Publications by authors named "Vandana Joshi"

40 Publications

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

Commun Biol 2021 Mar 19;4(1):359. Epub 2021 Mar 19.

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025, Evry, France.

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.
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http://dx.doi.org/10.1038/s42003-021-01862-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979924PMC
March 2021

Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus.

Cells 2019 Dec 31;9(1). Epub 2019 Dec 31.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia.

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.
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http://dx.doi.org/10.3390/cells9010104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016903PMC
December 2019

A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates.

J Mol Biol 2019 07 23;431(15):2655-2673. Epub 2019 May 23.

Institute of Chemical Biology and Fundamental Medicine (ICBFM) SB RAS, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia. Electronic address:

Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.
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http://dx.doi.org/10.1016/j.jmb.2019.05.028DOI Listing
July 2019

The impact of perceived stigma on psychiatric care and outcomes for correctional mental health patients.

Psychiatry Res 2019 06 10;276:191-195. Epub 2019 May 10.

Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California at Los Angeles, 11075 Santa Monica Blvd, Suite 200, Los Angeles, CA 90025, United States.

The purpose of this study was to examine factors related to the delivery and effectiveness of psychiatric care prior to and following prison release. Particular attention was placed on patients' self-reported needs, psychiatric medication adherence, and perceived stigma related to mental health treatment, and how these factors related to post-release clinical and recidivism outcomes. Participants (N = 103) with serious psychiatric disorders (SPD; global assessment of functioning scores below 50) were recruited within 60 days of scheduled release from prison, and provided pre-release and six monthly follow-up interviews. Seventy eight percent of the released sample had at least one follow-up contact. Baseline interviews revealed low social stability prior to the current term of incarceration, and forty five percent of the sample had been returned to jail or prison within six months of release. Regression models revealed that perceived psychiatric stigma was a significant (negative) predictor of medication adherence in the community and even in prison. A path analysis showed that perceived stigma predicted responses on the K-6 psychological distress measure and recidivism both directly and indirectly via its influence on medication adherence. Mitigating the effects of this real or perceived stigma may significantly improve post-release outcomes for this high-risk population.
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http://dx.doi.org/10.1016/j.psychres.2019.05.018DOI Listing
June 2019

PARP-1 Activation Directs FUS to DNA Damage Sites to Form PARG-Reversible Compartments Enriched in Damaged DNA.

Cell Rep 2019 05;27(6):1809-1821.e5

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France. Electronic address:

PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNA-rich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates.
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http://dx.doi.org/10.1016/j.celrep.2019.04.031DOI Listing
May 2019

Infant and young child feeding practices and nutritional status in Bhutan.

Matern Child Nutr 2018 11;14 Suppl 4:e12762

Center for Human Nutrition, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.

In South Asia, childhood undernutrition persists while overweight is increasing. Internationally recommended infant and young child feeding (IYCF) practices promote healthy nutritional status; however, little is known about IYCF in Bhutan, investigated here using 2015 National Nutrition Survey data. WHO/UNICEF IYCF indicators, anthropometry and household socio-economic status were available for 441 children <24 months. Stunting, wasting, and underweight prevalence (<-2Z length-for-age [LAZ], weight-for-age, [WAZ] and weight-for-length [WLZ], respectively) were 15%, 9%, and 5%, respectively, whereas overweight (WLZ >2) prevalence was 6%. In survey-design-adjusted analyses, 52% of mothers of 0- to 5-month olds reported exclusive breastfeeding (EBF), with EBF less common for girls than boys (OR: 0.2 [95% CI: 0.1-0.9]). Although 61% of children were breastfed at 2 years and 75% of children >6 months met a minimum daily meal frequency, only 18% of children 6-23 months met minimum dietary diversity. IYCF was unassociated with risk of stunting, wasting, or underweight, possibly due to relatively low prevalence of anthropometric failure and small sample size. However, currently-breastfed children were less often overweight [OR: ~0.1 (95% upper limit ≤1.0)]. Neither breastfeeding nor most complementary feeding practices differed by socio-economic status, but children in the highest two fifth of a wealth index had 7.8 (1.3-46.9) and 5.3 (1.1-25.2) times greater odds than children in the lowest fifth of meeting minimum dietary diversity criteria. Low rates of EBF, given possible protection of breastfeeding against overweight, and inadequate dietary diversity offer evidence to guide future program interventions to improve nutritional status of young children.
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http://dx.doi.org/10.1111/mcn.12762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587771PMC
November 2018

Epidemiology of anaemia in children, adolescent girls, and women in Bhutan.

