Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

SAMHD1 expression contributes to doxorubicin resistance and predicts survival outcomes in diffuse large B-cell lymphoma patients.

Diffuse large B-cell lymphoma (DLBCL) is a commonly diagnosed, aggressive non-Hodgkin's lymphoma. While R-CHOP chemoimmunotherapy is potentially curative, about 40% of DLBCL patients will fail, highlighting the need to identify biomarkers to optimize management. SAMHD1 has a dNTPase-independent role in promoting resection to facilitate DNA double-strand break (DSB) repair by homologous recombination. We evaluated the relationship of SAMHD1 levels with sensitivity to DSB-sensitizing agents in DLBCL cells and the association of SAMHD1 expression with clinical outcomes in 79 DLBCL patients treated with definitive therapy and an independent cohort dataset of 234 DLBCL patients. Low SAMHD1 expression, Vpx-mediated, or siRNA-mediated degradation/depletion in DLBCL cells was associated with greater sensitivity to doxorubicin and PARP inhibitors. On Kaplan-Meier log-rank survival analysis, low SAMHD1 expression was associated with improved overall survival (OS), which on subset analysis remained significant only in patients with advanced stage (III-IV) and moderate to high risk (2-5 International Prognostic Index (IPI)). The association of low SAMHD1 expression with improved OS remained significant on multivariate analysis independent of other adverse factors, including IPI, and was validated in an independent cohort. Our findings suggest that SAMHD1 expression mediates doxorubicin resistance and may be an important prognostic biomarker in advanced, higher-risk DLBCL patients.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis . Dyrk1a mRNA and protein was expressed throughout the developing head and was enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with DYRK1A mutations.

DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

Risk factors of stunting and wasting in Somali pre-school age children: results from the 2019 Somalia micronutrient survey.

BACKGROUND: Stunting and wasting in children less than 5 years of age are two key indicators of child malnutrition. Reducing their prevalence is a priority of the global public health community and for Somalia, a country suffering complex humanitarian emergencies such as drought, flooding, conflict and large-scale displacements. METHODS: Data from the nationally representative cross-sectional Somalia Micronutrient Survey (SMS 2019) on 1947 children were analyzed to assess the prevalence and potential risk factors of stunting and wasting. Bivariate and multivariable analyses were conducted separately for children 0-5 months and 6-59 months, and population attributable fractions were calculated using adjusted risk ratios produced by Poisson regression models. RESULTS: Among the 1947 children, the prevalence of stunting and wasting were 17.2% (95% CI: 15.0, 19.6) and 11.0% (95% CI: 9.3, 12.9), respectively. Among children 6-59 months of age, those residing in severely food insecure households had a higher risk of stunting (adjusted risk ratio [aRR] 1.47; CI: 1.12, 1.93) compared to those in food secure households. This risk of stunting was also higher in children with inflammation (aRR 1.75; CI: 1.35, 2.25) and iron deficiency (ID) (aRR 2.09; CI: 1.58, 2.80). For wasting, a dose-response relationship was found with household wealth, with the risk of wasting increasing significantly as the household wealth quintile decreased. On the other hand, the risk of wasting was lower in iron-deficient children (aRR 0.69; CI: 0.49, 0.98) than in iron-replete children. Among children 0-5 months of age no variables remained statistically significantly associated with stunting in the multivariable analysis. Wasting, however, was more common in children with recent diarrhea (aRR 3.51; CI: 1.68, 7.36). CONCLUSIONS: Nutritional status of children in Somalia may be improved by prevention of diarrhea and other infections and improvements in household food security.

Risk factors of anaemia and iron deficiency in Somali children and women: Findings from the 2019 Somalia Micronutrient Survey.

There are limited data on the prevalence of anaemia and iron deficiency (ID) in Somalia. To address this data gap, Somalia's 2019 micronutrient survey assessed the prevalence of anaemia and ID in children (6-59 months) and non-pregnant women of reproductive age (15-49 years). The survey also collected data on vitamin A deficiency, inflammation, malaria and other potential risk factors for anaemia and ID. Multivariable Poisson regressions models were used to identify the risk factors for anaemia and ID in children and women. Among children, the prevalence of anaemia and ID were 43.4% and 47.2%, respectively. Approximately 36% and 6% of anaemia were attributable to iron and vitamin A deficiencies, respectively, whereas household possession of soap was associated with approximately 11% fewer cases of anaemia. ID in children was associated with vitamin A deficiency and stunting, whereas inflammation was associated with iron sufficiency. Among women, 40.3% were anaemic, and 49.7% were iron deficient. In women, ID and number of births were significantly associated with anaemia in multivariate models, and approximately 42% of anaemia in women was attributable to ID. Increased parity was associated with ID, and incubation and early convalescent inflammation was associated with ID, whereas late convalescent inflammation was associated with iron sufficiency. ID is the main risk factor of anaemia in both women and children and contributed to a substantial portion of the anaemia cases. To tackle both anaemia and ID in Somalia, food assistance and micronutrient-specific programmes (e.g. micronutrient powders and iron supplements) should be enhanced.

