Communication between the stem cell niche and an adjacent differentiation niche through miRNA and EGFR signaling orchestrates exit from the stem cell state in the Drosophila ovary.

The signaling environment, or niche, often governs the initial difference in behavior of an adult stem cell and a derivative that initiates a path towards differentiation. The transition between an instructive stem cell niche and differentiation niche must generally have single-cell resolution, suggesting that multiple mechanisms might be necessary to sharpen the transition. Here, we examined the Drosophila ovary and found that Cap cells, which are key constituents of the germline stem cell (GSC) niche, express a conserved microRNA (miR-124). Surprisingly, loss of miR-124 activity in Cap cells leads to a defect in differentiation of GSC derivatives. We present evidence that the direct functional target of miR-124 in Cap cells is the epidermal growth factor receptor (EGFR) and that failure to limit EGFR expression leads to the ectopic expression of a key anti-differentiation BMP signal in neighboring somatic escort cells (ECs), which constitute a differentiation niche. We further found that Notch signaling connects EFGR activity in Cap cells to BMP expression in ECs. We deduce that the stem cell niche communicates with the differentiation niche through a mechanism that begins with the selective expression of a specific microRNA and culminates in the suppression of the major anti-differentiation signal in neighboring cells, with the functionally important overall role of sharpening the spatial distinction between self-renewal and differentiation environments.

Immunogenomic features of radiologically distinctive nodules in multiple primary lung cancer.

OBJECTIVES: To provide molecular and immunological attributes mechanistic insights for the management of radiologically distinctive multiple primary lung cancer (MPLC). METHODS: The Bulk RNA-seq data of MPLC were obtained from our center. The Bulk RNA-seq data and CT images of patients with single primary lung cancer (SPLC) were obtained from GSE103584. Immune infiltration algorithms were performed to investigate the disparities in the immunological microenvironment between the two groups. Single-cell gene analysis was used to explore immune cells composition and communication relationships between cells in MPLC. RESULTS: In MPLC, 11 pure ground-glass opacity nodules (pGGN) and 10 mixed GGN (mGGN) were identified, while in SPLC, the numbers were 18 pGGN and 22 mGGN, respectively. In MPLC, compared to pGGN, mGGN demonstrated a significantly elevated infiltration of CD8+ T cells. Single-cell gene analysis demonstrated that CD8+ T cells play a central role in the signaling among immune cells in MPLC. The transcription factors including MAFG, RUNX3, and TBX21 may play pivotal roles in regulation of CD8+ T cells. Notably, compared to SPLC nodules for both mGGN and pGGN, MPLC nodules demonstrated a significantly elevated degree of tumor-infiltrating immune cells, with this difference being particularly pronounced in mGGN. There was a positive correlation between the proportion of immune cells and consolidation/tumor ratio (CTR). CONCLUSIONS: Our findings provided a comprehensive description about the difference in the immune microenvironment between pGGN and mGGN in early-stage MPLC, as well as between MPLC and SPLC for both mGGN and pGGN. The findings may provide evidence for the design of immunotherapeutic strategies for MPLC.

Multidimensional engineering of Saccharomyces cerevisiae for the efficient production of heme by exploring the cytotoxicity and tolerance of heme.

Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on Saccharomyces cerevisiae was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H2O2 and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD600), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in S. cerevisiae. The combined overexpression of 5 genes (SPS22, REE1, PHO84, HEM4 and CLB2) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with S. cerevisiae strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts.

Selective Synthesis of Sulfonamides and Sulfenamides from Sodium Sulfinates and Amines.

An effective method was explored for the selective synthesis of sulfonamides and sulfenamides using sodium sulfinates and amines as starting materials. This method offers mild reaction conditions, a broad substrate scope, high efficiency, and readily accessible materials, making it suitable and an alternative strategy for the preparation of a variety of biologically or pharmaceutically active compounds.

Cobalt/Salicylaldehyde-Enabled C-H Alkoxylation of Benzamides with Secondary Alcohols under Solvothermal Conditions.

