services, insertion in the inadequate level of prenatal care, usual risk, while they should have been classified as high risk, and mean prenatal consultations below recommended. Conclusion: the study made it possible to advance in the contexts of the vulnerability of these pregnant women. Recognizing these contexts makes it possible to formulate strategies to reduce harm and damages to maternal and fetal health related to drug use during pregnancy, leading to a favorable gestational outcome.

Immune Reconstitution and Preliminary Safety Analysis Of 9 Patients Treated With Somatic Gene Therapy For X-Linked Severe Combined Immunodeficiency (SCID-X1) With a Self-Inactivating Gammaretroviral Vector

▪X-linked severe combined immunodeficiency (SCID-X1) is a fatal genetic disease currently treated with hematopoietic stem cell transplantation. Previous gene therapy trials using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (γc) controlled by the viral long-terminal repeat (LTR) induced T cell reconstitution in 18 of 20 boys with SCID-X1, but also resulted in insertional oncogenesis in 5 of 20, leading to leukemia, with 4 of the 5 demonstrating insertion at the LMO2 locus (Trial 1). We tested whether a self-inactivating (SIN) gammaretroviral vector, pSRS11.EFS.IL2RG.pre*, derived by removing the MLV LTR U3 (enhancer/promoter) region and driving expression of γc by an intronless elongation factor 1α (EF1-α) promoter, would promote T cell reconstitution in boys with X-SCID with an improved safety profile (Trial 2). Pre-clinical studies showed a lack of clonal skewing in primary and secondary transplant recipient mice in vivo and lack of insertions in either Lmo2 or Evi1 in these mice. The primary endpoints of the study are: 1. immune recovery defined as CD3+ T cell count >300/microliter and restoration of proliferation to phytohemagglutinin (PHA); and 2. Lack of insertional oncogenesis.To date 9 boys with SCID-X1 age 3.9 to 10.5 months were enrolled with 5-32 months follow-up on the current trial. All received bone marrow derived CD34+ cells transduced with the pSRS11.EFS.IL2RG.pre* vector, infused without conditioning. Transduction was performed at each site with one of two common protocols and the same GMP-produced vector supernatant. Transduction efficiency of infused CD34+ cells ranged from 0.25-2.92 vector copy number (VCN). One patient with a low initial transduction underwent a repeat procedure 1 month after infusion of the first product. Overall survival is excellent with 8 out of 9 patients alive and well; 1 patient died of overwhelming adenoviral infection, present at study entry, at 4 months prior to full reconstitution with genetically modified T cells. Excluding 1 patient with high level maternal engraftment who is unevaluable with regard to T cell number, 5 of 7 (71%) achieved CD3+ T cell count >300/microliter. The 2 patients who did not achieve T cell count >300/microliter received CD34+ cells with the lowest vector copy number (VCN). An early rise in CD16/56+ NK cells was also associated with T cell recovery. Of 8 surviving patients, 7 of 8 (87.5%) had PHA stimulation index >15 at or before 6 months (n=8; median 85.5; range 0.2-224); the patient who had no functional recovery received mismatched allogeneic cord blood transplant. At last follow-up, the remaining 7 patients have evidence of γc transgene expression in T cells, naïve T cell generation, and normal diversity. They are free of SCID related infections and humoral immune evaluation is ongoing.We compared the early immune recovery and peripheral blood insertion site analysis between 6 evaluable subjects on Trial 2 with the 20 subjects enrolled on the previous trials of MLV-based γc vector. The CD3+ T cell count at 6 months for Trial 2 (n=6; median 548/microliter; range 87-3960) does not appear to be lower than for Trial 1 (n=19; median 1755/microliter; range 200-4759; p=0.14 in 2-sided Wilcoxon rank sum test). However, we recognize that this test is underpowered, and results are preliminary. Because the latency time to leukemic events in the previous trial was long (33-60 months), we compared insertion sites as a surrogate measure of safety. Insertional activation of growth controlling genes has been demonstrated to be associated in some murine models with clonal skewing during in vitro expansion and in vivo reconstitution. Therefore, we hypothesized that removal of the transcription factor binding site-rich LTR U3 enhancer regions would change the insertion pattern detected in peripheral blood after transplantation away from the 5' ends of genes important for cell proliferation or persistence. Whereas in Trial 1, LMO2, EVI1 and other lymphoid proto-oncogenes showed prominent clusters of integration sites post-transplantation, these locations were not significant clusters in the second trial (p<10-4; comparison of n=19 Trial 1 versus n=6 Trial 2). Thus, we show promising early data that a modified gammaretroviral vector retains efficacy in treatment of SCID-X1 and is likely to have an improved safety profile. Disclosures:Off Label Use: Off-label use of CliniMACS device for purification of CD34+ bone marrow cells. Baum:Transatlantic Gene Therapy Consortium: Patents & Royalties.

