Pathogenic FLNA variants affecting the hinge region of filamin A are associated with male survival.

Pathogenic variants in FLNA cause a diversity of X-linked developmental disorders associated with either preserved or diminished levels of filamin A protein and are conceptualized dichotomously as relating to underlying gain- or loss-of-function pathogenic mechanisms. Hemizygosity for germline deletions or truncating variants in FLNA is generally considered to result in embryonic lethality. Structurally, filamin A is composed of an N-terminal actin-binding region, followed by 24 immunoglobulin-like repeat units. The repeat domains are separated into distinct segments by two regions of low-complexity known as hinge-1 and hinge-2. Hinge-1 is proposed to confer flexibility to the otherwise rigid protein and is a target for cleavage by calpain with the resultant filamin fragments mediating crucial cellular signaling processes. Here, three families with pathogenic variants in FLNA that impair the function of hinge-1 in males are described, leading to distinct clinical phenotypes. One large in-frame deletion that includes the hinge leads to frontometaphyseal dysplasia in affected males and females, while two germline truncating variants located within the exon encoding hinge 1 result in phenotypes in males that are explained by exon skipping and under-expression of a transcript that deletes hinge-1 from the resultant protein. These three variants affecting hinge-1 indicate that this domain does not mediate cellular functions that, when deficientresult in embryonic lethality in males and that germline truncating variants in this region of FLNA can result in viable phenotypes in males.

Impact of the COVID-19 Pandemic on Latino Families With Alzheimer Disease and Related Dementias: Qualitative Interviews With Family Caregivers and Primary Care Providers.

Background: Latino individuals experience disparities in the care of Alzheimer disease and related dementias (ADRD) and have disproportionately high COVID-19 infection and death outcomes. Objective: We aimed to gain an in-depth understanding of the impact of the COVID-19 pandemic among Latino families with ADRD in the United States. Methods: This was a qualitative study of 21 informal caregivers of Latino individuals with ADRD and 23 primary care providers who serve Latino patients. We recruited participants nationwide using convenience and snowball sampling methods and conducted remote interviews in English and Spanish. We organized the transcripts for qualitative review to identify codes and themes, using a pragmatic approach, a qualitative description methodology, and thematic analysis methods. Results: Qualitative analysis of transcripts revealed eight themes, including (1) the pandemic influenced mental and emotional health; (2) the pandemic impacted physical domains of health; (3) caregivers and care recipients lost access to engaging activities during the confinement; (4) the pandemic impacted Latino caregivers' working situation; (5) the pandemic impacted health care and community care systems; (6) health care and community care systems took measures to reduce the impact of the pandemic; (7) Latino families experienced barriers to remote communication during the pandemic; and (8) caregiver social support was critical for reducing social isolation and its sequalae. Conclusions: Latino families with ADRD experienced similar but also unique impacts compared to those reported in the general population. Unique impacts may result from Latino individuals' underserved status in the United States, commonly held cultural values, and their intersectionality with ADRD-related disability. Family caregiver social support was crucial during this time of adversity. These findings suggest the need for more equitable access, culturally appropriate and trustworthy content and delivery of health care and community services, as well as stronger financial and social supports for family caregivers.

Mycoplasma synoviae lipid-associated membrane proteins identification and expression changes when exposed to chicken cells.