C-O bond formation via C-H alkoxylation remains a challenge, especially coupling with a secondary alcohol, due to its low activity and sterically encumbered property. Here, we report a general and effective cobalt-catalyzed oxidative cross-coupling of benzamides with secondary alcohols via C-H alkoxylation reaction under solvothermal conditions, enabled by a salicylaldehyde/cobalt complex. The protocol features easy operation without additives, broad substrate scope, and excellent functional tolerance. The applicability is proven by the gram-scale synthesis and modification of natural products.

Selective Construction of C-S/S-N Bonds from N-Substituted O-Thiocarbamates and Indoles under Transition-Metal-Free Conditions.

A method for the selective construction of S-N/C(sp2)-S bonds using N-substituted O-thiocarbamates and indoles as substrates is reported. This protocol features good atom utilization, mild conditions, short reaction time, and wide substrate scope, which can provide a convenient path for the functionalization of indoles. In addition, the reaction could be scaled up on gram scale, showing potential application value in industry synthesis.

Small Conjugated Organic Ligand-Modified Polyoxometalate-Based Hybrid Materials for Boosting Photocatalytic CO2 Reduction.

Photocatalytic carbon dioxide (CO2) reduction to value-added chemicals is a multielectron transfer process, and the crucial step is the synthesis of photocatalysts. The introduction of small conjugated organic ligands can make the catalytic active site of the compound easier to be exposed in the reaction system and fully contact with the substrate, accelerating the photocatalytic reaction process. In this paper, we synthesized two isomorphic compounds, namely, {[Co(mtrz)3·(H2O)2]2·[SiW12O40]}·6H2O (1) and {[Ni(mtrz)3·(H2O)2]2·[SiW12O40]}·6H2O (2) (mtrz = 1-methyl-1,2,4-triazole). We found that compound 1 has a great photocatalytic performance through a series of experiments, with a CO reduction yield of 7364.92 µmol g-1 h-1 and a CO selectivity of 82.5%. Furthermore, the high catalytic activity can be maintained over four cycle experiments. The catalytic mechanism of its photocatalytic system is also elucidated, which provides an idea for realizing efficient catalytic reduction of CO2 to CO.

Selective α-oxidation of amides via visible-light-driven iron catalysis.

Hydroxyl radicals (˙OH) as one of the highly reactive species can react unselectively with a wide range of chemicals. The ˙OH radicals are typically generated under harsh conditions. Herein, we report hydroxyl radical-induced selective N-α C(sp3)-H bond oxidation of amides under greener and mild conditions via an Fe(NO3)3·9H2O catalyst inner sphere pathway upon irradiation with a 30 W blue LED light strip (λ = 455 nm) using NaBrO3 as the oxidant. This protocol exhibited high chemoselectivity and excellent functional group tolerance. A preliminary mechanism investigation demonstrated that the iron catalyst afforded hydroxyl radicals via the visible-light-induced homolysis (VLIH) of iron complexes followed by a hydrogen atom transfer (HAT) process to realize this transformation.

bHLH transcription factors Hes1, Ascl1 and Oligo2 exhibit different expression patterns in the process of physiological electric fields-induced neuronal differentiation.

BACKGROUND: Recent studies have shown that the expression of bHLH transcription factors Hes1, Ascl1, and Oligo2 has an oscillating balance in neural stem cells (NSCs) to maintain their self-proliferation and multi-directional differentiation potential. This balance can be disrupted by exogenous stimulation. Our previous work has identified that electrical stimulation could induce neuronal differentiation of mouse NSCs. METHODS: To further evaluate if physiological electric fields (EFs)-induced neuronal differentiation is related to the expression patterns of bHLH transcription factors Hes1, Ascl1, and Oligo2, mouse embryonic brain NSCs were used to investigate the expression changes of Ascl1, Hes1 and Oligo2 in mRNA and protein levels during EF-induced neuronal differentiation. RESULTS: Our results showed that NSCs expressed high level of Hes1, while expression of Ascl1 and Oligo2 stayed at very low levels. When NSCs exited proliferation, the expression of Hes1 in differentiated cells began to decrease and oscillated at the low expression level. Oligo2 showed irregular changes in low expression level. EF-stimulation significantly increased the expression of Ascl1 at mRNA and protein levels accompanied by an increased percentage of neuronal differentiation. What's more, over-expression of Hes1 inhibited the neuronal differentiation induced by EFs. CONCLUSION: EF-stimulation directed neuronal differentiation of NSCs by promoting the continuous accumulation of Ascl1 expression and decreasing the expression of Hes1.

