RNA and the RNA-binding protein FUS act in concert to prevent TDP-43 spatial segregation.

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.

Novel mutations found in Mycobacterium leprae DNA repair gene nth from central India.

INTRODUCTION: The importance of DNA repair enzymes in maintaining genomic integrity is highlighted by the hypothesis that DNA damage by reactive oxygen/nitrogen species produced inside the host cell is essential for the mutagenesis process. Endonuclease III (Nth), formamidopyrimide (Fpg) and endonuclease VIII (Nei) DNA glycosylases are essential components of the bacterial base excision repair process. Mycobacterium leprae lost both fpg/nei genes during the reductive evolution event and only has the nth (ML2301) gene. This study aims to characterize the mutations in the nth gene of M. leprae strains and explore its correlation with drug-resistance. METHOD: A total of 91 M. leprae positive DNA samples extracted from skin biopsy samples of newly diagnosed leprosy patients from NSCB Hospital Jabalpur were assessed for the nth gene as well as drug resistance-associated loci of the rpoB, gyrA and folP1 genes through PCR followed by Sanger sequencing. RESULTS: Of these 91 patients, a total of two insertion frameshift mutations, two synonymous and seven nonsynonymous mutations were found in nth in seven samples. Sixteen samples were found to be resistant to ofloxacin and one was found to be dapsone resistant as per the known DRDR mutations. No mutations were found in the rpoB region. Interestingly, none of the nth mutations were identified in the drug-resistant associated samples. CONCLUSION: The in-silico structural analysis of the non-synonymous mutations in the Nth predicted five of them were to be deleterious. Our results suggest that the mutations in the nth gene may be potential markers for phylogenetic and epidemiological studies.

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells.

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5'UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

The Effect of the Drug Medi-Cal Organized Delivery System 1115 Demonstration Waiver on Substance Use Disorder Treatment Access: Evidence from California.

BACKGROUND: In August 2015, the California Department of Health Care Services created the Drug Medi-Cal Organized Delivery System 1115 demonstration waiver (DMC-ODS waiver) to improve service delivery to Medi-Cal-eligible individuals with a substance use disorder (SUD). We examine if implementing the DMC-ODS waiver across California counties improved patient access to SUD treatment services. METHODS: We use administrative data from 2016 to 2020 from a reporting system for all publicly-funded SUD treatment services delivered in California and employ difference-in-differences and event study empirical strategies exploiting the differential timing of DMC-ODS waiver adoption across counties. RESULTS: Event study analyses show that eleven or more months after the introduction of the DMC-ODS waiver, the number of unique patient admissions significantly increase by nearly 20%. Residential treatment admissions significantly increase by roughly 25% in all months post-waiver introduction. CONCLUSIONS: This study provides valuable information for policymakers about implementing 1115 waivers, and the important public health implications. California's DMC-ODS waiver has demonstrated that 1115 waivers similar to it can likely increase access to SUD treatment.

FUS RRM regulates poly(ADP-ribose) levels after transcriptional arrest and PARP-1 activation on DNA damage.

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.

Targeting RNA:protein interactions with an integrative approach leads to the identification of potent YBX1 inhibitors.

RNA-protein interactions (RPIs) are promising targets for developing new molecules of therapeutic interest. Nevertheless, challenges arise from the lack of methods and feedback between computational and experimental techniques during the drug discovery process. Here, we tackle these challenges by developing a drug screening approach that integrates chemical, structural and cellular data from both advanced computational techniques and a method to score RPIs in cells for the development of small RPI inhibitors; and we demonstrate its robustness by targeting Y-box binding protein 1 (YB-1), a messenger RNA-binding protein involved in cancer progression and resistance to chemotherapy. This approach led to the identification of 22 hits validated by molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy of which 11 were found to significantly interfere with the binding of messenger RNA (mRNA) to YB-1 in cells. One of our leads is an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor. This work shows the potential of our integrative approach and paves the way for the rational development of RPI inhibitors.

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

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.

PARP1 Activation Controls Stress Granule Assembly after Oxidative Stress and DNA Damage.

DNA damage causes PARP1 activation in the nucleus to set up the machinery responsible for the DNA damage response. Here, we report that, in contrast to cytoplasmic PARPs, the synthesis of poly(ADP-ribose) by PARP1 opposes the formation of cytoplasmic mRNA-rich granules after arsenite exposure by reducing polysome dissociation. However, when mRNA-rich granules are pre-formed, whether in the cytoplasm or nucleus, PARP1 activation positively regulates their assembly, though without additional recruitment of poly(ADP-ribose) in stress granules. In addition, PARP1 promotes the formation of TDP-43- and FUS-rich granules in the cytoplasm, two RNA-binding proteins which form neuronal cytoplasmic inclusions observed in certain neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Together, the results therefore reveal a dual role of PARP1 activation which, on the one hand, prevents the early stage of stress granule assembly and, on the other hand, enables the persistence of cytoplasmic mRNA-rich granules in cells which may be detrimental in aging neurons.

Development and Measurement of the Treatment Perceptions Survey (TPS) for Clients with Substance Use Disorders.

Statistical reliability of the Treatment Perceptions Survey (TPS) questionnaire was examined using data from 19 California counties. The 14-item TPS was designed for clients receiving substance use disorder services at publicly funded community-based programs. The TPS is being used for evaluation of the State's 1115 Medicaid Waiver, external quality review of county-based systems of care, and quality improvement efforts. The survey addresses four domains of access to care, quality of care, care coordination, and general satisfaction that each include multiple items, plus a single item focused on self-reported outcome. Reliability test results of the four domains as composite measures were statistically significant. General satisfaction ratings were the best predictor of self-reported outcome in a path analysis model, followed by ratings of care coordination and quality of care. Separate analyses of TPS data from clients receiving specialty mental health services suggest the questionnaire can also be used reliably in mental health settings.

A Review of Vitamin A Supplementation in South Sudan: Successes, Challenges, and Opportunities for the Way Forward.

AIM: To identify vitamin A supplementation (VAS) trends in South Sudan and provide insights to refocus VAS programming vis a vis polio eradication campaigns recently phased out while access to health care, land, food, and markets remain challenging. METHOD: Review of data from survey and coverage reports; review of policy and program documents; key informant responses; general literature search. RESULTS: Vitamin A deficiency (VAD) is likely a severe public health problem among preschool-aged children in South Sudan based on a high under-5 mortality rate (96.2 deaths/1,000 live births) and high levels of undernutrition, infections, and food insecurity. Vitamin A capsules, with deworming tablets (VASD), have been delivered to preschool-aged children during national immunization days (NIDs) for the past decade. Although areas of South Sudan and certain populations continue to have low VAS coverage, when comparing national VAS coverage (reported in the last 6 months) between 2010 and August 2019, a large improvement is noted from 4% to 76%. In 2021, VAS coverage was more than 90% at the national level during 2 stand-alone distribution campaigns. Deworming coverage trends generally mimicked VAS coverage. VAS is provided to postpartum mothers who deliver at health facilities (approximately 12%-25%), but coverage data are not available. CONCLUSION: Twice-yearly VAS should remain a key lifesaving intervention to address VAD, but alternative delivery strategies will be needed. Conducting events, such as child health days, supported by promotional activities or community-based VASD distribution activities for the youngest children and those missed during campaigns, should be considered. For the long term, a hybrid approach targeting underserved areas with mass distribution events while integrating VASD into community-based programs such as quarterly screening for wasting should be tested further and gradually scaled up everywhere as this has the potential to sustainably reach all vulnerable children twice yearly.

A central role for polyamines in microtubule assembly in cells.

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.