Matern Child Nutr 2018 11;14 Suppl 4:e12740

Center for Human Nutrition, Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland.

Anaemia inhibits health and development in Bhutan. We estimated anaemia prevalence and explored risk factors in children and women using data from Bhutan's National Nutrition Survey 2015. Prevalence was calculated using life-stage-specific cut-offs adjusted for altitude and survey design. Risk factors were evaluated in modified Poisson regressions. Anaemia affected 42%, 29%, 36%, and 28% of children, adolescent girls, and non-pregnant and pregnant women, respectively. Risk of anaemia was greater in children who were younger (RR 2.0, 95% CI [1.7, 2.3] and RR 1.9, 95% CI [1.6, 2.3], respectively, for 12-23 and 6-11 vs. 24-59 months), male (1.2, 1.1-1.4, ref.: female), and stunted (1.2, 1.0-1.3, ref.: height-for-age ≥ -2z). Older (15-19 years) versus younger (10-14 years) adolescents were at higher risk (1.5, 1.2-1.8), as were adolescents living at home versus at school (1.2, 0.9-1.6) and those working versus studying (1.3, 1.0-1.7). Among adult women, anaemia risk increased with age (1.2, 1.0-1.4 and 1.3, 1.1-1.5, for 30-39 and 40-49, respectively, vs. 20-29 years) and was higher for women without schooling (1.1, 1.0-1.3, vs. primary schooling), who were unmarried or separated (1.4, 1.2-1.7 and 1.3, 1.1-1.6, respectively, vs. married), without a child <5 years (1.1, 1.0-1.3), and lacking improved sanitation (1.1, 1.0-1.3). High coverage of antennal iron and folic acid supplementation may contribute to the lower prevalence of anaemia among pregnant women and women with young children. Expansion of iron supplementation programmes, fortification, and other strategies to improve dietary iron intake may reduce the prevalence of anaemia, but causes of anaemia other than iron deficiency (e.g., thalassemias) should also be investigated.
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http://dx.doi.org/10.1111/mcn.12740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948218PMC
November 2018

Nutritional status and risk factors for stunting in preschool children in Bhutan.

Matern Child Nutr 2018 11 9;14 Suppl 4:e12653. Epub 2018 Nov 9.

Center for Human Nutrition, Johns Hopkins School of Public Health, Baltimore, MD.

Childhood malnutrition remains endemic in South Asia, although the burden varies by country. We examined the anthropometric status and risk factors for malnutrition among children aged 0-59 months through the 2015 National Nutrition Survey in Bhutan. We assessed in 1,506 children nutritional status (by z-scores of height-for-age [HAZ], weight-for-height [WHZ], and weight-for-age [WAZ]), estimating prevalence, adjusted for survey design, of stunting, wasting, underweight, and overweight (<-2 for HAZ, WHZ, and WAZ and >2 for WHZ). Children were also assessed for pedal oedema. We conducted multivariable linear/logistic regression analysis to identify child, maternal, and household risk factors for childhood undernutrition and overweight, excluding children with oedema (1.7%). Mean (SE) HAZ, WHZ, and WAZ were -0.82 (0.13), 0.10 (0.04), and -0.42 (0.05), respectively. Prevalence of stunting, wasting, underweight, and overweight were 21.2%, 2.6%, 7.4%, and 2.6%, respectively. In multivariable regressions, risk of stunting significantly increased by age: 5.3% at <6 months (reference), 16.8% at 6-23 months (OR = 3.06, 95% CI [0.63, 14.8]), and 25.0% at 24-59 months (OR = 5.07, [1.16, 22.2]). Risk of stunting also decreased in a dose-response manner with improved maternal education. None of the examined variables were significantly associated with wasting or overweight. Despite a WHZ distribution comparable with the World Health Organization reference (with ~2.6% vs. an expected 2.5% of children beyond 2 z in each tail), stunting persists in one fifth of preschool Bhutanese children, suggesting that other nutrient deficits or nonnutritional factors may be constraining linear growth for a substantial proportion of children.
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http://dx.doi.org/10.1111/mcn.12653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587444PMC
November 2018

Functional differences of short and long isoforms of spastin harboring missense mutation.