RNAi efficiency is enhanced through knockdown of double-stranded RNA-degrading enzymes in butterfly Papilio xuthus.

The efficiency of RNA interference (RNAi) has always limited the research on the phenotype innovation of Lepidoptera insects. Previous studies have found that double-stranded RNA-degrading enzyme (dsRNase) is an important factor in RNAi efficiency, but there have been no relevant reports in butterflies (Papilionoidea). Papilio xuthus is one of the important models in butterflies with an extensive experimental application value. To explore the effect of dsRNase in the RNAi efficiency on butterflies, six dsRNase genes (PxdsRNase 1-6) were identified in P. xuthus genome, and their dsRNA-degrading activities were subsequently detected by ex vivo assays. The result shows that the dsRNA-degrading ability of gut content (<1 h) was higher than hemolymph content (>12 h). We then investigated the expression patterns of these PxdsRNase genes during different tissues and developmental stages, and related RNAi experiments were carried out. Our results show that different PxdsRNase genes had different expression levels at different developmental stages and tissues. The expression of PxdsRNase2, PxdsRNase3, and PxdsRNase6 were upregulated significantly through dsGFP injection, and PxdsRNase genes can be silenced effectively by injecting their corresponding dsRNA. RNAi-of-RNAi studies with PxEbony, which acts as a reporter gene, observed that silencing PxdsRNase genes can increase RNAi efficiency significantly. These results confirm that silencing dsRNase genes can improve RNAi efficiency in P. xuthus significantly, providing a reference for the functional study of insects such as butterflies with low RNAi efficiency.

Enhanced chlorobenzene removal by internal magnetic field through initial cell adhesion and biofilm formation.

Magnetic fields (MF) have been proven efficient in bioaugmentation, and the internal MFs have become competitive because they require no configuration, despite their application in waste gas treatment remaining largely unexplored. In this study, we firstly developed an intensity-regulable bioaugmentation with internal MF for gaseous chlorobenzene (CB) treatment with modified packing in batch bioreactors, and the elimination capacity increased by up to 26%, surpassing that of the external MF. Additionally, the microbial affinity to CB and the packing surface was enhanced, which was correlated with the ninefold increased secreted ratio of proteins/polysaccharides, 43% promoted cell surface hydrophobicity, and half reduced zeta potential. Furthermore, the dehydrogenase content was promoted over 3 times, and CB removal steadily increased with the rising intensity indicating enhanced biofilm activity and reduced CB bioimpedance; this was further supported by kinetic analysis, which resulted in improved cell adhesive ability and biological utilisation of CB. The results introduced a novel concept of adjustable magnetic bioaugmentation and provided technical support for industrial waste gas treatments. KEY POINTS: • Regulable magnetic bioaugmentation was developed to promote 26% chlorobenzene removal • Chlorobenzene mineralisation was enhanced under the magnetic field • Microbial adhesion was promoted through weakening repulsive forces.

A novel MED12 pathogenic variant in a female fetus with facial cleft and cardiac defects identified in the first trimester.

Trio exome sequencing was performed on a female fetus with an increased nuchal translucency, along with nasal bone hypoplasia, suspected cleft palate and abnormal outflow tract of the heart. A de novo heterozygous variant c.5500_5507del, p.(Tyr1834Argfs × 58) in the MED12 gene was detected. Loss-of-function variants in MED12 in females are associated with Hardikar syndrome (HS). A follow-up ultrasound at 15+5 weeks of gestation identified multiple fetal anomalies including bilateral cleft lip and palate, diaphragmatic hernia, atrioventricular septal defect, persistent truncus arteriosus, and bilateral renal pelvis dilation. Fetal autopsy confirmed the prenatal sonographic findings, and the MED12 variant was discussed by our multidisciplinary team to be the cause of fetal anomalies. Our case is the first prenatal one in which HS was diagnosed due to first trimester structural malformations. This case report presents another example of early identification of a major anomaly which allows earlier genetic diagnosis and more time for clinical management.