The effectiveness of madurese culture wisdom on science learning sound material in primary school students’ critical thinking skills

Lack of insertion or understanding of local culture and traditions in classroom learning will cause students to appreciate the existing culture and grow in their environment. This research is a development research. The stages that has been through to make a valid, practical, and effective calculus learning tool are: 1) defining stage, 2) planning stage, and 3) developing stage. Trials that had been done are expert trials/expert validation, limited trials, and field trials. Field trials were conducted at SD Negeri 5 Patokan in two classes, 4A and 4B class. The research instrumentswere validation sheet, teacher’s evaluation sheet, student’s evaluation sheet, observation sheet for learning implementation, test of learning outcomes, and questionnaire for independence student’s learning. Validation and practicality data analysis are done by converting quantitative data in the form of an assessment score into qualitative data in the form of a standard scale of five. Analysis of effectiveness data is done by determining the percentage of mastery learning and student learning independence. Validity and practicality are shown by the results of expert validation which show a value of 4.46; while the effectiveness of learning is indicated by calculating the normal gain which shows 48.8% students get an effective score; character values in students show good categories, and student responses to learning are positive with 96% of students assessing positively towards the implementation of learning. It can be concluded that 1) developing science teaching materials based on Madura culture (sound material) for class 4 belongs to valid category, 2) developing science teaching materials based on Madura culture (sound material) for class 4 belongs topractical category and 3) developing science teaching materials based on Madura culture (sound material) for class 4 belongs toeffective category.

O trabalho do assistente social na saúde mental: revisão narrativa

Objetivo: Discutir, por meio de revisão narrativa da literatura científica, a atuação do Assistente Social na Saúde Mental. Revisão bibliográfica: A partir da Reforma Psiquiátrica em 1978, asilos e hospitais psiquiátricos são substituídos gradualmente por serviços psicossociais abertos e comunitários, com vistas ao atendimento das necessidades individuais dos usuários em sofrimento psíquico utilizando-se de abordagem humanizada. Sendo a violação dos direitos sociais um grande desafio lançado ao Assistente Social demandado pela Saúde Mental, faz-se necessário uma análise sobre o seu fazer profissional nesse espaço de trabalho, seu papel de atuação, entender como se dá o acolhimento no campo da saúde, além de situar a população usuária enquanto sujeitos de direitos, ressaltando a importância da escuta qualificada junto ao serviço social. Considerações finais: As expressões da Questão Social na Saúde Mental são verificadas a partir da exclusão social dos usuários em sofrimento psíquico através da privação de seu convívio social, na falta de inserção nas redes intersetoriais, além da estigmatização que enfrentam diariamente, acarretando-lhes prejuízos sociais e emocionais.

Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a ‘phenotype of interest’ (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with ‘Fuzzy-Spreader’-like morphologies were also identified through a visual screen. The second, a ‘mutant of interest’ (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

Human diversity in Jordan: polymorphic Alu insertions in general Jordanian and Bedouin groups.

Jordan, located in the Levant region, is an area crucial for the investigation of human migration between Africa and Eurasia. However, the genetic history of Jordanians has yet to be clarified, including the origin of the Bedouins today resident in Jordan. Here, we provide new genetic data on autosomal independent markers in two Jordanian population samples (Bedouins and the general population) to begin to examine the genetic diversity inside this country and to provide new information about the genetic position of these populations in the context of the Mediterranean and Middle East area. The markers analyzed were 18 Alu polymorphic insertions characterized by their identity by descent, known ancestral state (lack of insertion), and apparent selective neutrality. The results indicate significant genetic diffferences between Bedouins and general Jordanians (p = 0.038). Whereas Bedouins show a close genetic proximity to North Africans, general Jordanians appear genetically more similar to other Middle East populations. In general, these data are consistent with the hypothesis that Bedouins had an important role in the peopling of Jordan and constitute the original substrate of the current population. However, migration into Jordan in recent years likely has contributed to the diversity among current Jordanian population groups.