Dis Model Mech 2018 09 10;11(9). Epub 2018 Sep 10.

Structure and Activity of Normal and Pathological Biomolecules, INSERM U1204, Université Paris Saclay, Université d'Evry, 91000 Evry, France

Mutations of the () gene encoding for spastin protein are the main causes of hereditary spastic paraplegia. Spastin binds to microtubules and severs them through the enzymatic activity of its AAA domain. Several missense mutations located in this domain lead to stable, nonsevering spastins that decorate a subset of microtubules, suggesting a possible negative gain-of-function mechanism for these mutants. Of the two main isoforms of spastin, only mutations of the long isoform, M1, are supposed to be involved in the onset of the pathology, leaving the role of the ubiquitously expressed shorter one, M87, not fully investigated and understood. Here, we show that two isoforms of spastin harboring the same missense mutation bind and bundle different subsets of microtubules in HeLa cells, and likely stabilize them by increasing the level of acetylated tubulin. However, only mutated M1 has the ability to interact with wild-type M1, and decorates a subset of perinuclear microtubules associated with the endoplasmic reticulum that display higher resistance to microtubule depolymerization and increased intracellular ionic strength, compared with those decorated by mutated M87. We further show that only mutated M1 decorates microtubules of proximal axons and dendrites, and strongly impairs axonal transport in cortical neurons through a mechanism likely independent of the microtubule-severing activity of this protein.
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http://dx.doi.org/10.1242/dmm.033704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177001PMC
September 2018

Microtubules as platforms for probing liquid-liquid phase separation in cells - application to RNA-binding proteins.

J Cell Sci 2018 06 11;131(11). Epub 2018 Jun 11.

SABNP Lab, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases.
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http://dx.doi.org/10.1242/jcs.214692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031325PMC
June 2018

Infant and young child feeding practices and nutritional status in Bhutan.

Matern Child Nutr 2018 07 21;14(3):e12580. Epub 2017 Dec 21.

Center for Human Nutrition, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.

In South Asia, childhood undernutrition persists while overweight is increasing. Internationally recommended infant and young child feeding (IYCF) practices promote healthy nutritional status; however, little is known about IYCF in Bhutan, investigated here using 2015 National Nutrition Survey data. WHO/UNICEF IYCF indicators, anthropometry and household socio-economic status were available for 441 children <24 months. Stunting, wasting, and underweight prevalence (<-2Z length-for-age [LAZ], weight-for-age, [WAZ] and weight-for-length [WLZ], respectively) were 15%, 9%, and 5%, respectively, whereas overweight (WLZ >2) prevalence was 6%. In survey-design-adjusted analyses, 52% of mothers of 0- to 5-month olds reported exclusive breastfeeding (EBF), with EBF less common for girls than boys (OR: 0.2 [95% CI: 0.1-0.9]). Although 61% of children were breastfed at 2 years and 75% of children >6 months met a minimum daily meal frequency, only 18% of children 6-23 months met minimum dietary diversity. IYCF was unassociated with risk of stunting, wasting, or underweight, possibly due to relatively low prevalence of anthropometric failure and small sample size. However, currently-breastfed children were less often overweight [OR: ~0.1 (95% upper limit ≤1.0)]. Neither breastfeeding nor most complementary feeding practices differed by socio-economic status, but children in the highest two fifth of a wealth index had 7.8 (1.3-46.9) and 5.3 (1.1-25.2) times greater odds than children in the lowest fifth of meeting minimum dietary diversity criteria. Low rates of EBF, given possible protection of breastfeeding against overweight, and inadequate dietary diversity offer evidence to guide future program interventions to improve nutritional status of young children.
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http://dx.doi.org/10.1111/mcn.12580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6866121PMC
July 2018

Multiplex epithelium dysfunction due to CLDN10 mutation: the HELIX syndrome.