Phenotypic and genotypic analysis of 11 fetal cases with Bardet-Biedl syndrome.

OBJECTIVE: To present the prenatal sonographic features and genomic spectrum of pregnancies with fetal Bardet-Biedl syndrome (BBS). METHODS: This was a retrospective study of 11 cases with BBS diagnosed by prenatal ultrasound and confirmed by genetic testing. Clinical and laboratory data were collected and reviewed for these cases, including maternal demographics, prenatal sonographic findings, molecular testing sequencing results, and pregnancy outcomes. RESULTS: All cases had unremarkable first-trimester ultrasound scans without reporting limb malformations. All had second-trimester abnormal ultrasounds: postaxial polydactyly in nine cases (9/11), renal abnormalities in seven (7/11), reduced amniotic fluid volume in two (2/11), central nervous system anomalies in two (2/11), and ascites in three (3/11). Ten fetuses presented with at least two-system anomalies, and one (Case 11) presented with only postaxial polydactyly. Variants were detected in five genes, including BBS2, ARL6/BBS3, BBS7, CEP290/BBS14 and IFT74/BBS22. Ten pregnancies were terminated in the second trimester, while one continued to term. CONCLUSION: Enlarged hyperechogenic kidneys and postaxial polydactyly are the two most common sonographic features of fetal BBS. Prenatal diagnosis of BBS can be done with ultrasound and genetic testing although the diagnosis may be made in the second trimester.

Performance, kinetic characteristics and bacterial community of short-cut nitrification and denitrification system at different ferrous ion conditions.

In order to explore the operation performance, kinetic characteristics and bacterial community of the short-cut nitrification and denitrification (SND) system, the SND system with pre-cultured short cut nitrification and denitrification sludge was established and operated under different ferrous ion (Fe (II)) conditions. Experimental results showed that the average NH4+-N removal efficiency (ARE) of SND system was 97.3% on Day 5 and maintained a high level of 94.9% ± 1.3% for a long operation period. When the influent Fe(II) concentration increased from 2.3 to 7.3 mg L-1, the sedimentation performance, sludge concentration and organic matter removal performance were improved. However, higher Fe(II) of 12.3 mg L-1 decreased the removal of nitrogen and CODCr with the relative abundance (RA) of Proteobacteria and Bacteroidetes decreased to 30.28% and 19.41%, respectively. Proteobacteria, Bacteroidetes and Firmicutes were the dominant phyla in SND system. Higher Fe(II) level of 12.3 mg L-1 increase the RA of denitrifying genus Trichococcus (33.93%), and the denitrifying genus Thauera and Tolumonas dominant at Fe(II) level of no more than 7.3 mg L-1.

A2AR-mediated lymphangiogenesis via VEGFR2 signaling prevents salt-sensitive hypertension.

AIMS: Excess dietary sodium intake and retention lead to hypertension. Impaired dermal lymphangiogenesis and lymphatic dysfunction-mediated sodium and fluid imbalance are pathological mechanisms. The adenosine A2A receptor (A2AR) is expressed in lymphatic endothelial cells (LECs), while the roles and mechanisms of LEC-A2AR in skin lymphangiogenesis during salt-induced hypertension are not clear. METHODS AND RESULTS: The expression of LEC-A2AR correlated with lymphatic vessel density in both high-salt diet (HSD)-induced hypertensive mice and hypertensive patients. Lymphatic endothelial cell-specific A2AR knockout mice fed HSD exhibited 17 ± 2% increase in blood pressure and 17 ± 3% increase in Na+ content associated with decreased lymphatic density (-19 ± 2%) compared with HSD-WT mice. A2AR activation by agonist CGS21680 increased lymphatic capillary density and decreased blood pressure in HSD-WT mice. Furthermore, this A2AR agonist activated MSK1 directly to promote VEGFR2 activation and endocytosis independently of VEGF as assessed by phosphoprotein profiling and immunoprecipitation assays in LECs. VEGFR2 kinase activity inhibitor fruquintinib or VEGFR2 knockout in LECs but not VEGF-neutralizing antibody bevacizumab suppressed A2AR activation-mediated decrease in blood pressure. Immunostaining revealed phosphorylated VEGFR2 and MSK1 expression in the LECs were positively correlated with skin lymphatic vessel density and A2AR level in hypertensive patients. CONCLUSION: The study highlights a novel A2AR-mediated VEGF-independent activation of VEGFR2 signaling in dermal lymphangiogenesis and sodium balance, which might be a potential therapeutic target in salt-sensitive hypertension.