Progesterone Receptors Promote Quiescence and Ovarian Cancer Cell Phenotypes via DREAM in p53-Mutant Fallopian Tube Models.

CONTEXT: The ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited. OBJECTIVE: This study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype. METHODS: PR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established. RESULTS: STICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes. CONCLUSION: Activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.

Restoring the DREAM Complex Inhibits the Proliferation of High-Risk HPV Positive Human Cells.

High-risk (HR) human papillomaviruses are known causative agents in 5% of human cancers including cervical, ano-genital and head and neck carcinomas. In part, HR-HPV causes cancer by targeting host-cell tumor suppressors including retinoblastoma protein (pRb) and RB-like proteins p107 and p130. HR-HPV E7 uses a LxCxE motif to bind RB proteins, impairing their ability to control cell-cycle dependent transcription. E7 disrupts DREAM (Dimerization partner, RB-like, E2F and MuvB), a transcriptional repressor complex that can include p130 or p107, but not pRb, which regulates genes required for cell cycle progression. However, it is not known whether disruption of DREAM plays a significant role in HPV-driven tumorigenesis. In the DREAM complex, LIN52 is an adaptor that binds directly to p130 via an E7-like LxSxE motif. Replacement of the LxSxE sequence in LIN52 with LxCxE (LIN52-S20C) increases p130 binding and partially restores DREAM assembly in HPV-positive keratinocytes and human cervical cancer cells, inhibiting proliferation. Our findings demonstrate that disruption of the DREAM complex by E7 is an important process promoting cellular proliferation by HR-HPV. Restoration of the DREAM complex in HR-HPV positive cells may therefore have therapeutic benefits in HR-HPV positive cancers.

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169.

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.

The cell cycle regulatory DREAM complex is disrupted by high expression of oncogenic B-Myb.

Overexpression of the oncogene MYBL2 (B-Myb) is associated with increased cell proliferation and serves as a marker of poor prognosis in cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adapter for assembly of both the DREAM and MMB complexes, by a mechanism that requires S28 phosphorylation site in LIN52. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification, and establish the rationale for targeting B-Myb to restore cell cycle control.

Anemia, Micronutrient Deficiencies, and Malaria in Children and Women in Sierra Leone Prior to the Ebola Outbreak - Findings of a Cross-Sectional Study.

To identify the factors associated with anemia and to document the severity of micronutrient deficiencies, malaria and inflammation, a nationally representative cross-sectional survey was conducted. A three-stage sampling procedure was used to randomly select children <5 years of age and adult women from households in two strata (urban and rural). Household and individual data were collected, and blood samples from children and women were used to measure the prevalence of malaria, inflammation, and deficiencies of iron, vitamin A, folate, and vitamin B12. 839 children and 945 non-pregnant women were included in the survey. In children, the prevalence rates of anemia (76.3%; 95% CI: 71.8, 80.4), malaria (52.6%; 95% CI: 46.0, 59.0), and acute and chronic inflammation (72.6%; 95% CI: 67.5, 77.1) were high. However, the prevalence of vitamin A deficiency (17.4%; 95% CI: 13.9, 21.6) was moderate, and the prevalence of iron deficiency (5.2%; 95% CI: 3.3, 8.1) and iron-deficiency anemia (3.8%; 95% CI: 2.5, 5.8) were low. Malaria and inflammation were associated with anemia, yet they explained only 25% of the population-attributable risk. In women, 44.8% (95% CI: 40.1, 49.5), 35.1% (95% CI: 30.1, 40.4), and 23.6% (95% CI: 20.4, 27.3) were affected by anemia, malaria, or inflammation, respectively. The prevalence rates of iron deficiency (8.3%; 95% CI: 6.2, 11.1), iron-deficiency anemia (6.1%; 95% CI: 4.4, 8.6), vitamin A deficiency (2.1%; 95% CI: 1.1, 3.1) and vitamin B12 deficiency (0.5%; 95% CI: 0.2, 1.4) were low, while folate deficiency was high (79.2%; 95% CI: 74.1, 83.5). Iron deficiency, malaria, and inflammation were significantly associated with anemia, but explained only 25% of cases of anemia. Anemia in children and women is a severe public health problem in Sierra Leone. Since malaria and inflammation only contributed to 25% of anemia, other causes of anemia, such as hemoglobinopathies, should also be explored.

Iodine Status of Women of Reproductive Age in Sierra Leone and Its Association with Household Coverage with Adequately Iodized Salt.