Genet Med 2018 02 3;20(2):190-201. Epub 2017 Aug 3.

Qatar Biomedical Research Institute, Hamad Ben Khalifa University, Doha, Qatar.

PurposeWe aimed to identify the genetic cause to a clinical syndrome encompassing hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX syndrome), and to comprehensively delineate the phenotype.MethodsWe performed homozygosity mapping, whole-genome sequencing, gene sequencing, expression studies, functional tests, protein bioinformatics, and histological characterization in two unrelated families with HELIX syndrome.ResultsWe identified biallelic missense mutations (c.386C>T, p.S131L and c.2T>C, p.M1T) in CLDN10B in six patients from two unrelated families. CLDN10B encodes Claudin-10b, an integral tight junction (TJ) membrane-spanning protein expressed in the kidney, skin, and salivary glands. All patients had hypohidrosis, renal loss of NaCl with secondary hyperaldosteronism and hypokalemia, as well as hypolacrymia, ichthyosis, xerostomia, and severe enamel wear. Functional testing revealed that patients had a decreased NaCl absorption in the thick ascending limb of the loop of Henle and a severely decreased secretion of saliva. Both mutations resulted in reduced or absent Claudin-10 at the plasma membrane of epithelial cells.ConclusionCLDN10 mutations cause a dysfunction in TJs in several tissues and, subsequently, abnormalities in renal ion transport, ectodermal gland homeostasis, and epidermal integrity.
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http://dx.doi.org/10.1038/gim.2017.71DOI Listing
February 2018

Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

Biochem Pharmacol 2016 Feb 18;102:45-63. Epub 2015 Dec 18.

Institut National de la Santé et de la Recherche Médicale, UMR1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val d'Essonne, Evry 91025, France. Electronic address:

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.
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http://dx.doi.org/10.1016/j.bcp.2015.12.013DOI Listing
February 2016

Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

Nucleic Acids Res 2016 Apr 15;44(6):e60. Epub 2015 Dec 15.

Institute of Chemical Biology and Fundamental Medicine, 630090, Novosibirsk, Russian Federation Novosibirsk State University, 630090, Novosibirsk, Russian Federation

PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.
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http://dx.doi.org/10.1093/nar/gkv1476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824093PMC
April 2016

Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition.

BMC Med 2015 Jun 17;13:144. Epub 2015 Jun 17.

Institut National de la Santé et de la Recherche Médicale (INSERM) - UMR 1204, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry, France.

Background: Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to alum particles have emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion in patients with myalgic encephalomyelitis, revealing an unexpectedly long-lasting biopersistence of alum within immune cells and a fundamental misconception of its biodisposition. Evidence that aluminum-coated particles phagocytozed in the injected muscle and its draining lymph nodes can disseminate within phagocytes throughout the body and slowly accumulate in the brain further suggested that alum safety should be evaluated in the long term. However, lack of specific staining makes difficult the assessment of low quantities of bona fide alum adjuvant particles in tissues.

Methods: We explored the feasibility of using fluorescent functionalized nanodiamonds (mfNDs) as a permanent label of alum (Alhydrogel(®)). mfNDs have a specific and perfectly photostable fluorescence based on the presence within the diamond lattice of nitrogen-vacancy centers (NV centers). As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term. We thus developed fluorescent nanodiamonds functionalized by hyperbranched polyglycerol (mfNDs) allowing good coupling and stability of alum:mfNDs (AluDia) complexes. Specificities of AluDia complexes were comparable to the whole reference vaccine (anti-hepatitis B vaccine) in terms of particle size and zeta potential.