Single-cell expression profile of Drosophila ovarian follicle stem cells illuminates spatial differentiation in the germarium.

BACKGROUND: How stem cell populations are organized and regulated within adult tissues is important for understanding cancer origins and for developing cell replacement strategies. Paradigms such as mammalian gut stem cells and Drosophila ovarian follicle stem cells (FSC) are characterized by population asymmetry, in which stem cell division and differentiation are separately regulated processes. These stem cells behave stochastically regarding their contributions to derivative cells and also exhibit dynamic spatial heterogeneity. Drosophila FSCs provide an excellent model for understanding how a community of active stem cells maintained by population asymmetry is regulated. Here, we use single-cell RNA sequencing to profile the gene expression patterns of FSCs and their immediate derivatives to investigate heterogeneity within the stem cell population and changes associated with differentiation. RESULTS: We describe single-cell RNA sequencing studies of a pre-sorted population of cells that include FSCs and the neighboring cell types, escort cells (ECs) and follicle cells (FCs), which they support. Cell-type assignment relies on anterior-posterior (AP) location within the germarium. We clarify the previously determined location of FSCs and use spatially targeted lineage studies as further confirmation. The scRNA profiles among four clusters are consistent with an AP progression from anterior ECs through posterior ECs and then FSCs, to early FCs. The relative proportion of EC and FSC clusters are in good agreement with the prevalence of those cell types in a germarium. Several genes with graded profiles from ECs to FCs are highlighted as candidate effectors of the inverse gradients of the two principal signaling pathways, Wnt and JAK-STAT, that guide FSC differentiation and division. CONCLUSIONS: Our data establishes an important resource of scRNA-seq profiles for FSCs and their immediate derivatives that is based on precise spatial location and functionally established stem cell identity, and facilitates future genetic investigation of regulatory interactions guiding FSC behavior.

RNA-seq revealed the protective effect of Huangqi Guizhi Wuwu Decoction against cisplatin induced PC12 cell injury.

BACKGROUND: Chemotherapy-induced peripheral neuropathy not only affects the tolerability of chemotherapy, but also causes intolerable and prolonged neuropathic pain in cancer patients. Currently, duloxetine is the only drug used to treat chemotherapy-induced peripheral neuropathy. However, the clinical use of this drug still faces several challenges. Therefore, we focused on traditional Chinese medicine to find an effective and safe alternative medicine. Huangqi Guizhi Wuwu Decoction is a traditional Chinese medicine that has been clinically used for treating nerve pain for thousands of years. This study aimed to investigate the neuroprotective effect of Huangqi Guizhi Wuwu Decoction on cisplatin-induced nerve injury in PC12 cells and to elucidate its potential mechanism of action. METHODS: Huangqi Guizhi Wuwu Decoction-containing serum and blank serum were prepared from a rat model. The protective effects of Huangqi Guizhi Wuwu Decoction on cisplatin (10 µmol/L)-induced PC12 cell injury were assessed by a Cell Counting Kit-8 assay. RNA expression in Huangqi Guizhi Wuwu Decoction-protected PC12 cells was analyzed using RNA-seq, and subsequently, differentially expressed genes were further analyzed using Gene Ontology and Gene Set Enrichment Analysis. RESULTS: The Cell Counting Kit-8 results showed that pretreatment of PC12 cells with Huangqi Guizhi Wuwu Decoction-containing serum (5%, 10%, 15%) significantly increased cells' viability to 10 µmol/L cisplatin-induced cell death. RNA-seq analysis revealed 843 differentially expressed genes in the chemotherapy-induced peripheral neuropathy group and 249 in the Huangqi Guizhi Wuwu Decoction group. The gene set enrichment analysis results in this study suggest that Huangqi Guizhi Wuwu Decoction may treat chemotherapy-induced peripheral neuropathy by enhancing axon guidance. CONCLUSIONS: This study provides valuable evidence for using Huangqi Guizhi Wuwu Decoction in treating chemotherapy-induced peripheral neuropathy, partially achieved by improving axon guidance pathways.