Human Diversity in Jordan: Polymorphic Alu Insertions in General Jordanian and Bedouin Groups

Human Diversity in Jordan:Polymorphic Alu Insertions in General Jordanian and Bedouin Groups Daniela Zanetti, May Sadiq, Robert Carreras-Torres, Omar Khabour, Almuthanna Alkaraki, Esther Esteban, Marc Via, and Pedro Moral abstract Jordan, located in the Levant region, is an area crucial for the investigation of human migration between Africa and Eurasia. However, the genetic history of Jordanians has yet to be clarified, including the origin of the Bedouins today resident in Jordan. Here, we provide new genetic data on autosomal independent markers in two Jordanian population samples (Bedouins and the general population) to begin to examine the genetic diversity inside this country and to provide new information about the genetic position of these populations in the context of the Mediterranean and Middle East area. The markers analyzed were 18 Alu polymorphic insertions characterized by their identity by descent, known ancestral state (lack of insertion), and apparent selective neutrality. The results indicate significant genetic differences between Bedouins and general Jordanians (p = 0.038). Whereas Bedouins show a close genetic proximity to North Africans, general Jordanians appear genetically more similar to other Middle East populations. In general, these data are consistent with the hypothesis that Bedouins had an important role in the peopling of Jordan and constitute the original substrate of the current population. However, migration into Jordan in recent years likely has contributed to the diversity among current Jordanian population groups. key words Alu Insertion Polymorphisms, Jordan, Bedouins, Population Genetics The State of Jordan emerged in 1946 as the Hashemite Kingdom of Transjordan when Britain and France divided the Middle East after World War II. Since 1948 it has officially been known as the Hashemite Kingdom of Jordan. Jordan is a predominantly Arab nation, whose capital and largest city is Amman. It is located on the East Bank of the Jordan River and the Dead Sea and borders Palestine and Israel states to the west, Syria to the north, Saudi Arabia to the south and east, and Iraq to the northeast. Because of its position in the Levant region, Jordan represents one of the major pathways for human movement. Since antiquity, traders traversed this area carrying products from the lands of the Indian Ocean basin to Syria, to be distributed from there to other parts of the Mediterranean world. Jordan was a crossroads for people from all over what is known today as the Middle East. Because of its strategic position connecting Asia, Africa, and Europe in the ancient world, Jordan was a major transit zone and thus an object of [End Page 131] contention among the rival empires of ancient Persians, Macedonian Greeks, and many others (Salibi 1998). Current inhabitants of Jordan are mostly Arab descendants of Transjordan or Palestine, and Bedouins, part of a predominantly desert-dwelling Arabian ethnic group traditionally divided into tribes. Historically, the inhabitants of this desert, which spreads northward into Syria, eastward into Iraq, and southward into Saudi Arabia, were Bedouin pastoralists (Salibi 1998). Today around 98% of the 7.9 million Jordanians are of Arab origin, along with other small minorities such as Circassians (1%) and Armenians (1%). Culturally, the official language is Arabic; in terms of religion, over 92% of the people are Sunni Muslims, around 6% are Christians (mostly Greek Orthodox, but some Greek and Roman Catholics, Syrian Orthodox, Coptic Orthodox, Armenian Orthodox, and Protestant denominations), and the remaining 2% are Shia Muslim and Druze populations (Central Intelligence Agency 2013–2014). Historically, the term “Bedouin” has denoted both a nomadic way of life and a group identity. Bedouins were the original settlers in the Middle East. From the Arabian Peninsula, their original home, they spread out and now live in desert regions of all the countries between the Arabian Gulf and the Atlantic. The Arab conquest of North Africa in the seventh century AD caused a wide dispersion, such that today the Arab culture is extended over North Africa and beyond. The availability of historical and ethnical information about Jordanian peoples (Salibi 1998) contrasts with the lack of information about the genetic background of these groups. As far as we know, previous genetic information about Jordanian populations includes two studies on uniparental markers analyzed in Bedouins and general Jordanians (Flores et...

Somaclonal variations of Nicotiana tabacum transgenic plants

There are difficulties in studying morphological and genetic features of transgenic plants which associate with the instability of transformation, the inactivation of insertion, and the derivation of somaclonal variations. This study describes the lines of tobacco plants that were obtained by Agrobacterium-mediated transformation and subsequent micropropagation in sterile conditions. Plants are characterized by dwarf length, increased proportion of sterile pollen grains, and the lack of insertion, which was detected before.

A dual role of the GTPase Rac in cardiac differentiation of stem cells.

The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.

A Highly Conserved Glutamate Residue (Glu-270) Is Essential for Plant Alternative Oxidase Activity

We have previously demonstrated that expression of a Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe confers cyanide-resistant respiratory activity on these cells (Albury, M. S., Dudley, P., Watts, F. Z., and Moore, A. L. (1996) J. Biol. Chem. 271, 17062-17066). Using this functional expression system we have investigated the active site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center. Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand within the active site, to asparagine abolishes catalytic activity because mitochondria containing the E270N mutant protein do not exhibit antimycin A-resistant respiration. Western blot analysis, using antibodies specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed by S. pombe mitochondria in a manner similar to that of the wild-type protein. It is possible that lack of antimycin A-insensitive respiration observed in mitochondria containing the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane. However, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lack of insertion is unlikely. These results provide the first experimental evidence to support the structural model in which the active site of the alternative oxidase contains a coupled binuclear iron center.