Results: In vivo, AluDia injection was followed by prompt phagocytosis and AluDia particles remained easily detectable by the specific signal of the fND particles in the injected muscle, draining lymph nodes, spleen, liver and brain. In vitro, mfNDs had low toxicity on THP-1 cells and AluDia showed cell toxicity similar to alum alone. Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.

Conclusions: The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.
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http://dx.doi.org/10.1186/s12916-015-0388-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482291PMC
June 2015

Detection of single DNA molecule hybridization on a surface by atomic force microscopy.

Small 2013 Nov 15;9(21):3630-8. Epub 2013 May 15.

Inserm U829, Laboratoire Structure-Activité des, Biomolécules Normales et Pathologiques, Université d'Evry-Val d'Essonne, Evry 91025, France.

Improving the detection of DNA hybridization is a critical issue for several challenging applications encountered in microarray and biosensor domains. Herein, it is demonstrated that hybridization between complementary single-stranded DNA (ssDNA) molecules loosely adsorbed on a mica surface can be achieved thanks to fine-tuning of the composition of the hybridization buffer. Single-molecule DNA hybridization occurs in only a few minutes upon encounters of freely diffusing complementary strands on the mica surface. Interestingly, the specific hybridization between complementary ssDNA is not altered in the presence of large amounts of nonrelated DNA. The detection of single-molecule DNA hybridization events is performed by measuring the contour length of DNA in atomic force microscopy images. Besides the advantage provided by facilitated diffusion, which promotes hybridization between probes and targets on mica, the present approach also allows the detection of single isolated DNA duplexes and thus requires a very low amount of both probe and target molecules.
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http://dx.doi.org/10.1002/smll.201300546DOI Listing
November 2013

Maternalism, race, class and citizenship: aspects of illegitimate motherhood in Nazi Germany.

Authors:
Vandana Joshi

J Contemp Hist 2011 ;46(4):832-53

University of Delhi, India.

This article juxtaposes three types of illegitimate motherhood that came in the wake of the Second World War in Nazi Germany. The first found institutional support in the Lebensborn project, an elite effort to raise the flagging birth-rates, which at the same time turned a new page in the history of sexuality. The second came before the lower courts in the form of paternity and guardianship suits that had a long precedent, and the third was a social practice that the regime considered a ‘mass crime' among its female citizenry: namely, forbidden unions between German women and prisoners of war. Through these cases the article addresses issues such as morality, sexuality, paternity, citizenship and welfarism. The flesh-and-blood stories have been culled from the Lebensborn Dossiers and Special Court files, as well as cases from the lower courts.
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http://dx.doi.org/10.1177/0022009411413409DOI Listing
October 2012

The C terminus of tubulin, a versatile partner for cationic molecules: binding of Tau, polyamines, and calcium.

J Biol Chem 2011 Jan 9;286(4):3065-78. Epub 2010 Nov 9.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM-Université d'Evry-Val d'Essonne U829, Université Evry-Val d'Essonne, 91025 Evry, France.

The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau, spermine, and calcium, three representative partners of the tubulin C-terminal region, with a peptide composed of the last 42 residues of α1a-tubulin. The results show that their binding involves overlapping amino acid stretches in the C-terminal tubulin region: amino acid residues 421-441 for Tau, 430-432 and 444-451 for spermine, and 421-443 for calcium. Isothermal titration calorimetry, NMR, and cosedimentation experiments show that Tau and spermine have similar micromolar binding affinities, whereas their binding stoichiometry differs (C-terminal tubulin peptide/spermine stoichiometry 1:2, and C-terminal tubulin peptide/Tau stoichiometry 8:1). Interestingly, calcium, known as a negative regulator of microtubule assembly, can compete with the binding of Tau and spermine with the C-terminal domain of tubulin and with the positive effect of these two partners on microtubule assembly in vitro. This observation opens up the possibility that calcium may participate in the regulation of microtubule assembly in vivo through direct (still unknown) or indirect mechanism (displacement of microtubule partners). The functional importance of this part of tubulin was also underlined by the observation that an α-tubulin mutant deleted from the last 23 amino acid residues does not incorporate properly into the microtubule network of HeLa cells. Together, these results provide a structural basis for a better understanding of the complex interactions and putative competition of tubulin cationic partners with the C-terminal region of tubulin.
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http://dx.doi.org/10.1074/jbc.M110.144089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3024800PMC
January 2011

A central role for polyamines in microtubule assembly in cells.