Fetal Phenotype of CHARGE Syndrome with a Molecular Confirmation: A Series of 13 Cases.

INTRODUCTION: CHARGE syndrome is an autosomal dominant genetic disorder with known pattern of features. The aim of the study was to present the fetal features of CHARGE syndrome to gain awareness that the antenatal characteristics can be very nonspecific. CASE PRESENTATION: This was a retrospective study of 13 cases with CHARGE syndrome diagnosed by prenatal or postnatal genetic testing and physical examination. Two (15.4%; 2/13) had normal ultrasound scans during pregnancy. One (7.7%; 1/13) with first-trimester cystic hygroma presented intrauterine fetal demise at 16 weeks gestation. The remaining 10 (76.9%; 10/13) cases had abnormal ultrasound features in utero; among these, 1 had an increased nuchal translucency in the first trimester, 5 had second-trimester abnormal ultrasounds including micrognathia, cardiac defects, and facial defects, and 4 third-trimester abnormal ultrasounds including micrognathia, isolated fetal growth restriction, and polyhydramnios. Among the 11 cases with abnormal prenatal ultrasound scans, no fetus could reach the diagnostic criteria of CHARGE syndrome if only based on the results of ultrasound. However, the diagnosis was made in all cases when CHD7 defects were detected. DISCUSSION/CONCLUSION: The CHARGE syndrome presents non-specific abnormal ultrasound markers in utero. Exome sequencing in the genetic workup will aid in prenatal diagnosis of this syndrome.

Early Onset of Severe Anemia Caused by Hb Calgary (HBB: C.194G > T): Another Case Report.

Unstable hemoglobin (Hb) variants are a rare cause of congenital hemolytic anemia. We describe a Chinese girl who presented with transfusion-dependent anemia in early infancy. Her diagnosis of Hb Calgary [ß64(E8)Gly > Val; HBB:c.194G > T] was not made until molecular testing was performed at the age of 5 years. Our case highlights the importance of early genetic testing in order to make the diagnosis, which may not only be useful for patient management and family counseling, but also for avoiding further unnecessary investigative attempts.

A 6-Year Follow-up of a Chinese Child with Homozygous ß0-Thalaasemia and a Heterozygous KLF1 Mutation.

Patients with the genotype of ß0/ß0 for ß-thalassemia (ß-thal) usually behave as ß-thal major (ß-TM) phenotype which is transfusion-dependent. The pathophysiology of ß-thal is the imbalance between α/ß-globin chains. The degree of α/ß-globin imbalance can be reduced by the more effective synthesis of γ-globin chains, and increased Hb F levels, modifying clinical severity of ß-TM. We report a Chinese child who had homozygous ß0-thal and a heterozygous KLF1 mutation. The patient had a moderate anemia since 6 months old, keeping a baseline Hb value of 8.0-9.0 g/dL. She had normal development except for a short stature (3rd percentile) until 6 years old, when splenomegaly and facial bone deformities occurred. Although genetic alteration of KLF1 expression in ß0/ß0 patients can result in some degree of disease alleviation, our case shows that it is insufficient to ameliorate satisfactorily the presentation. This point should be borne in mind for physicians who provide the genetic counseling and prenatal diagnosis to at-risk families.