Mycoplasma synoviae is a significant cause of respiratory disease and synovitis among chickens, and has an adverse economic impact on broiler breeding efforts. The present study was designed to develop a systematic understanding of the role that M. synoviae lipid-associated membrane proteins (LAMPs) may play in the virulence of this pathogen. Bioinformatics tools were used to identify 146 predicted membrane proteins and lipoproteins in the M. synoviae proteome. Then, Triton X-114 was used to extract LAMPs that were subsequently identified via LC-MS/MS. This approach enabled the detection of potential LAMPs, and the top 200 most abundant proteins detected using this strategy were subject to further analysis. M. synoviae cells (100 MOI) were exposed to chicken fibroblasts (DF-1) and macrophages (HD-11) in a 1:1 mixed culture. Analysis of LAMP transcripts identified 72 up-regulated LAMP genes which were analyzed in depth by bioinformatics. GO analysis revealed these genes to be enriched in the nucleotide binding, sulfur amino acid transmembrane transporter activity, tRNA binding, rRNA modification, and transition metal ion transport pathways. Moreover, KEGG enrichment analysis suggested that these genes were enriched in the biosynthesis of secondary metabolites, carbon metabolism, glycolysis/gluconeogenesis, and nitrogen metabolism pathways.

Spinal muscular atrophy type I associated with a novel SMN1 splicing variant that disrupts the expression of the functional transcript.

Introduction: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by pathogenic variants in the SMN1 gene. The majority of SMA patients harbor a homozygous deletion of SMN1 exon 7 (95%). Heterozygosity for a conventional variant and a deletion is rare (5%) and not easily detected, due to the highly homologous SMN2 gene interference. SMN2 mainly produces a truncated non-functional protein (SMN-d7) instead of the full-length functional (SMN-FL). We hereby report a novel SMN1 splicing variant in an infant with severe SMA. Methods: MLPA was used for SMN1/2 exon dosage determination. Sanger sequencing approaches and long-range PCR were employed to search for an SMN1 variant. Conventional and improved Real-time PCR assays were developed for the qualitative and quantitative SMN1/2 RNA analysis. Results: The novel SMN1 splice-site variant c.835-8_835-5delinsG, was identified in compound heterozygosity with SMN1 exons 7/8 deletion. RNA studies revealed complete absence of SMN1 exon 7, thus confirming a disruptive effect of the variant on SMN1 splicing. No expression of the functional SMN1-FL transcript, remarkable expression of the SMN1-d7 and increased levels of the SMN2-FL/SMN2-d7 transcripts were observed. Discussion: We verified the occurrence of a non-deletion SMN1 variant and supported its pathogenicity, thus expanding the SMN1 variants spectrum. We discuss the updated SMA genetic findings in the Cypriot population, highlighting an increased percentage of intragenic variants compared to other populations.

Selection and Validation of Optimal RT-qPCR Reference Genes for the Normalization of Gene Expression under Different Experimental Conditions in Lindera megaphylla.

Lindera megaphylla, a broad-leaved evergreen that is used as a landscape ornamental plant and medicinal plant, is an ecologically important and dominant tree species. However, little is known about the molecular mechanisms of its growth, development, and metabolism. The selection of suitable reference genes is critical for molecular biological analyses. To date, no research on reference genes as a foundation for gene expression analysis has been undertaken in L. megaphylla. In this study, 14 candidate genes were selected from the transcriptome database of L. megaphylla for RT-qPCR assay under different conditions. Results showed that helicase-15 and UBC28 were most stable in different tissues of seedlings and adult trees. For different leaf developmental stages, the best combination of reference genes was ACT7 and UBC36. UBC36 and TCTP were the best under cold treatment, while PAB2 and CYP20-2 were the best under heat treatment. Finally, a RT-qPCR assay of LmNAC83 and LmERF60 genes were used to further verify the reliability of selected reference genes above. This work is the first to select and evaluate the stability of reference genes for the normalization of gene expression analysis in L. megaphylla and will provide an important foundation for future genetic studies of this species.

Molecular Analysis of the Global Population of Potato Virus S Redefines Its Phylogeny, and Has Crop Biosecurity Implications.