Salt iodization programs are a public health success in tackling iodine deficiency. Yet, a large proportion of the world's population remains at risk for iodine deficiency. In a nationally representative cross-sectional survey in Sierra Leone, household salt samples and women's urine samples were quantitatively analyzed for iodine content. Salt was collected from 1123 households, and urine samples from 817 non-pregnant and 154 pregnant women. Household coverage with adequately iodized salt (≥15 mg/kg iodine) was 80.7%. The median urinary iodine concentration (UIC) of pregnant women was 175.8 µg/L and of non-pregnant women 190.8 µg/L. Women living in households with adequately iodized salt had higher median UIC (for pregnant women: 180.6 µg/L vs. 100.8 µg/L, respectively, p < 0.05; and for non-pregnant women: 211.3 µg/L vs. 97.8 µg/L, p < 0.001). Differences in UIC by residence, region, household wealth, and women's education were much smaller in women living in households with adequately iodized salt than in households without. Despite the high household coverage of iodized salt in Sierra Leone, it is important to reach the 20% of households not consuming adequately iodized salt. Salt iodization has the potential for increasing equity in iodine status even with the persistence of other risk factors for deficiency.

Integrating Vitamin A Supplementation at 6 months into the Expanded Program of Immunization in Sierra Leone.

Since 2004, twice-yearly mass vitamin A supplementation (VAS) has equitably reached over 85% of children 6-59 months old in Sierra Leone. However infants who turn 6 months after the event may wait until they are 11 months old to receive their first dose. The effectiveness of integrating VAS at 6 months into the Expanded Program of Immunization (EPI) in a revised child health card was studied. Health facilities matched according to staff cadre and work load were assigned to provide either a 'mini package' of VAS and infant and young child feeding (IYCF), a 'full package' of VAS, IYCF and family planning (FP), or 'child health card' only. 400 neonates were enrolled into each group, caregivers given the new child health card and followed until they were 12 months old. More infants in the full: 74.5% and mini: 71.7% group received VAS between 6 and 7 months of age compared with the new CH card only group: 60.2% (p = 0.002, p < 0.001 respectively). FP commodities were provided to 44.5% of caregivers in the full compared with <2.5% in the mini and new child health card only groups (p < 0.0001). Integration of VAS within the EPI schedule achieved >60% coverage for infants between 6 and 7 months of age. Provision of FP and/or IYCF further improved coverage. Funding was provided by the Canadian Department of Foreign Affairs, Trade and Development who had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

High coverage of vitamin A supplementation and measles vaccination during an integrated Maternal and Child Health Week in Sierra Leone.

BACKGROUND: In May 2012, the twice-yearly Maternal and Child Health Week (MCHW) integrated vitamin A supplementation (VAS) and supplementary measles vaccination to reach all children 6-59 months in Sierra Leone. Following the MCHW, a post event coverage survey was conducted to validate VAS coverage and assess adverse events following immunization. METHODS: Using the WHO Expanded Program on Immunization sampling methodology, 30 clusters were randomly selected using population proportionate to size sampling. Fourteen caregivers of children 6-59 months were interviewed per cluster for precision of ±5%. Responses were collected via mobile phones using EpiSurveyor. RESULTS: Overall VAS and measles coverage was 91.9% and 91.6%, respectively, with no significant differences by age group, sex, religion or occupation. Major reasons given for not receiving VAS and measles vaccination were not knowing about the MCHW or being out of the area. Significantly more mild adverse events (fever, pain at injection site) were reported via the post event coverage survey (29.1%) than MCHW (0.01%) (p<0.0001). CONCLUSION: The MCHW reached >90% of children in Sierra Leone with equitable coverage. Increased reporting of mild adverse events during the survey may be attributed to delayed onset after measles vaccination and/or direct inquiry from enumerators. Even mild adverse events following immunization requires strengthened reporting during and after vaccination campaigns.

High and equitable mass vitamin A supplementation coverage in Sierra Leone: a post-event coverage survey.

BACKGROUND: In Sierra Leone, children ages 6-59 months receive twice-yearly vitamin A supplementation (VAS) through Maternal and Child Health Week (MCHW) events. VAS coverage in 2011 was calculated using government tally sheets of vitamin A capsule distribution and outdated population projections from the 2004 census. We conducted a national post-event coverage (PEC) survey to validate coverage and inform strategies to reach universal coverage of VAS in Sierra Leone. METHODOLOGY: Immediately following the November 2011 MCHW event, we conducted a national PEC survey by interviewing caregivers with children ages 6-59 months using a randomized 30X30 cluster design (N = 900). We also interviewed one health worker and one community health worker in each cluster to determine their knowledge about VAS (N = 60). RESULTS: VAS coverage was 91.8% among children ages 6-59 months, which was lower than the 105.1% reported through tally sheets. Coverage was high and equitable among all districts and between age groups (98.5% for infants ages 6-11 months and 90.5% for children ages 12-59 months). Major reasons for not receiving VAS were that the child was out of the area (42.4%), the household was not visited by community health workers (28.0%), and the caretaker was not aware of the event (11.9%). CONCLUSION: Twice-yearly delivery of VAS through MCHW events achieved consistently high and equitable coverage in Sierra Leone. Universal coverage may be achieved through continued focus on communication and targeted outreach to hard-to-reach areas during the MCHWs.