Patient- and caregiver-reported factors associated with school absenteeism in children with chronic kidney disease.

BACKGROUND: Children with chronic kidney disease (CKD) are at risk for neurocognitive deficits while simultaneously being at risk for chronic school absenteeism (≥ 18 school days per school year). Chronic school absenteeism compounds the negative impacts of CKD on academic achievement. In this study, we examined patient- and caregiver-reported factors associated with school absenteeism in children with non-dialysis- or transplant-dependent CKD in order to help identify which factors could be modifiable and ultimately improve school attendance. METHODS: We utilized a combination of chart review and questionnaires distributed in person to patients and caregivers at a pediatric nephrology clinic between November 2018 and August 2019 to gather data. We used descriptive statistics to illustrate clinical characteristics of the children included in the study, caregiver characteristics, and examined reported reasons for missing school. RESULTS: Twenty-one percent of participants (10/48) missed 18 full days of school or more, categorizing them as chronically absent. The top three reasons for missing school were doctor appointments, feeling sick, and being bullied. More specific sequelae of CKD were not highly reported as reasons for missing school. CONCLUSIONS: Chronic absenteeism is a highly reported phenomenon among children with pediatric CKD. Given that missing school for doctor appointments was a top reason for absenteeism, this data suggests alternative appointment hours and virtual appointments may reduce chronic school absenteeism in children, and by extension improve their health, behavioral, and academic outcomes. A higher resolution version of the Graphical abstract is available as Supplementary information.

Discovery of a gene involved in a third bacterial protoporphyrinogen oxidase activity through comparative genomic analysis and functional complementation.

Tetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained "missing" in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizing Acinetobacter baylyi ADP1, to identify one of the missing enzymes, a new protoporphyrinogen oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known protoporphyrinogen oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome of A. baylyi, a bacterium that lacks both hemG and hemY, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked protoporphyrinogen oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne protoporphyrinogen oxidase gene from a number of different organisms, such as hemG from Escherichia coli, hemY from Myxococcus xanthus, or the human gene for protoporphyrinogen oxidase.

Full-length structures of BenM and two variants reveal different oligomerization schemes for LysR-type transcriptional regulators.

BenM, a LysR-type transcriptional regulator (LTTR) from the bacterium Acinetobacter baylyi, responds synergistically to benzoate and cis,cis-muconate. With these effectors, BenM activates gene expression during benzoate consumption. Without effectors, BenM represses transcription. Here, X-ray crystallography was used to determine the full-length structures of BenM and two variants that activate transcription without benzoate or cis,cis-muconate: BenM(R156H) and BenM(E226K). Previous studies indicate that these regulators function as tetramers. Here, interconnections between subunits in the crystals prevented the formation of a closed oligomer and highlighted the inherent flexibility of this multidomain regulator. Nevertheless, analysis of subunit interfaces suggested the functional significance of key interactions. The structures of BenM and its variants were nearly identical, implying that transcriptional differences rely on factors beyond major conformational changes defined solely by sequence. Comparisons of BenM with other LTTRs, including unpublished structures in the Protein Data Bank, revealed extensive variation in the relative orientations of DNA-binding domains (DBDs) and effector-binding domains (EBDs). To form dimers, different LTTRs used similar interfaces between two EBDs, each containing two subdomains: EBD-I and EBD-II. Surprisingly, the dimers used three substantially different schemes to form higher-order oligomers. In one scheme used by BenM, oligomer assembly involved contacts between the EBD-II regions and the DBD regions of adjacent subunits. In another scheme, there were no contacts between the EBDs; only the DBDs were involved in tetramer formation. In the third scheme, the oligomer interface involved DBD and EBD-I/EBD-II contacts. These diverse schemes demonstrate novel variation in the oligomeric structures of individual LTTRs within this large and important family.

Inducer responses of BenM, a LysR-type transcriptional regulator from Acinetobacter baylyi ADP1.

BenM and CatM control transcription of a complex regulon for aromatic compound degradation. These Acinetobacter baylyi paralogues belong to the largest family of prokaryotic transcriptional regulators, the LysR-type proteins. Whereas BenM activates transcription synergistically in response to two effectors, benzoate and cis,cis-muconate, CatM responds only to cis,cis-muconate. Here, site-directed mutagenesis was used to determine the physiological significance of an unexpected benzoate-binding pocket in BenM discovered during structural studies. Residues in BenM were changed to match those of CatM in this hydrophobic pocket. Two BenM residues, R160 and Y293, were found to mediate the response to benzoate. Additionally, alteration of these residues caused benzoate to inhibit activation by cis,cis-muconate, positioned in a separate primary effector-binding site of BenM. The location of the primary site, in an interdomain cleft, is conserved in diverse LysR-type regulators. To improve understanding of this important family, additional regulatory mutants were analysed. The atomic-level structures were characterized of the effector-binding domains of variants that do not require inducers for activation, CatM(R156H) and BenM(R156H,T157S). These structures clearly resemble those of the wild-type proteins in their activated muconate-bound complexes. Amino acid replacements that enable activation without effectors reside at protein interfaces that may impact transcription through effects on oligomerization.

Double trouble: medical implications of genetic duplication and amplification in bacteria.