In 2020, 264 samples were collected from potato fields in the Turkish provinces of Bolu, Afyon, Kayseri and Nigde. RT-PCR tests, with primers which amplified its coat protein (CP), detected potato virus S (PVS) in 35 samples. Complete CP sequences were obtained from 14 samples. Phylogenetic analysis using non-recombinant sequences of (i) the 14 CP's, another 8 from Tokat province and 73 others from GenBank; and (ii) 130 complete ORF, RdRp and TGB sequences from GenBank, found that they fitted within phylogroups, PVSI, PVSII or PVSIII. All Turkish CP sequences were in PVSI, clustering within five subclades. Subclades 1 and 4 were in three to four provinces, whereas 2, 3 and 5 were in one province each. All four genome regions were under strong negative selection constraints (ω = 0.0603-0.1825). Considerable genetic variation existed amongst PVSI and PVSII isolates. Three neutrality test methods showed PVSIII remained balanced whilst PVSI and PVSII underwent population expansion. The high fixation index values assigned to all PVSI, PVSII and PVSIII comparisons supported subdivision into three phylogroups. As it spreads more readily by aphid and contact transmission, and may elicit more severe symptoms in potato, PVSII spread constitutes a biosecurity threat for countries still free from it.

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris).

BACKGROUND: The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. RESULTS: We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. CONCLUSION: The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.

Genome-wide analysis and identification of microRNAs in Medicago truncatula under aluminum stress.

Numerous studies have shown that plant microRNAs (miRNAs) play key roles in plant growth and development, as well as in response to biotic and abiotic stresses; however, the role of miRNA in legumes under aluminum (Al) stress have rarely been reported. Therefore, here, we aimed to investigate the role of miRNAs in and their mechanism of Al tolerance in legumes. To this end, we sequenced a 12-strand-specific library of Medicago truncatula under Al stress. A total of 195.80 M clean reads were obtained, and 876 miRNAs were identified, of which, 673 were known miRNAs and 203 were unknown. A total of 55 miRNAs and their corresponding 2,502 target genes were differentially expressed at various time points during Al stress. Further analysis revealed that mtr-miR156g-3p was the only miRNA that was significantly upregulated at all time points under Al stress and could directly regulate the expression of genes associated with root cell growth. Three miRNAs, novel_miR_135, novel_miR_182, and novel_miR_36, simultaneously regulated the expression of four Al-tolerant transcription factors, GRAS, MYB, WRKY, and bHLH, at an early stage of Al stress, indicating a response to Al stress. In addition, legume-specific miR2119 and miR5213 were involved in the tolerance mechanism to Al stress by regulating F-box proteins that have protective effects against stress. Our results contribute to an improved understanding of the role of miRNAs in Al stress in legumes and provide a basis for studying the molecular mechanisms of Al stress regulation.

Genome-Wide Identification and Characterisation of Stress-Associated Protein Gene Family to Biotic and Abiotic Stresses of Grapevine.

Grapevine is one of the earliest domesticated fruit crops and prized for its table fruits and wine worldwide. However, the concurrence of a number of biotic/abiotic stresses affects their yield. Stress-associated proteins (SAPs) play important roles in response to both biotic and abiotic stresses in plants. Despite the growing number of studies on the genomic organisation of SAP gene family in various species, little is known about this family in grapevines (Vitis vinifera L.). In this study, a total of 15 genes encoding proteins possessing A20/AN1 zinc-finger were identified based on the analysis of several genomic and proteomic grapevine databases. According to their structural and phylogenetics features, the identified SAPs were classified into three main groups. Results from sequence alignments, phylogenetics, genomics structure and conserved domains indicated that grapevine SAPs are highly and structurally conserved. In order to shed light on their regulatory roles in growth and development, as well as the responses to biotic/abiotic stresses in grapevine, the expression profiles of SAPs were examined in publicly available microarray data. Bioinformatics analysis revealed distinct temporal and spatial expression patterns of SAPs in various tissues, organs and developmental stages, as well as in response to biotic/abiotic stresses. This study provides insight into the evolution of SAP genes in grapevine and may aid in efforts for further functional identification of A20/AN1-type proteins in the signalling cross-talking induced by biotic/abiotic stresses.

The tobacco chloroplast YCF4 gene is essential for transcriptional gene regulation and plants photoautotrophic growth.