Biochem J 2010 Aug;430(1):151-9

Institut National de la Santé et de la Recherche Médicale, UMR, Université Evry-Val d'Essonne, Evry, France.

Owing to preferential electrostatic adsorption of multivalent cations on highly anionic surfaces, natural multivalent polyamines and especially quadrivalent spermine can be considered as potential regulators of the complex dynamical properties of anionic MTs (microtubules). Indeed, the C-terminal tails of tubulin display many negative residues in a row which should enable the formation of a correlated liquid-like phase of multivalent counterions on its surface. Although it is known that polyamine counterions promote MT assembly in vitro, little is known about the relevance of this interaction in vivo. In the present study, we have explored the relationship between polyamine levels and MT assembly in HeLa and epithelial NRK (normal rat kidney) cells using DFMO (alpha-difluoromethylornithine), an irreversible inhibitor of ornithine decarboxylase, and APCHA [N-(3-aminopropyl)-N-cyclohexylamine], a spermine synthase inhibitor. Under conditions of intracellular polyamine depletion, the MT network is clearly disrupted and the MT mass decreases. Addition of spermine to polyamine-depleted cells reverses this phenotype and rapidly promotes the extensions of the MT network. Finally, we show that polyamine levels modulate the coating of MTs with MAP4 (MT-associated protein 4), an MT-stabilizing protein, and the spatial distribution of EB1 (end-binding protein 1), an MT plus-end-binding protein. In addition, polyamines favour the formation of gap junctions in NRK cells, a process which requires MT extensions at the cell periphery. The present study provides a basis for a better understanding of the role played by polyamines in MT assembly and establishes polyamine metabolism as a potential cellular target for modulating MT functions.
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http://dx.doi.org/10.1042/BJ20091811DOI Listing
August 2010

Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells.

ACS Nano 2009 Dec;3(12):3955-62

Laboratoire de Photonique Quantique et Moleculaire, Ecole Normale Superieure de Cachan and CNRS UMR 8537, Cachan, France.

Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm), resonant laser scattering and Raman scattering cross sections are too small to allow single nanoparticle observation. Nanodiamonds can, however, be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time scales. In this work, we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells. By blocking selectively different uptake processes, we show that nanodiamonds enter cells mainly by endocytosis, and converging data indicate that it is clathrin-mediated. We also examine nanodiamond intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule delivery.
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http://dx.doi.org/10.1021/nn901014jDOI Listing
December 2009

The stathmin-derived I19L peptide interacts with FtsZ and alters its bundling.

Biochemistry 2009 Oct;48(41):9734-44

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM/UEVE U829, Evry, 91025 France.

FtsZ is a prokaryotic tubulin-like protein. Despite a low degree of sequence identity with tubulin, it presents the same folding pattern and some similar functions, notably in cell division. Indeed, FtsZ and tubulin polymerize to form bundles and microtubules, respectively, which are essential for cell cytokinesis. We previously demonstrated that peptides derived from the N-terminal stathmin domain interact with tubulin and impede microtubule formation. We demonstrated here that I19L, the most efficient of these peptides, also alters FtsZ bundling assembly in vitro. STD-NMR and TRNOESY experiments revealed that I19L interacts with FtsZ and folds upon its binding but in a way different from what we observed with tubulin. These NMR data were used in molecular modeling calculations to propose models of the I19L-FtsZ complex. Interestingly, two models, consistent with NMR data, show an interaction of I19L near the T7 loop or near the GTP binding site of FtsZ, explaining the modifications of the bundling assembly observed with this peptide. The fine analysis of the structural differences of the complexes of I19L with FtsZ and tubulin should help for the rational development of new specific antibiotic agents.
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http://dx.doi.org/10.1021/bi900556aDOI Listing
October 2009

Mica surface promotes the assembly of cytoskeletal proteins.