Gene amplification allows organisms to adapt to changing environmental conditions. This type of increased gene dosage confers selectable benefits, typically by augmenting protein production. Gene amplification is a reversible process that does not require permanent genetic change. Although transient, altered gene dosage has significant medical impact. Recent examples of amplification in bacteria, described here, affect human disease by modifying antibiotic resistance, the virulence of pathogens, vaccine efficacy and antibiotic biosynthesis. Amplification is usually a two-step process whereby genetic duplication (step one) promotes further increases in copy number (step two). Both steps have important evolutionary significance for the emergence of innovative gene functions. Recent genome sequence analyses illustrate how genome plasticity can affect the evolution and immunogenic properties of bacterial pathogens.

CD8+ T-Cell responses to Trypanosoma cruzi are highly focused on strain-variant trans-sialidase epitopes.

CD8+ T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8+ T cells in T. cruzi-infected mice and humans. This analysis demonstrates that in both hosts the CD8+ T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi-infected mice, with more than 30% of the CD8+ T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level.

Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region.

The paired domain transcription factor Pax2 is required for the formation of the isthmic organizer (IsO) at the midbrain-hindbrain boundary, where it initiates expression of the IsO signal Fgf8. To gain further insight into the role of Pax2 in mid-hindbrain patterning, we searched for novel Pax2-regulated genes by cDNA microarray analysis of FACS-sorted GFP+ mid-hindbrain cells from wild-type and Pax2-/- embryos carrying a Pax2(GFP) BAC transgene. Here, we report the identification of five genes that depend on Pax2 function for their expression in the mid-hindbrain boundary region. These genes code for the transcription factors En2 and Brn1 (Pou3f3), the intracellular signaling modifiers Sef and Tapp1, and the non-coding RNA Ncrms. The Brn1 gene was further identified as a direct target of Pax2, as two functional Pax2-binding sites in the promoter and in an upstream regulatory element of Brn1 were essential for lacZ transgene expression at the mid-hindbrain boundary. Moreover, ectopic expression of a dominant-negative Brn1 protein in chick embryos implicated Brn1 in Fgf8 gene regulation. Together, these data defined novel functions of Pax2 in the establishment of distinct transcriptional programs and in the control of intracellular signaling during mid-hindbrain development.

Loss of Hspa9b in zebrafish recapitulates the ineffective hematopoiesis of the myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) comprises a heterogeneous group of often fatal hematopoietic stem cell disorders for which neither curative nor standard treatment exists. The complex karyotypes and multistep nature of MDS have severely restricted the identification of causative genetic mutations and thus limited insight into new and more effective therapies. Here we describe a zebrafish mutant crimsonless (crs) with a developmental blood defect that closely recapitulates the ineffective hematopoiesis of MDS including anemia, dysplasia, increased blood cell apoptosis, and multilineage cytopenia. By positional cloning, rescue, and morpholino knockdown experiments, we demonstrate that crs encodes a conserved mitochondrial matrix chaperone HSPA9B containing a glycine-to-glutamate substitution within the substrate-binding domain. This mutation compromises mitochondrial function, producing oxidative stress and apoptosis distinctly in blood cells. Thus, we identify an essential role for Hspa9b in hematopoiesis and implicate both loss of HSPA9B specifically and mitochondrial dysfunction generally in the pathogenesis of the MDS.

Gata2 specifies serotonergic neurons downstream of sonic hedgehog.

Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebrate hindbrain. Here, we identify a Sonic hedgehog (Shh)-regulated cascade of transcription factors that acts to generate a specific subset of 5-HT neurons. This transcriptional cascade is sufficient for the induction of rostral 5-HT neurons within rhombomere 1 (r1), which project to the forebrain, but not for the induction of caudal 5-HT neurons, which largely terminate in the spinal cord. Within the rostral hindbrain, the Shh-activated homeodomain proteins Nkx2.2 and Nkx6.1 cooperate to induce the closely related zinc-finger transcription factors Gata2 and Gata3. Gata2 in turn is necessary and sufficient to activate the transcription factors Lmx1b and Pet1, and to induce 5-HT neurons within r1. In contrast to Gata2, Gata3 is not required for the specification of rostral 5-HT neurons and appears unable to substitute for the loss of Gata2. Our findings reveal that the identity of closely related 5-HT subclasses occurs through distinct responses of adjacent rostrocaudal progenitor domains to broad ventral inducers.

Lipid- and protein-mediated multimerization of PSD-95: implications for receptor clustering and assembly of synaptic protein networks.

Postsynaptic density protein 95 (PSD-95/SAP-90) is a palmitoylated membrane-associated guanylate kinase that oligomerizes and clusters ion channels and associated signaling machinery at excitatory synapses in brain. However, the mechanism for PSD-95 oligomerization and its relationship to ion channel clustering remain uncertain. Here, we find that multimerization of PSD-95 is determined by only its first 13 amino acids, which also have a remarkable capacity to oligomerize heterologous proteins. Multimerization does not involve a covalent linkage but rather palmitoylation of two cysteine residues in the 13 amino acid motif. This lipid-mediated oligomerization is a specific property of the PSD-95 motif, because it is not observed with other palmitoylated domains. Clustering K+ channel Kv1.4 requires interaction of palmitoylated PSD-95 with tetrameric K+ channel subunits but, surprisingly, does not require multimerization of PSD-95. Finally, disrupting palmitoylation with 2-bromopalmitate disperses PSD-95/K+-channel clusters. These data suggest new models for K+ channel clustering by PSD-95 - a reversible process regulated by protein palmitoylation.