A tobacco chloroplast hypothetical open reading frame 4 (YCF4) has been reported as a non-essential assembly factor for photosynthesis based on an incomplete knockout of YCF4, just 93 of 184 amino acids from the N-terminus were knocked out. On the other hand, we removed the complete sequence of YCF4 from tobacco chloroplasts and observed that ΔYCF4 plants were unable to survive photoautotrophically as their growth was hampered in the absence of an external carbon supply, clearly showing that the YCF4 is essential for photosynthesis. Initially, the aadA gene was introduced into the tobacco plastome replacing the complete YCF4 gene through homologous recombination events. The replacement of YCF4 with aadA was confirmed by PCR and Southern blot analysis in ΔYCF4 plants. Homoplasmic ΔYCF4 plants had a light green phenotype, and the leaves became pale yellow as the plants grew older. The structure of chloroplasts of ΔYCF4 mutants of light green phenotype was studied using a transmission electron microscope (TEM), and the micrographs demonstrated structural anomalies in the chloroplasts; including shape, size, and grana stacking compared to the wild-type plants. Further, transcriptome analysis revealed that the expression of PSI, PSII, and ribosomal genes remained unchanged in ∆YCF4 plants. On the other hand, transcriptome levels of rbcL (Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit), LHC (Light-Harvesting Complex), and ATP Synthase (atpB and atpL) decreased, indicating that the YCF4 has the function(s) in addition to assembling the photosynthetic complex. This was confirmed by in-silico protein-protein interactions of full-length YCF4 as well as 93 and 91 of 184 amino acids from N- and C-termini of the full-length protein, which revealed that the C-terminus (91 aa) of YCF4 is important in interacting with other chloroplast proteins. These findings provide genetic support for the plastid YCF4 gene's critical role in regulating the plastid gene expression and assembling the photosynthetic complex.

Charged Gold Nanoparticles Promote In Vitro Proliferation in Nardostachys jatamansi by Differentially Regulating Chlorophyll Content, Hormone Concentration, and Antioxidant Activity.

Nardostachys jatamansi is a critically endangered medicinal plant and endemic to the Himalayas, having high commercial demand globally. The accumulation of various secondary metabolites in its shoots and roots with antioxidant potential are well-documented in traditional as well as modern medicine systems. In the present study, we first attempted to investigate the impact of citrate (-ve charge, 11.1 ± 1.9 nm) and CTAB (+ve charge, 19.5 ± 3.2 nm) coated gold nanoparticles (AuNPs) on the in vitro proliferation and antioxidant activities of N. jatamansi. Both the nanoparticles differentially affected the morphological and biochemical parameters, chlorophyll content, internal hormone concentration, and antioxidant activities in a concentration-dependent (10-100 µM) manner. Vigorous shooting was observed in half strength MS medium supplemented with IAA (1 mg/L) with 60 µM citrate-AuNPs (46.4 ± 3.7 mm) and 40 µM CTAB-AuNPs (42.2 ± 3.2 mm). Similarly, the maximum number of roots (5.00 ± 0.67 and 5.33 ± 0.58) and root length (29.9 ± 1.5 mm and 27.3 ± 4.8 mm) was reported in half-strength MS medium with IAA (1 mg/L) supplemented with 60 µM citrate-AuNPs and 40 µM CTAB-AuNPs, respectively. In addition, plants growing on MS medium supplemented with 60 µM citrate-AuNPs and 40 µM CTAB-AuNPs showed significantly enhanced photosynthetic pigments (chlorophyll a and b, carotenoids, and total chlorophyll), internal hormone concentration (GA3, IAA, and ABA), and antioxidant activities (total phenolics, flavonoids, DPPH, and SOD enzyme activity). Moreover, the transcript analysis of ANR1, ARF18, PLY9, SAUR28, GID1A, GRF1, SOD, and CAT further confirmed the role of 60 µM citrate-AuNPs and 40 µM CTAB-AuNPs in the improvement in the growth and antioxidant activities of N. jatamansi. Bearing in mind the urgent requirements of the effective conservation measures of this endangered species, the present findings suggest the elicitation of citrate-AuNPs and CTAB-AuNPs would significantly improve the potential applications of N. jatamansi in the medicinal plant-based industry.