Langmuir 2009 Apr;25(6):3331-5

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM/UEVE U829, Evry 91025 France.

We report the surface-mediated polymerization of FtsZ protein, the prokaryote homologue of tubulin, by AFM. FtsZ protein can form filaments on mica whereas the bulk FtsZ concentration is orders of magnitude lower than the critical concentration. Surface polymerization is favored by a local increase in protein concentration and requires a high mobility of proteins on the surface. To generalize to other cytoskeleton protein, we also show that mica can initiate the formation of tubulin protofilaments. This study is of particular interest for studying cytoskeletal protein dynamics by AFM but also for the surface autoassembly of nanostructures.
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http://dx.doi.org/10.1021/la8035743DOI Listing
April 2009

Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway.

Small 2008 Dec;4(12):2236-9

Laboratoire de Photonique Quantique et Moléculaire, UMR CNRS 8537, Cachan, France.

Diamond nanoparticles are promising photoluminescent probes for tracking intracellular processes, due to embedded, perfectly photostable color centers. In this work, the spontaneous internalization of such nanoparticles (diameter 25 nm) in HeLa cancer cells is investigated by confocal microscopy and time-resolved techniques. Nanoparticles are observed inside the cell cytoplasm at the single-particle and single-color-center level, assessed by time-correlation intensity measurements. Improvement of the nanoparticle signal-to-noise ratio inside the cell is achieved using a pulsed-excitation laser and time-resolved detection taking advantage of the long radiative lifetime of the color-center excited state as compared to cell autofluorescence. The internalization pathways are also investigated, with endosomal marking and colocalization analyses. The low colocalization ratio observed proves that nanodiamonds are not trapped in endosomes, a promising result in prospect of drug delivery by these nanoparticles. Low cytotoxicity of these nanoparticles in this cell line is also shown.
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http://dx.doi.org/10.1002/smll.200800655DOI Listing
December 2008

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules.

BMC Biochem 2008 Sep 15;9:23. Epub 2008 Sep 15.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM/UEVE U829 Evry, 91025 France.

Background: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.

Results: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.

Conclusion: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.
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http://dx.doi.org/10.1186/1471-2091-9-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557009PMC
September 2008

Prevalence of Salmonella species in various raw meat samples of a local market in Kathmandu.

Ann N Y Acad Sci 2006 Oct;1081:249-56

Central Department of Zoology (Parasitology), Tribhuvan University, Kirtipur, Kathmandu, Nepal.

A cross-sectional study of raw meat samples from the local meat market of Kathmandu Metropolitan City was carried out during September 2002 to May 2003 with special emphasis on isolation and identification of Salmonella bacteria. A total of 123 raw meat samples (55 chicken, 37 buffalo, and 31 goat) were collected and analyzed relative to season. Salmonella spp was found in 11.4% (14/123) meat samples. Eight samples of chicken, that is, 14.5%, five samples of buffalo (13.5%), and one sample of goat (3.3%) were found to be positive for Salmonella. Salmonella prevalence revealed Salmonella (S.) pullorum in 3.3% samples, S. gallinarum in 0.8%, S. typhi in 1.6%, S. choleraesuis in 0.8%, and Salmonella of subgenus I or II group in 4.9% samples. More than 80% meat samples microbiologically processed indicated coliform contamination. Seasonal prevalence of Salmonella was highest in the months of April/May. Surveys revealed unsatisfactory conditions of sanitation in the local meat markets of Kathmandu.
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http://dx.doi.org/10.1196/annals.1373.031DOI Listing
October 2006

Correlates of anxiety and depression among HIV test-seekers at a Voluntary Counseling and Testing facility in Pune, India.

Qual Life Res 2007 Feb 8;16(1):41-52. Epub 2006 Nov 8.

Division of Social and Behavioral Sciences, National AIDS Research Institute, Pune, India.

Objective: We assessed the extent of anxiety/depression/distress using Hospital Anxiety and Depression Scale (HADS) among a cross-section of HIV test-seekers at a Voluntary Counseling and Testing (VCT) facility in Pune, India.