Transcript analysis and expression of the glbO gene, encoding truncated hemoglobin,O, of M. Smegmatis implicate its role under hypoxia and oxidative stress.

Although truncated hemoglobin O, (trHbO), is ubiquitous among mycobacteria, its physiological function is not very obvious and may be diverse. In an attempt to understand role of trHbO in cellular metabolism of a non-pathogenic mycobacterium, we analysed expression profile of the glbO gene, encoding trHbO, in M. smegmatis and studied implications of its overexpression on physiology of its host under different environmental conditions. Quantitative RT-PCR indicated that transcript level of the glbO gene remains low at a basal level under aerobic growth cycle of M. smegmatis but its level gets induced significantly during low oxygen, oxidative stress and macrophage infection. Overexpression of the glbO gene enhanced growth of M. smegmatis under hypoxia, promoted pellicle biofilm formation and provided resistance towards oxidative stress. Additionally, glbO gene overexpressing M. smegmatis exhibited enhanced cell survival over isogenic control cells and altered the level of pro- and anti- inflammatory cytokines during intracellular infection. These results suggested important role of trHbO, in supporting the cellular metabolism and survival of M, smegmatis both under low oxygen and oxidative stress.

Spatial Resolution of Mycobacterium tuberculosis Bacteria and Their Surrounding Immune Environments Based on Selected Key Transcripts in Mouse Lungs.

Mycobacterium tuberculosis (Mtb) bacilli are the causative agent of tuberculosis (TB), a major killer of mankind. Although it is widely accepted that local interactions between Mtb and the immune system in the tuberculous granuloma determine whether the outcome of infection is controlled or disseminated, these have been poorly studied due to methodological constraints. We have recently used a spatial transcriptomic technique, in situ sequencing (ISS), to define the spatial distribution of immune transcripts in TB mouse lungs. To further contribute to the understanding of the immune microenvironments of Mtb and their local diversity, we here present two complementary automated bacteria-guided analysis pipelines. These position 33 ISS-identified immune transcripts in relation to single bacteria and bacteria clusters. The analysis was applied on new ISS data from lung sections of Mtb-infected C57BL/6 and C3HeB/FeJ mice. In lungs from C57BL/6 mice early and late post infection, transcripts that define inflammatory macrophages were enriched at subcellular distances to bacteria, indicating the activation of infected macrophages. In contrast, expression patterns associated to antigen presentation were enriched in non-infected cells at 12 weeks post infection. T-cell transcripts were evenly distributed in the tissue. In Mtb-infected C3HeB/FeJ mice, transcripts characterizing activated macrophages localized in apposition to small bacteria clusters, but not in organized granulomas. Despite differences in the susceptibility to Mtb, the transcript patterns found around small bacteria clusters of C3HeB/FeJ and C57BL/6 mice were similar. Altogether, the presented tools allow us to characterize in depth the immune cell populations and their activation that interact with Mtb in the infected lung.

Mechanism of Sugarbeet Seed Germination Enhanced by Hydrogen Peroxide.

Seed germination is a critical first stage of plant development but can be arrested by factors including dormancy and environmental conditions. Strategies to enhance germination are of interest to plant breeders to ensure the ability to utilize the genetic potential residing inside a dormant seed. In this study, seed germination in two sugarbeet (Beta vulgaris ssp. vulgaris L.) lines F1004 and F1015 through incubating seeds in hydrogen peroxide (H2O2) solution was improved over 70% relative to germinating seeds through water incubation. It was further found that low germination from water incubation was caused by physical dormancy in F1015 seeds with initial seed imbibition blocked by the seed pericarp, and physiological dormancy in F1004 seeds with germination compromised due to the physiological condition of the embryo. To identify genes that are differentially expressed in response to cellular activities promoted by H2O2 during overcoming different type of dormancies, an RNA-Seq study was carried out and found H2O2 treatment during germination accelerated the degradation of seed stored mRNAs that were synthesized before or during seed storage to provide protections and maintain the dormant state. Comparison of transcripts in H2O2-treated seeds between the two sugarbeet lines identified differentially expressed genes (DEGs) that were higher in F1004 for alleviating physiological dormancy were known to relative to gene expression regulation. The research established that H2O2 overcomes both physical and physiological dormancies by hastening the transition of seeds from dormancy into germination. More DEGs related to gene expression regulation were involved in relieving physiological dormancy which provides new knowledge about the role of exogenous H2O2 as a signaling molecule for regulating gene activities during germination. Moreover, the protocol using H2O2 to promote germination will be useful for rescuing plant germplasms with poor germination.

Contribution of Whole-Genome Sequencing and Transcript Analysis to Decipher Retinal Diseases Associated with MFSD8 Variants.

Biallelic gene defects in MFSD8 are not only a cause of the late-infantile form of neuronal ceroid lipofuscinosis, but also of rare isolated retinal degeneration. We report clinical and genetic data of seven patients compound heterozygous or homozygous for variants in MFSD8, issued from a French cohort with inherited retinal degeneration, and two additional patients retrieved from a Swiss cohort. Next-generation sequencing of large panels combined with whole-genome sequencing allowed for the identification of twelve variants from which seven were novel. Among them were one deep intronic variant c.998+1669A>G, one large deletion encompassing exon 9 and 10, and a silent change c.750A>G. Transcript analysis performed on patients' lymphoblastoid cell lines revealed the creation of a donor splice site by c.998+1669A>G, resulting in a 140 bp pseudoexon insertion in intron 10. Variant c.750A>G produced exon 8 skipping. In silico and in cellulo studies of these variants allowed us to assign the pathogenic effect, and showed that the combination of at least one severe variant with a moderate one leads to isolated retinal dystrophy, whereas the combination in trans of two severe variants is responsible for early onset severe retinal dystrophy in the context of late-infantile neuronal ceroid lipofuscinosis.

Identification of the key functional genes in salt-stress tolerance of Cyanobacterium Phormidium tenue using in silico analysis.

The development of artificial biocrust using cyanobacterium Phormidium tenue has been suggested as an effective strategy to prevent soil degradation. Here, a combination of in silico approaches with growth rate, photosynthetic pigment, morphology, and transcript analysis was used to identify specific genes and their protein products in response to 500 mM NaCl in P. tenue. The results show that 500 mM NaCl induces the expression of genes encoding glycerol-3-phosphate dehydrogenase (glpD) as a Flavoprotein, ribosomal protein S12 methylthiotransferase (rimO), and a hypothetical protein (sll0939). The constructed co-expression network revealed a group of abiotic stress-responsive genes. Using the Basic Local Alignment Search Tool (BLAST), the homologous proteins of rimO, glpD, and sll0939 were identified in the P. tenue genome. Encoded proteins of glpD, rimO, and DUF1622 genes, respectively, contain (DAO and DAO C), (UPF0004, Radical SAM and TRAM 2), and (DUF1622) domains. The predicted ligand included 22B and MG for DUF1622, FS5 for rimO, and FAD for glpD protein. There was no direct disruption in ligand-binding sites of these proteins by Na+, Cl-, or NaCl. The growth rate, photosynthetic pigment, and morphology of P. tenue were investigated, and the result showed an acceptable tolerance rate of this microorganism under salt stress. The quantitative real-time polymerase chain reaction (qRT-PCR) results revealed the up-regulation of glpD, rimO, and DUF1622 genes under salt stress. This is the first report on computational and experimental analyses of the glpD, rimO, and DUF1622 genes in P. tenue under salt stress to the best of our knowledge. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03050-w.