Methods: HADS has 14 items for uniscale with 7 items each for anxiety and depression rated on a four-point Likert scale. Between September 2002 and March 2003, HADS was administered to 150 consecutive HIV tests-seekers attending NARI-Talera VCT facility. Subsequently, HIV testing was done after obtaining informed consent.

Results: HADS showed strong internal consistency (Cronbach-alpha 0.77). The prevalence of risk behavior (73.3%) and HIV (45.5%) were high. Education levels influenced anxiety (p = 0.033; 0.008), more so in women (p = 0.044). Repeat test-seekers exhibited significant depression (AOR: 2.9; 95% CI: 1.4-6.1; p = 0.004) and distress (AOR: 2.5; 95% CI: 1.2-5.3; p = 0.017). Marital status influenced the uniscale scores. The HIV positive repeat test-seekers were more anxious (p = 0.035) and depressed (0.037).

Conclusions: Existence of emotional distress among HIV test-seekers, particularly among repeat test-seekers, possibly 'AIDS-anxious' individuals indicates additional counseling needs specifically by introducing gender and education sensitive interventions. VCT staff can be trained to assess emotional distress among HIV test-seekers to formulate long-term intervention.
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http://dx.doi.org/10.1007/s11136-006-9112-1DOI Listing
February 2007

Activation of RNA metabolism-related genes in mouse but not human tissues deficient in SMN.

Physiol Genomics 2006 Jan 23;24(2):97-104. Epub 2005 Aug 23.

Molecular Neurogenetics Laboratory, Institut National de la Santé et de la Recherche Médicale (INSERM), E-223, University of Evry, Genopole, Evry, France.

Mutations of the survival of motor neuron gene (SMN1) are responsible for spinal muscular atrophies (SMA), a frequent recessive autosomal motor neuron disease. SMN is involved in various processes including RNA metabolism. However, the molecular pathway linking marked deficiency of SMN to SMA phenotype remains unclear. Homozygous deletion of murine Smn exon 7 directed to neurons or skeletal muscle causes severe motor axonal or myofiber degeneration, respectively. With the use of cDNA microarrays, expression profiles of 8,400 genes were analyzed in skeletal muscle and spinal cord of muscular and neuronal mutants, respectively, and compared with age-matched controls. A high proportion of genes (20 of 429, 5%) was involved in pre-mRNA splicing, ribosomal RNA processing, or RNA decay, and 18 of them were upregulated in mutant tissues. By analyzing other neuromuscular disorders, we showed that most of them (14 of 18) were specific to the SMN defect. Quantitative PCR analysis of these transcripts showed that gene activation was an early adaptive response to the lack but not reduced amount of full-length SMN in mouse mutant tissues. In human SMA tissues, activation of this program was not observed, which could be ascribed to the reduction but not the absence of full-length SMN.
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http://dx.doi.org/10.1152/physiolgenomics.00134.2005DOI Listing
January 2006

Childhood sexual abuse patterns, psychosocial correlates, and treatment outcomes among adults in drug abuse treatment.

J Child Sex Abus 2005 ;14(1):39-55

UCLA Integrated Substance Abuse Program, Los Angeles, CA, USA.

This study reports on the effects of having a history of childhood sexual abuse (CSA) on treatment outcomes among substance abusing men and women (N = 2,434) in a national, multisite study of drug treatment outcomes. A history of CSA was reported by 27.2% of the women and 9.2% of the men. Controlling for gender, compared to patients without CSA, patients reporting CSA were younger at entry into the current drug treatment, were more likely to be White, were more likely to have a comorbid mental disorder, be alcohol or cocaine dependent, had higher levels of criminal activities, had a higher level of problem recognition, and had a more negative peer influence. Controlling for these correlates, a history of abuse was related to a lower likelihood of posttreatment abstinence.
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http://dx.doi.org/10.1300/J070v14n01_03DOI Listing
September 2005

A survey of tuberculosis hospitals in India.

Authors:
Vandana Joshi

Int J Tuberc Lung Dis 2005 Apr;9(4):467; author reply 467

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April 2005