Emotion Words in Early Childhood: A Language Transcript Analysis.

Children learn the abstract, challenging categories of emotions from young ages, and it has recently been suggested that language (and more specifically emotion words) may aid this learning. To examine the language that young children hear and produce as they're learning emotion categories, the present study examined nearly 2,000 transcripts from 179 children ranging from 15- to 47-months from the Child Language Data Exchange System (CHILDES). Results provide key descriptive, developmental, and predictive information regarding child emotion language production, including the finding that child emotion word production was predicted by mothers' emotion word production (ß=.21, p<.001), but not by child or mother language complexity (ß=.01, p=.690; ß=.00, p=.872). Frequency of specific emotion words are presented, as are developmental trends in early emotion language production and input. These results improve the understanding of children's daily emotional language environments and may inform theories of emotional development.

Combined microbial transcript and metabolic analysis reveals the different roles of hydrochar and biochar in promoting anaerobic digestion of waste activated sludge.

Hydrothermal pretreatment of waste activated sludge (WAS) could eliminate the rate limiting step of anaerobic digestion (AD) -hydrolysis. However, the high organic loading rate may cause acid accumulation, thus leading to an unstable system. This study compared the effect of different hydrochar (HC2-260°C and HC3-320°C) and biochar (BC5-500°C and BC7-700°C) on AD of hydrothermal pretreated WAS (HPS). Results demonstrated that hydrochar was superior to biochar in the methane yield and production rate, especially HC2. HC2 had the highest surface oxygen-containing functional groups that could facilitate direct interspecies electron transfer (DIET). The enhanced methane yield was related with the increased protein utilization, and hydrochar and biochar enriched different microbes related to protein degradation. Metabolomic analysis showed the significantly changed metabolites induced by hydrochar and biochar were involved in fatty acids and amino acids-related metabolism, indicating the rapid conversion of intermediated products, which was consistent with the microbial community structure results. Hydrochar and biochar also induced upregulation of metabolites related to microbial metabolic activity and extracellular electron transfer. Although biochar induced the same metabolic changes, the alterations of these metabolites were weaker than those of hydrochar. The results of this study offered new insights into the molecular mechanisms of enhanced AD of HPS by hydrochar and biochar.

Systematic Review of Genotype-Phenotype Correlations in Frasier Syndrome.

INTRODUCTION: Frasier syndrome (FS) is a rare inherited kidney disease caused by intron 9 splicing variants of WT1. For wild-type WT1, 2 active splice donor sites in intron 9 cause a mixture of 2 essential transcripts (with or without lysine-threonine-serine [+/KTS or -KTS]), and imbalance of the +KTS/-KTS ratio results in the development of FS. To date, 6 causative intron 9 variants have been identified; however, detailed transcript analysis has not yet been conducted and the genotype-phenotype correlation also remains to be elucidated. METHODS: We conducted an in vitro minigene splicing assay for 6 reported causative variants and in vivo RNA sequencing to determine the +KTS/-KTS ratio using patients' samples. We also performed a systematic review of reported FS cases with a description of the renal phenotype. RESULTS: The in vitro assay revealed that although all mutant alleles produced -KTS transcripts only, the wild-type allele produced both +KTS and -KTS transcripts at a 1:1 ratio. In vivo RNA sequencing showed that patients' samples with all heterozygous variants produced similar ratios of +KTS to -KTS (1:3.2-1:3.5) and wild-type kidney showed almost a 1:1 ratio (1:0.85). A systematic review of 126 cases clarified that the median age of developing ESKD was 16 years in all FS patients, and there were no statistically significant differences between the genotypes or sex chromosome karyotypes in terms of the renal survival period. CONCLUSION: Our study suggested no differences in splicing pattern or renal survival period among reported intron 9 variants causative of FS.